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The Howard Hughes Medical Institute is a United States non-profit medical research organization based in Chevy Chase, Maryland. It was founded by the American businessman Howard Hughes in 1953. It is one of the largest private funding organizations for biological and medical research in the United States. HHMI spends about $1 million per HHMI Investigator per year, which amounts to annual investment in biomedical research of about $825 million. The institute has an endowment of $16.9 billion, making it the second-wealthiest philanthropic organization in the United States and the second best endowed medical research foundation in the world. HHMI is the former owner of the Hughes Aircraft Company - an American aerospace firm which was divested to various firms over time. Wikipedia.

Mani R.-S.,University of Michigan | Chinnaiyan A.M.,University of Michigan | Chinnaiyan A.M.,Howard Hughes Medical Institute
Nature Reviews Genetics | Year: 2010

Genomic rearrangements are associated with many human genomic disorders, including cancers. It was previously thought that most genomic rearrangements formed randomly but emerging data suggest that many are nonrandom, cell type-, cell stage- and locus-specific events. Recent studies have revealed novel cellular mechanisms and environmental cues that influence genomic rearrangements. In this Review, we consider the multitude of influences on genomic rearrangements by grouping these influences into four categories: proximity of chromosomal regions in the nucleus, cellular stress, inappropriate DNA repair or recombination, and DNA sequence and chromatin features. The synergy of these triggers can poise a cell for rearrangements and here we aim to provide a conceptual framework for understanding the genesis of genomic rearrangements. © 2010 Macmillan Publishers Limited. All rights reserved.

Sternberg S.H.,University of California at Berkeley | Doudna J.A.,University of California at Berkeley | Doudna J.A.,Howard Hughes Medical Institute | Doudna J.A.,Lawrence Berkeley National Laboratory
Molecular Cell | Year: 2015

Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, anRNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control. © 2015 Elsevier Inc.

Bergmann A.,University of Texas M. D. Anderson Cancer Center | Steller H.,Howard Hughes Medical Institute
Science Signaling | Year: 2010

Most metazoans have at least some ability to regenerate damaged cells and tissues, although the regenerative capacity varies depending on the species, organ, or developmental stage. Cell replacement and regeneration occur in two contexts: renewal of spent cells during tissue homeostasis (homeostatic growth), and in response to external injury, wounding, or amputation (epimorphic regeneration). Model organisms that display remarkable regenerative capacity include amphibians, planarians, Hydra, and the vertebrate liver. In addition, several mammalian organs - including the skin, gut, kidney, muscle, and even the human nervous system - have some ability to replace spent or damaged cells. Although the regenerative response is complex, it typically involves the induction of new cell proliferation through formation of a blastema, followed by cell specification, differentiation, and patterning. Stem cells and undifferentiated progenitor cells play an important role in both tissue homeostasis and tissue regeneration. Stem cells are typically quiescent or passing slowly through the cell cycle in adult tissues, but they can be activated in response to cell loss and wounding. A series of studies, mostly performed in Drosophila as well as in Hydra, Xenopus, and mouse, has revealed an unexpected role of apoptotic caspases in the production of mitogenic signals that stimulate the proliferation of stem and progenitor cells to aid in tissue regeneration. This Review summarizes some of the key findings and discusses links to stem cell biology and cancer.

Frank J.,Howard Hughes Medical Institute | Frank J.,Columbia University | Gonzalez Jr. R.L.,Columbia University
Annual Review of Biochemistry | Year: 2010

There is mounting evidence indicating that protein synthesis is driven and regulated by mechanisms that direct stochastic, large-scale conformational fluctuations of the translational apparatus. This mechanistic paradigm implies that a free-energy landscape governs the conformational states that are accessible to and sampled by the translating ribosome. This scenario presents interdependent opportunities and challenges for structural and dynamic studies of protein synthesis. Indeed, the synergism between cryogenic electron microscopic and X-ray crystallographic structural studies, on the one hand, and single-molecule fluorescence resonance energy transfer (smFRET) dynamic studies, on the other, is emerging as a powerful means for investigating the complex free-energy landscape of the translating ribosome and uncovering the mechanisms that direct the stochastic conformational fluctuations of the translational machinery. In this review, we highlight the principal insights obtained from cryogenic electron microscopic, X-ray crystallographic, and smFRET studies of the elongation stage of protein synthesis and outline the emerging themes, questions, and challenges that lie ahead in mechanistic studies of translation. © 2010 by Annual Reviews. All rights reserved.

Pang Z.P.,Stanford University | Sudhof T.C.,Stanford University | Sudhof T.C.,Howard Hughes Medical Institute
Current Opinion in Cell Biology | Year: 2010

Ca2+ triggers many forms of exocytosis in different types of eukaryotic cells, for example synaptic vesicle exocytosis in neurons, granule exocytosis in mast cells, and hormone exocytosis in endocrine cells. Work over the past two decades has shown that synaptotagmins function as the primary Ca2+-sensors for most of these forms of exocytosis, and that synaptotagmins act via Ca2+-dependent interactions with both the fusing phospholipid membranes and the membrane fusion machinery. However, some forms of Ca2+-induced exocytosis may utilize other, as yet unidentified Ca2+-sensors, for example, slow synaptic exocytosis mediating asynchronous neurotransmitter release. In the following overview, we will discuss the synaptotagmin-based mechanism of Ca2+-triggered exocytosis in neurons and neuroendocrine cells, and its potential extension to other types of Ca2+-stimulated exocytosis for which no synaptotagmin Ca2+-sensor has been identified. © 2010 Elsevier Ltd.

Zhou H.-X.,Florida State University | McCammon J.A.,Howard Hughes Medical Institute
Trends in Biochemical Sciences | Year: 2010

Protein dynamics are essential for virtually all protein functions, certainly for gating mechanisms of ion channels and regulation of enzyme catalysis. Ion channels usually feature a gate in the channel pore that prevents ion permeation in the closed state. Some bifunctional enzymes with two distant active sites use a tunnel to transport intermediate products; a gate can help prevent premature leakage. Enzymes with a buried active site also require a tunnel for substrate entrance; a gate along the tunnel can contribute to selectivity. The gates in these different contexts show distinct characteristics in sequence, structure and dynamics, but they also have common features. In particular, aromatic residues often appear to serve as gates, probably because of their ability, through side chain rotation, to effect large changes in cross section. © 2009 Elsevier Ltd. All rights reserved.

Jorgensen E.M.,Howard Hughes Medical Institute
Current Biology | Year: 2014

The human brain is easily the most baffling bit of biology on the planet. How did the nervous system evolve? What came first: neurons or synaptic proteins? A new paper studying the pancake-shaped Trichoplax suggests it was not the neurons. © 2014 Elsevier Ltd All rights reserved.

Sudhof T.C.,Howard Hughes Medical Institute
Angewandte Chemie - International Edition | Year: 2014

The most important property of synaptic transmission is its speed, which is crucial for the overall workings of the brain. In his Nobel Lecture, T. C. Südhof explains how the synaptic vesicle and the plasma membrane undergo rapid fusion during neurotransmitter release and how this process is spatially organized, such that opening of Ca2+-channels allows rapid translation of the entering Ca2+ signal into a fusion event. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Korennykh A.,Princeton University | Walter P.,Howard Hughes Medical Institute | Walter P.,University of California at San Francisco
Annual Review of Cell and Developmental Biology | Year: 2012

The unfolded protein response (UPR) is a network of intracellular signaling pathways that maintain the protein-folding capacity of the endoplasmic reticulum (ER) in eukaryotic cells. Dedicated molecular sensors embedded in the ER membrane detect incompletely folded or unfolded proteins in the ER lumen and activate a transcriptional program that increases the abundance of the ER according to need. In metazoans the UPR additionally regulates translation and thus relieves unfolded protein load by globally reducing protein synthesis. If homeostasis in the ER cannot be reestablished, the metazoan UPR switches from the prosurvival to the apoptotic mode. The UPR involves a complex, coordinated action of many genes that is controlled by one ER-embedded sensor, Ire1, in yeasts, and three sensors, Ire1, PERK, and ATF6, in higher eukaryotes, including human. We discuss the emerging molecular understanding of the UPR and focus on the structural biology of Ire1 and PERK, the two recently crystallized UPR sensors. Copyright © 2012 by Annual Reviews. All rights reserved.

Rajan A.,Harvard University | Perrimon N.,Harvard University | Perrimon N.,Howard Hughes Medical Institute
Cell | Year: 2012

In Drosophila, the fat body (FB), a functional analog of the vertebrate adipose tissue, is the nutrient sensor that conveys the nutrient status to the insulin-producing cells (IPCs) in the fly brain to release Drosophila insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. We identify Unpaired 2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the fed state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, we find that human Leptin can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin. © 2012 Elsevier Inc.

Mabb A.M.,Duke University | Ehlers M.D.,Duke University | Ehlers M.D.,Howard Hughes Medical Institute
Annual Review of Cell and Developmental Biology | Year: 2010

Neurons are highly specialized cells whose connectivity at synapses subserves rapid information transfer in the brain. Proper information processing, learning, and memory storage in the brain requires continuous remodeling of synaptic networks. Such remodeling includes synapse formation, elimination, synaptic protein turnover, and changes in synaptic transmission. An emergent mechanism for regulating synapse function is posttranslational modification through the ubiquitin pathway at the postsynaptic membrane. Here, we discuss recent findings implicating ubiquitination and protein degradation in postsynaptic function and plasticity. We describe postsynaptic ubiquitination pathways and their role in brain development, neuronal physiology, and brain disorders. Copyright © 2010 by Annual Reviews. All rights reserved.

Cabeen M.T.,Yale University | Jacobs-Wagner C.,Yale University | Jacobs-Wagner C.,Howard Hughes Medical Institute
Annual Review of Genetics | Year: 2010

Bacteria, like eukaryotes, employ cytoskeletal elements to perform many functions, including cell morphogenesis, cell division, DNA partitioning, and cell motility. They not only possess counterparts of eukaryotic actin, tubulin, and intermediate filament proteins, but they also have cytoskeletal elements of their own. Unlike the rigid sequence and structural conservation often observed for eukaryotic cytoskeletal proteins, the bacterial counterparts can display considerable diversity in sequence and function across species. Their wide range of function highlights the flexibility of core cytoskeletal protein motifs, such that one type of cytoskeletal element can perform various functions, and one function can be performed by different types of cytoskeletal elements. © 2010 by Annual Reviews. All rights reserved.

Pyle A.M.,Howard Hughes Medical Institute
Critical Reviews in Biochemistry and Molecular Biology | Year: 2010

Group II introns are some of the largest ribozymes in nature, and they are a major source of information about RNA assembly and tertiary structural organization. These introns are of biological significance because they are self-splicing mobile elements that have migrated into diverse genomes and played a major role in the genomic organization and metabolism of most life forms. The tertiary structure of group II introns has been the subject of many phylogenetic, genetic, biochemical and biophysical investigations, all of which are consistent with the recent crystal structure of an intact group IIC intron from the alkaliphilic eubacterium Oceanobacillus iheyensis. The crystal structure reveals that catalytic intron domain V is enfolded within the other intronic domains through an elaborate network of diverse tertiary interactions. Within the folded core, DV adopts an activated conformation that readily binds catalytic metal ions and positions them in a manner appropriate for reaction with nucleic acid targets. The tertiary structure of the group II intron reveals new information on motifs for RNA architectural organization, mechanisms of group II intron catalysis, and the evolutionary relationships among RNA processing systems. Guided by the structure and the wealth of previous genetic and biochemical work, it is now possible to deduce the probable location of DVI and the site of additional domains that contribute to the function of the highly derived group IIB and IIA introns. © 2010 Informa UK Ltd.

Zheng Y.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2010

Eukaryotic cell division uses morphologically different forms of mitosis, referred to as open, partially open and closed mitosis, for accurate chromosome segregation and proper partitioning of other cellular components such as endomembranes and cell fate determinants. Recent studies suggest that the spindle matrix provides a conserved strategy to coordinate the segregation of genetic material and the partitioning of the rest of the cellular contents in all three forms of mitosis. © 20 Macmillan Publishers Limited. All rights reserved.

Kelly A.,University of California at Berkeley | Wickliffe K.,University of California at Berkeley | Song L.,University of California at Berkeley | Fedrigo I.,University of California at Berkeley | Rape M.,Howard Hughes Medical Institute
Molecular Cell | Year: 2014

Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood. Here, we show that the RING-E3 APC/C catalyzes chain elongation by strongly increasing the affinity of its E2 for the distal acceptor ubiquitin in a growing conjugate. This function of the APC/C requires its coactivator as well as conserved residues of the E2 and ubiquitin. APC/C's ability to track the tip of an emerging conjugate is required for APC/C-substrate degradation and accurate cell division. Our results suggest that RING-E3s tether the distal ubiquitin of a growing chain in proximity to the active site of their E2s, allowing them to assemble polymeric conjugates without altering their binding to substrate or E2. Kelly et al. demonstrate how a RING-dependent E3 ligase, the human APC/C, assembles long ubiquitin chains by tethering the distal ubiquitin of the growing chain close to the active site of its associated E2 conjugating enzyme. © 2014 Elsevier Inc.

Hobert O.,Howard Hughes Medical Institute
Genetics | Year: 2010

Much of our understanding of how organisms develop and function is derived from the extraordinarily powerful, classic approach of screening for mutant organisms in which a specific biological process is disrupted. Reaping the fruits of such forward genetic screens in metazoan model systems like Drosophila, Caenorhabditis elegans, or zebrafish traditionally involves time-consuming positional cloning strategies that result in the identification of the mutant locus. Whole genome sequencing (WGS) has begun to provide an effective alternative to this approach through direct pinpointing of the molecular lesion in a mutated strain isolated from a genetic screen. Apart from significantly altering the pace and costs of genetic analysis, WGS also provides new perspectives on solving genetic problems that are difficult to tackle with conventional approaches, such as identifying the molecular basis of multigenic and complex traits. Copyright © 2010 by the Genetics Society of America.

Lindquist S.L.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2011

Maintaining the proteome to preserve the health of an organism in the face of developmental changes, environmental insults, infectious diseases, and rigors of aging is a formidable task. The challenge is magnified by the inheritance of mutations that render individual proteins subject to misfolding and/or aggregation. Maintenance of the proteome requires the orchestration of protein synthesis, folding, degradation, and trafficking by highly conserved/deeply integrated cellular networks. In humans, no less than 2000 genes are involved. Stress sensors detect the misfolding and aggregation of proteins in specific organelles and respond by activating stress-responsive signaling pathways. These culminate in transcriptional and posttranscriptional programs that up-regulate the homeostatic mechanisms unique to that organelle. Proteostasis is also strongly influenced by the general properties of protein folding that are intrinsic to every proteome. These include the kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We then introduce chemical approaches to prevent the misfolding or aggregation of specific proteins through direct binding interactions. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organismal proteostasis.

Sudhof T.C.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2011

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for "soluble NSF-attachment protein receptor") and SM (for "Sec1/Munc18-like") proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for "N-ethylmaleimide sensitive factor"). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the "active zone") that includes Munc13 and RIM proteins as central components.

Zoghbi H.Y.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2012

The discovery of the genetic causes of syndromic autism spectrum disorders and intellectual disabilities has greatly informed our understanding of the molecular pathways critical for normal synaptic function. The top-down approaches using human phenotypes and genetics helped identify causative genes and uncovered the broad spectrum of neuropsychiatric features that can result from various mutations in the same gene. Importantly, the human studies unveiled the exquisite sensitivity of cognitive function to precise levels of many diverse proteins. Bottom-up approaches applying molecular, biochemical, and neurophysiological studies to genetic models of these disorders revealed unsuspected pathogenic mechanisms and identified potential therapeutic targets. Moreover, studies in model organisms showed that symptoms of these devastating disorders can be reversed, which brings hope that affected individuals might benefit from interventions even after symptoms set in. Scientists predict that insights gained from studying these rare syndromic disorders will have an impact on the more common nonsyndromic autism and mild cognitive deficits.

Nussenzweig A.,U.S. National Institutes of Health | Nussenzweig M.C.,Rockefeller University | Nussenzweig M.C.,Howard Hughes Medical Institute
Cell | Year: 2010

Aberrant fusions between heterologous chromosomes are among the most prevalent cytogenetic abnormalities found in cancer cells. Oncogenic chromosomal translocations provide cells with a proliferative or survival advantage. They may either initiate transformation or be acquired secondarily as a result of genomic instability. Here, we highlight recent advances toward understanding the origin of chromosomal translocations in incipient lymphoid cancers and how tumor-suppressive pathways normally limit the frequency of these aberrant recombination events. Deciphering the mechanisms that mediate chromosomal fusions will open new avenues for developing therapeutic strategies aimed at eliminating lesions that lead to the initiation, maintenance, and progression of cancer. © 2010 Elsevier Inc.

Sudhof T.C.,Howard Hughes Medical Institute
Nature | Year: 2015

The finding that acute and chronic manipulations of the same neural circuit can produce different behavioural outcomes poses new questions about how best to analyse these circuits. © 2015 Macmillan Publishers Limited. All rights reserved.

Kim Y.,Howard Hughes Medical Institute
Nucleus (Austin, Tex.) | Year: 2012

Lamins are the major structural components of the nuclear lamina found in metazoan organisms. Extensive studies using tissue culture cells have shown that lamins are involved in a wide range of basic cell functions. This has led to the prevailing idea that a given animal cell needs at least one lamin protein for its basic proliferation and survival. However, recent studies have shown that lamins are dispensable for the proliferation and survival of mouse embryonic stem cells (ESC). In contrast to a lack of essential functions in ESCs, certain differentiated cells lacking B-type lamins exhibit increased cell cycle exit rates and enhanced senescence. In this Extra View, we discuss how studies using animal models and cell cultures have begun to reveal cell-type specific functions of lamins in tissue building and homeostasis.

Law J.A.,University of California at Los Angeles | Jacobsen S.E.,University of California at Los Angeles | Jacobsen S.E.,Howard Hughes Medical Institute
Nature Reviews Genetics | Year: 2010

Cytosine DNA methylation is a stable epigenetic mark that is crucial for diverse biological processes, including gene and transposon silencing, imprinting and X chromosome inactivation. Recent findings in plants and animals have greatly increased our understanding of the pathways used to accurately target, maintain and modify patterns of DNA methylation and have revealed unanticipated mechanistic similarities between these organisms. Key roles have emerged for small RNAs, proteins with domains that bind methylated DNA and DNA glycosylases in these processes. Drawing on insights from both plants and animals should deepen our understanding of the regulation and biological significance of DNA methylation. © 2010 Macmillan Publishers Limited. All rights reserved.

Abraham C.,Yale University | Medzhitov R.,Yale University | Medzhitov R.,Howard Hughes Medical Institute
Gastroenterology | Year: 2011

The intestinal immune system defends against pathogens and entry of excessive intestinal microbes; simultaneously, a state of immune tolerance to resident intestinal microbes must be maintained. Perturbation of this balance is associated with intestinal inflammation in various mouse models and is thought to predispose humans to inflammatory bowel disease (IBD). The innate immune system senses microbes; dendritic cells, macrophages, and epithelial cells produce an initial, rapid response. The immune system continuously monitors resident microbiota and utilizes constitutive antimicrobial mechanisms to maintain immune homeostasis. associations between IBD and genes that regulate microbial recognition and innate immune pathways, such as nucleotide oligomerization domain 2 (Nod2), genes that control autophagy (eg, ATG16L1, IRGM), and genes in the interleukin-23T helper cell 17 pathway indicate the important roles of host-microbe interactions in regulating intestinal immune homeostasis. There is increasing evidence that intestinal microbes influence host immune development, immune responses, and susceptibility to human diseases such as IBD, diabetes mellitus, and obesity. Conversely, host factors can affect microbes, which in turn modulate disease susceptibility. We review the cell populations and mechanisms that mediate interactions between host defense and tolerance and how the dysregulation of host-microbe interactions leads to intestinal inflammation and IBD. © 2011 AGA Institute.

Kolodkin A.L.,Howard Hughes Medical Institute | Tessier-Lavigne M.,Genentech | Tessier-Lavigne M.,Rockefeller University
Cold Spring Harbor Perspectives in Biology | Year: 2011

The complex patterns of neuronal wiring in the adult nervous system depend on a series of guidance events during neural development that establish a framework on which functional circuits can be built. In this subject collection the cellular and molecular mechanisms that underlie neuronal guidance are considered from several perspectives ranging from how cytoskeletal dynamics within extending neuronal growth cones steer axons to howguidance cues influence synaptogenesis. We introduce here some basic topics to frame the more detailed reviews in following articles including the cellular strategies that define basic themes governing neuronal wiring throughout life an enumeration of the molecular cues and receptors knownto play key guidance roles during neural development and an overview of the signaling mechanisms that transduce guidance information into growth-cone steering. © 2011 Cold Spring Harbor Laboratory Press.

Green A.A.,Wyss Institute for Biologically Inspired Engineering | Silver P.A.,Wyss Institute for Biologically Inspired Engineering | Silver P.A.,Harvard University | Collins J.J.,Wyss Institute for Biologically Inspired Engineering | And 3 more authors.
Cell | Year: 2014

Efforts to construct synthetic networks in living cells have been hindered by the limited number of regulatory components that provide wide dynamic range and low crosstalk. Here, we report a class of de-novo-designed prokaryotic riboregulators called toehold switches that activate gene expression in response to cognate RNAs with arbitrary sequences. Toehold switches provide a high level of orthogonality and can be forward engineered to provide average dynamic range above 400. We show that switches can be integrated into the genome to regulate endogenous genes and use them as sensors that respond to endogenous RNAs. We exploit the orthogonality of toehold switches to regulate 12 genes independently and to construct a genetic circuit that evaluates 4-input AND logic. Toehold switches, with their wide dynamic range, orthogonality, and programmability, represent a versatile and powerful platform for regulation of translation, offering diverse applications in molecular biology, synthetic biology, and biotechnology. © 2014 Elsevier Inc.

Deal R.B.,Fred Hutchinson Cancer Research Center | Henikoff S.,Fred Hutchinson Cancer Research Center | Henikoff S.,Howard Hughes Medical Institute
Developmental Cell | Year: 2010

Understanding the production and function of specialized cells during development requires the isolation of individual cell types for analysis, but this is currently a major technical challenge. Here we describe a method for cell type-specific RNA and chromatin profiling that circumvents many of the limitations of current methods for cell isolation. We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. We identified hundreds of genes that are preferentially expressed in each cell type and show that genes with the largest expression differences between hair and non-hair cells also show differences between cell types in the trimethylation of histone H3 at lysines 4 and 27. This method should be applicable to any organism that is amenable to transformation. © 2010 Elsevier Inc.

Greene E.C.,Howard Hughes Medical Institute
Methods in enzymology | Year: 2010

Single-molecule approaches provide a valuable tool in the arsenal of the modern biologist, and new discoveries continue to be made possible through the use of these state-of-the-art technologies. However, it can be inherently difficult to obtain statistically relevant data from experimental approaches specifically designed to probe individual reactions. This problem is compounded with more complex biochemical reactions, heterogeneous systems, and/or reactions requiring the use of long DNA substrates. Here we give an overview of a technology developed in our laboratory, which relies upon simple micro- or nanofabricated structures in combination with "bio-friendly" lipid bilayers, to align thousands of long DNA molecules into defined patterns on the surface of a microfluidic sample chamber. We call these "DNA curtains," and we have developed several different versions varying in complexity and DNA substrate configuration, which are designed to meet different experimental needs. This novel approach to single-molecule imaging provides a powerful experimental platform that offers the potential for concurrent observation of hundreds or even thousands of protein-DNA interactions in real time. Copyright 2010 Elsevier Inc. All rights reserved.

Su L.,Stanford University | Kidd B.,Stanford University | Han A.,Stanford University | Kotzin J.,Stanford University | And 2 more authors.
Immunity | Year: 2013

Although T cell memory is generally thought to require direct antigen exposure, we found an abundance of memory-phenotype cells (20%-90%, averaging over 50%) of CD4+ T cells specific to viral antigens in adults who had never been infected. These cells express the appropriate memory markers and genes, rapidly produce cytokines, and have clonally expanded. In contrast, the same T cell receptor (TCR) specificities in newborns are almost entirely naïve, which might explain the vulnerability of young children to infections. One mechanism for this phenomenon is TCR cross-reactivity to environmental antigens, and in support of this, we found extensive cross-recognition by HIV-1 and influenza-reactive T lymphocytes to other microbial peptides and expansion of one of these after influenza vaccination. Thus, the presence of these memory-phenotype T cells has significant implications for immunity to novel pathogens, child and adult health, and the influence of pathogen-rich versus hygienic environments. © 2013 Elsevier Inc.

Schekman R.,Howard Hughes Medical Institute
Molecular biology of the cell | Year: 2010

George Palade, a founding father of cell biology and of the American Society for Cell Biology (ASCB), established the ultrastructural framework for an analysis of how proteins are secreted and membranes are assembled in eukaryotic cells. His vision inspired a generation of investigators to probe the molecular mechanisms of protein transport. My laboratory has dissected these pathways with complementary genetic and biochemical approaches. Peter Novick, one of my first graduate students, isolated secretion mutants of Saccharomyces cerevisiae, and through cytological analysis of single and double mutants and molecular cloning of the corresponding SEC genes, we established that yeast cells use a secretory pathway fundamentally conserved in all eukaryotes. A biochemical reaction that recapitulates the first half of the secretory pathway was used to characterize Sec proteins that comprise the polypeptide translocation channel in the endoplasmic reticulum (ER) membrane (Sec61) and the cytoplasmic coat protein complex (COPII) that captures cargo proteins into transport vesicles that bud from the ER.

Schuldiner M.,Weizmann Institute of Science | Weissman J.S.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2013

The endoplasmic reticulum (ER) is a complex organelle responsible for a range of functions including protein folding and secretion, lipid biosynthesis, and ion homeostasis. Despite its central and essential roles in eukaryotic cells during development, growth, and disease, many ER proteins are poorly characterized. Moreover, the range of biochemical reactions that occur within the ER membranes, let alone how these different activities are coordinated, is not yet defined. In recent years, focused studies on specific ER functions have been complemented by systematic approaches and innovative technologies for high-throughput analysis of the location, levels, and biological impact of given components. This article focuses on the recent progress of these efforts, largely pioneered in the budding yeast Saccharomyces cerevisiae, and also addresses how future systematic studies can be geared to uncover the "dark matter" of uncharted ER functions. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

Castel S.E.,Cold Spring Harbor Laboratory | Martienssen R.A.,Cold Spring Harbor Laboratory | Martienssen R.A.,Howard Hughes Medical Institute
Nature Reviews Genetics | Year: 2013

A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability. © 2013 Macmillan Publishers Limited. All rights reserved.

Molkentin J.D.,Howard Hughes Medical Institute
Circulation Research | Year: 2011

Transient receptor potential (TRP) channels of multiple subclasses are expressed in the heart, although their functions are only now beginning to emerge, especially for the TRPC subclass that appears to regulate the cardiac hypertrophic response. Although TRP channels permeate many different cations, they are most often ascribed a specific biological function because of Ca 2+ influx, either for microdomain signaling or to reload internal Ca2+ stores in the endoplasmic reticulum through a store-operated mechanism. However, adult cardiac myocytes arguably do not require store-operated Ca2+ entry to regulate sarcoplasmic reticulum Ca 2+ levels and excitation-contraction coupling; hence, TRP channels expressed in the heart most likely coordinate signaling within local domains or through direct interaction with Ca-dependent regulatory proteins. Here, we review the emerging evidence that TRP channels, especially TRPCs, are critical regulators of microdomain signaling in the heart to control pathological hypertrophy in coordination with signaling through effectors such as calcineurin and NFAT (nuclear factor of activated T cells). © 2011 American Heart Association, Inc.

Lian Y.,Yale University | Bergman R.G.,Lawrence Berkeley National Laboratory | Lavis L.D.,Howard Hughes Medical Institute | Ellman J.A.,Yale University
Journal of the American Chemical Society | Year: 2013

An efficient, one-step, and highly functional group-compatible synthesis of substituted N-aryl-2H-indazoles is reported via the rhodium(III)-catalyzed C-H bond addition of azobenzenes to aldehydes. The regioselective coupling of unsymmetrical azobenzenes was further demonstrated and led to the development of a new removable aryl group that allows for the preparation of indazoles without N-substitution. The 2-aryl-2H-indazole products also represent a new class of readily prepared fluorophores for which initial spectroscopic characterization has been performed. © 2013 American Chemical Society.

Kimble J.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2011

A major step in the journey from germline stem cell to differentiated gamete is the decision to leave the mitotic cell cycle and begin progression through the meiotic cell cycle. Over the past decade, molecular regulators of the mitosis/meiosis decision have been discovered in most of the major model multicellular organisms. Historically, the mitosis/meiosis decision has been closely linked with controls of germline self-renewal and the sperm/egg decision, especially in nematodes and mice. Molecular explanations of those linkages clarify our understanding of this fundamental germ cell decision, and unifying themes have begun to emerge. Although the complete circuitry of the decision is not known in any organism, the recent advances promise to impact key issues in human reproduction and agriculture.

Hollenhorst P.C.,Indiana University | McIntosh L.P.,University of British Columbia | Graves B.J.,University of Utah | Graves B.J.,Howard Hughes Medical Institute
Annual Review of Biochemistry | Year: 2011

ETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5′-GGA(A/T)-3′. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells. © 2011 by Annual Reviews. All rights reserved.

Alegado R.A.,University of the Sea | King N.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2014

Animals evolved in seas teeming with bacteria, yet the influences of bacteria on animal origins are poorly understood. Comparisons among modern animals and their closest living relatives, the choanoflagellates, suggest that the first animals used flagellated collar cells to capture bacterial prey. The cell biology of prey capture, such as cell adhesion between predator and prey, involves mechanisms that may have been co-opted to mediate intercellular interactions during the evolution of animal multicellularity. Moreover, a history of bacterivory may have influenced the evolution of animal genomes by driving the evolution of genetic pathways for immunity and facilitating lateral gene transfer. Understanding the interactions between bacteria and the progenitors of animals may help to explain themyriad ways in which bacteria shape the biology of modern animals, including ourselves. © 2014 Cold Spring Harbor Laboratory Press. All rights reserved.

Stroud J.C.,Howard Hughes Medical Institute
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

Fibrous proteins in the amyloid state are found both associated with numerous diseases and in the normal functions of cells. Amyloid fibers contain a repetitive spine, commonly built from a pair of β-sheets whose β-strands run perpendicular to the fiber direction and whose side chains interdigitate, much like the teeth of a zipper. In fiber spines known as homosteric zippers, identical protein segments sharing identical packing environments make the two β-sheets. In previous work based on atomic resolution crystal structures of homosteric zippers derived from a dozen proteins, the symmetries of homosteric zippers were categorized into eight classes. Here, it is shown through a formal derivation that each homosteric zipper class corresponds to a unique set of symmetry groups termed 'zipper groups'. Furthermore, the eight previously identified classes do not account for all of the 15 possible zipper groups, which may be categorized into the complete set of ten classes. Because of their foundations in group theory, the 15 zipper groups provide a mathematically rigorous classification for homosteric zippers. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved.

Parker R.,Howard Hughes Medical Institute
Genetics | Year: 2012

All RNA species in yeast cells are subject to turnover. Work over the past 20 years has defined degradation mechanisms for messenger RNAs, transfer RNAs, ribosomal RNAs, and noncoding RNAs. In addition, numerous quality control mechanisms that target aberrant RNAs have been identified. Generally, each decay mechanism contains factors that funnel RNA substrates to abundant exoand/or endonucleases. Key issues for future work include determining the mechanisms that control the specificity of RNA degradation and how RNA degradation processes interact with translation, RNA transport, and other cellular processes. © 2012 by the Genetics Society of America.

Song L.,University of California at Berkeley | Craney A.,University of California at Berkeley | Rape M.,University of California at Berkeley | Rape M.,Howard Hughes Medical Institute
Molecular Cell | Year: 2014

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex (APC/C). Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C substrates provides a mechanism for the selective disposal of cell-cycle regulators that have fulfilled their mitotic roles. © 2014 Elsevier Inc.

Pavri R.,Rockefeller University | Nussenzweig M.C.,Rockefeller University | Nussenzweig M.C.,Howard Hughes Medical Institute
Advances in Immunology | Year: 2011

Antibody maturation requires class switch recombination (CSR) and somatic hypermutation (SHM), both of which are initiated by activation-induced cytidine deaminase (AID). AID deaminates cytosine residues resulting in mismatches that are differentially processed to produce double-strand breaks in Ig switch (S) regions that lead to CSR, or to point mutations in variable (V) exons resulting in SHM. Although AID was first thought to be Ig-specific, recent work indicates that it also targets a diverse group of non- Ig loci, including genes such as Bcl6 and c-myc, whose modification by AID results in lymphoma-associated mutations and translocations. Here, we review the recent literature on AID targeting and the role for transcriptional stalling in recruitment of this enzyme to Ig and non- Ig loci. We propose a model for AID recruitment based on transcriptional stalling, which reconciles several of the key features of SHM, CSR, and lymphoma-associated translocation. © 2011 Elsevier Inc.

Xi L.,University of Colorado at Boulder | Cech T.R.,University of Colorado at Boulder | Cech T.R.,Howard Hughes Medical Institute
Nucleic Acids Research | Year: 2014

Telomerase is the ribonucleoprotein (RNP) enzyme that elongates telomeric DNA to compensate for the attrition occurring during each cycle of DNA replication. Knowing the levels of telomerase in continuously dividing cells is important for understanding how much telomerase is required for cell immortality. In this study, we measured the endogenous levels of the human telomerase RNP and its two key components, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). We estimate ∼240 telomerase monomers per cell for HEK 293T and HeLa, a number similar to that of telomeres in late S phase. The subunits were in excess of RNPs (e.g. ∼1150 hTR and ∼500 hTERT molecules per HeLa cell), suggesting the existence of unassembled components. This hypothesis was tested by overexpressing individual subunits, which increased total telomerase activity as measured by the direct enzyme assay. Thus, there are subpopulations of both hTR and hTERT not assembled into telomerase but capable of being recruited. We also determined the specific activity of endogenous telomerase and of overexpressed super-telomerase both to be ∼60 nt incorporated per telomerase per minute, with Km(dGTP) ∼17 μM, indicating super-telomerase is as catalytically active as endogenous telomerase and is thus a good model for biochemical studies. © 2014 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Kim P.B.,Yale University | Nelson J.W.,Yale University | Breaker R.R.,Yale University | Breaker R.R.,Howard Hughes Medical Institute
Molecular Cell | Year: 2015

Over 30 years ago, ZTP (5-aminoimidazole-4-carboxamide riboside 5'-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein, we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one-carbon metabolism. Biochemical data confirm that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches invivo. The wide distribution ofthis riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages. © 2015 Elsevier Inc.

Kirchhausen T.,Harvard University | Owen D.,University of Cambridge | Harrison S.C.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2014

Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation. © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Eisele E.,Johns Hopkins University | Siliciano R.,Johns Hopkins University | Siliciano R.,Howard Hughes Medical Institute
Immunity | Year: 2012

This Perspective proposes definitions for key terms in the field of HIV-1 latency and eradication. In the context of eradication, a reservoir is a cell type that allows persistence of replication-competent HIV-1 on a timescale of years in patients on optimal antiretroviral therapy. Reservoirs act as a barrier to eradication in the patient population in which cure attempts will likely be made. Halting viral replication is essential to eradication, and definitions and criteria for assessing whether this goal has been achieved are proposed. The cell types that may serve as reservoirs for HIV-1 are discussed. Currently, only latently infected resting CD4+ T cells fit the proposed definition of a reservoir, and more evidence is necessary to demonstrate that other cell types, including hematopoietic stem cells and macrophages, fit this definition. Further research is urgently required on potential reservoirs in the gut-associated lymphoid tissue and the central nervous system. © 2012 Elsevier Inc.

Vanharanta S.,Sloan Kettering Cancer Center | Massague J.,Sloan Kettering Cancer Center | Massague J.,Howard Hughes Medical Institute
Cancer Cell | Year: 2013

How cancer cells acquire the competence to colonize distant organs remains a central question in cancer biology. Tumors can release large numbers of cancer cells into the circulation, but only a small proportion of these cells survive on infiltrating distant organs and even fewer form clinically meaningful metastases. During the past decade, many predictive gene signatures and specific mediators of metastasis have been identified, yet how cancer cells acquire these traits has remained obscure. Recent experimental work and high-resolution sequencing of human tissues have started to reveal the molecular and tumor evolutionary principles that underlie the emergence of metastatic traits. © 2013 Elsevier Inc.

Zuo Y.,Yale University | Steitz T.A.,Yale University | Steitz T.A.,Howard Hughes Medical Institute
Molecular Cell | Year: 2015

During transcription initiation, RNA polymerase binds to promoter DNA to form an initiation complex containing a DNA bubble and enters into abortive cycles of RNA synthesis before escaping the promoter to transit into the elongation phase for processive RNA synthesis. Here we present the crystal structures of E.coli transcription initiation complexes containing a complete transcription bubble and de novo synthesized RNA oligonucleotides at about 6-Å resolution. The structures show how RNA polymerase recognizes DNA promoters that contain spacers of different lengths and reveal a bridging interaction between the 5'-triphosphate of the nascent RNA and the σ factor that may function to stabilize the short RNA-DNA hybrids during the early stage of transcription initiation. The conformation of the RNA oligonucleotides and the paths of the DNA strands in the complete initiation complexes provide insights into the mechanism that controls both the abortive and productive RNA synthesis. © 2015 Elsevier Inc.

Tanaka E.,TU Dresden | Reddien P.,Howard Hughes Medical Institute
Developmental Cell | Year: 2011

The ability of animals to regenerate missing parts is a dramatic and poorly understood aspect of biology. The sources of new cells for these regenerative phenomena have been sought for decades. Recent advances involving cell fate tracking in complex tissues have shed new light on the cellular underpinnings of regeneration in Hydra, planarians, zebrafish, Xenopus, and Axolotl. Planarians accomplish regeneration with use of adult pluripotent stem cells, whereas several vertebrates utilize a collection of lineage-restricted progenitors from different tissues. Together, an array of cellular strategies-from pluripotent stem cells to tissue-specific stem cells and dedifferentiation-are utilized for regeneration. © 2011 Elsevier Inc.

Cheung N.-K.V.,Sloan Kettering Cancer Center | Dyer M.A.,St Jude Childrens Research Hospital | Dyer M.A.,University of Tennessee Health Science Center | Dyer M.A.,Howard Hughes Medical Institute
Nature Reviews Cancer | Year: 2013

Neuroblastoma is a solid tumour that arises from the developing sympathetic nervous system. Over the past decade, our understanding of this disease has advanced tremendously. The future challenge is to apply the knowledge gained to developing risk-based therapies and, ultimately, improving outcome. In this Review we discuss the key discoveries in the developmental biology, molecular genetics and immunology of neuroblastoma, as well as new translational tools for bringing these promising scientific advances into the clinic. © 2013 Macmillan Publishers Limited. All rights reserved.

Suzanne M.,Toulouse 1 University Capitole | Suzanne M.,French National Center for Scientific Research | Steller H.,Howard Hughes Medical Institute
Cell Death and Differentiation | Year: 2013

Programmed cell death is an important process during development that serves to remove superfluous cells and tissues, such as larval organs during metamorphosis, supernumerary cells during nervous system development, muscle patterning and cardiac morphogenesis. Different kinds of cell death have been observed and were originally classified based on distinct morphological features: (1) type I programmed cell death (PCD) or apoptosis is recognized by cell rounding, DNA fragmentation, externalization of phosphatidyl serine, caspase activation and the absence of inflammatory reaction, (2) type II PCD or autophagy is characterized by the presence of large vacuoles and the fact that cells can recover until very late in the process and (3) necrosis is associated with an uncontrolled release of the intracellular content after cell swelling and rupture of the membrane, which commonly induces an inflammatory response. In this review, we will focus exclusively on developmental cell death by apoptosis and its role in tissue remodeling. © 2013 Macmillan Publishers Limited All rights reserved.

Lippert A.R.,University of California at Berkeley | New E.J.,University of California at Berkeley | Chang C.J.,University of California at Berkeley | Chang C.J.,Howard Hughes Medical Institute
Journal of the American Chemical Society | Year: 2011

Hydrogen sulfide (H 2S) is emerging as an important mediator of human physiology and pathology but remains difficult to study, in large part because of the lack of methods for selective monitoring of this small signaling molecule in live biological specimens. We now report a pair of new reaction-based fluorescent probes for selective imaging of H 2S in living cells that exploit the H 2S-mediated reduction of azides to fluorescent amines. Sulfidefluor-1 (SF1) and Sulfidefluor-2 (SF2) respond to H 2S by a turn-on fluorescence signal enhancement and display high selectivity for H 2S over other biologically relevant reactive sulfur, oxygen, and nitrogen species. In addition, SF1 and SF2 can be used to detect H 2S in both water and live cells, providing a potentially powerful approach for probing H 2S chemistry in biological systems. © 2011 American Chemical Society.

Kaelin Jr. W.G.,Harvard University | Kaelin Jr. W.G.,Howard Hughes Medical Institute | McKnight S.L.,University of Texas Southwestern Medical Center
Cell | Year: 2013

Chemical modifications of histones and DNA, such as histone methylation, histone acetylation, and DNA methylation, play critical roles in epigenetic gene regulation. Many of the enzymes that add or remove such chemical modifications are known, or might be suspected, to be sensitive to changes in intracellular metabolism. This knowledge provides a conceptual foundation for understanding how mutations in the metabolic enzymes SDH, FH, and IDH can result in cancer and, more broadly, for how alterations in metabolism and nutrition might contribute to disease. Here, we review literature pertinent to hypothetical connections between metabolic and epigenetic states in eukaryotic cells. © 2013 Elsevier Inc.

Lee J.T.,Howard Hughes Medical Institute | Lee J.T.,Harvard University | Bartolomei M.S.,University of Pennsylvania
Cell | Year: 2013

X chromosome inactivation and genomic imprinting are classic epigenetic processes that cause disease when not appropriately regulated in mammals. Whereas X chromosome inactivation evolved to solve the problem of gene dosage, the purpose of genomic imprinting remains controversial. Nevertheless, the two phenomena are united by allelic control of large gene clusters, such that only one copy of a gene is expressed in every cell. Allelic regulation poses significant challenges because it requires coordinated long-range control in cis and stable propagation over time. Long noncoding RNAs have emerged as a common theme, and their contributions to diseases of imprinting and the X chromosome have become apparent. Here, we review recent advances in basic biology, the connections to disease, and preview potential therapeutic strategies for future development. © 2013 Elsevier Inc.

Maillet M.,University of Cincinnati | Van Berlo J.H.,University of Cincinnati | Molkentin J.D.,University of Cincinnati | Molkentin J.D.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2013

The heart hypertrophies in response to developmental signals as well as increased workload. Although adult-onset hypertrophy can ultimately lead to disease, cardiac hypertrophy is not necessarily maladaptive and can even be beneficial. Progress has been made in our understanding of the structural and molecular characteristics of physiological cardiac hypertrophy, as well as of the endocrine effectors and associated signalling pathways that regulate it. Physiological hypertrophy is initiated by finite signals, which include growth hormones (such as thyroid hormone, insulin, insulin-like growth factor 1 and vascular endothelial growth factor) and mechanical forces that converge on a limited number of intracellular signalling pathways (such as PI3K, AKT, AMP-activated protein kinase and mTOR) to affect gene transcription, protein translation and metabolism. Harnessing adaptive signalling mediators to reinvigorate the diseased heart could have important medical ramifications. © 2012 Macmillan Publishers Limited. All rights reserved.

Duxin J.P.,Harvard University | Dewar J.M.,Harvard University | Yardimci H.,Cancer Research UK Research Institute | Walter J.C.,Harvard University | Walter J.C.,Howard Hughes Medical Institute
Cell | Year: 2014

DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair in vitro and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then progresses past the adduct using DNA polymerase ζ. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse. © 2014 Elsevier Inc.

Gilmore E.C.,Case Western Reserve University | Walsh C.A.,Howard Hughes Medical Institute
Wiley Interdisciplinary Reviews: Developmental Biology | Year: 2013

The study of human developmental microcephaly is providing important insights into brain development. It has become clear that developmental microcephalies are associated with abnormalities in cellular production, and that the pathophysiology of microcephaly provides remarkable insights into how the brain generates the proper number of neurons that determine brain size. Most of the genetic causes of 'primary' developmental microcephaly (i.e., not associated with other syndromic features) are associated with centrosomal abnormalities. In addition to other functions, centrosomal proteins control the mitotic spindle, which is essential for normal cell proliferation during mitosis. However, the brain is often uniquely affected when microcephaly genes are mutated implying special centrosomal-related functions in neuronal production. Although models explaining how this could occur have some compelling data, they are not without controversy. Interestingly, some of the microcephaly genes show evidence that they were targets of evolutionary selection in primates and human ancestors, suggesting potential evolutionary roles in controlling neuronal number and brain volume across species. Mutations in DNA repair pathway genes also lead to microcephaly. Double-stranded DNA breaks appear to be a prominent type of damage that needs to be repaired during brain development, yet why defects in DNA repair affect the brain preferentially and if DNA repair relates to centrosome function, are not clearly understood. © 2012 Wiley Periodicals, Inc.

Siolas D.,New York University | Hannon G.J.,Howard Hughes Medical Institute
Cancer Research | Year: 2013

Tumor graft models (also known as patient-derived xenografts or PDX) are based on the transfer of primary tumors directly from the patient into an immunodeficient mouse. Because PDX mice are derived from human tumors, they offer a tool for developing anticancer therapies and personalizedmedicine for patients with cancer. In addition, these models can be used to study metastasis and tumor genetic evolution. This review examines the development, challenges, and broad use of these attractive preclinical models. ©2013 AACR.

Eisenberg D.,Howard Hughes Medical Institute | Jucker M.,University of Tubingen | Jucker M.,German Center for Neurodegenerative Diseases
Cell | Year: 2012

Amyloid fibers and oligomers are associated with a great variety of human diseases including Alzheimer's disease and the prion conditions. Here we attempt to connect recent discoveries on the molecular properties of proteins in the amyloid state with observations about pathological tissues and disease states. We summarize studies of structure and nucleation of amyloid and relate these to observations on amyloid polymorphism, prion strains, coaggregation of pathogenic proteins in tissues, and mechanisms of toxicity and transmissibility. Molecular studies have also led to numerous strategies for biological and chemical interventions against amyloid diseases. © 2012 Elsevier Inc.

Rossman J.S.,University of Kent | Lamb R.A.,Northwestern University | Lamb R.A.,Howard Hughes Medical Institute
Annual Review of Cell and Developmental Biology | Year: 2013

Virus budding is a complex, multistep process in which viral proteins make specific alterations in membrane curvature. Many different viral proteins can deform the membrane and form a budding virion, but very few can mediate membrane scission to complete the budding process. As a result, enveloped viruses have developed numerous ways of facilitating membrane scission, including hijacking host cellular scission machinery and expressing their own scission proteins. These proteins mediate scission in very different ways, though the biophysical mechanics underlying their actions may be similar. In this review, we explore the mechanisms of membrane scission and the ways in which enveloped viruses use these systems to mediate the release of budding virions. © 2013 by Annual Reviews. All rights reserved.

Hobert O.,Howard Hughes Medical Institute
WormBook : the online review of C. elegans biology | Year: 2010

The nervous system represents the most complex tissue of C. elegans both in terms of numbers (302 neurons and 56 glial cells = 37% of the somatic cells in a hermaphrodite) and diversity (118 morphologically distinct neuron classes). The lineage and morphology of each neuron type has been described in detail and neuronal fate markers exists for virtually all neurons in the form of fluorescent reporter genes. The ability to "phenotype" neurons at high resolution combined with the amenability of C. elegans to genetic mutant analysis make the C. elegans nervous system a prime model system to elucidate the nature of the gene regulatory programs that build a nervous system-a central question of developmental neurobiology. Discussing a number of regulatory genes involved in neuronal lineage determination and neuronal differentiation, I will try to carve out in this review a few general principles of neuronal development in C. elegans. These principles may be conserved across phylogeny.

Schrum J.P.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2010

Understanding the origin of cellular life on Earth requires the discovery of plausible pathways for the transition from complex prebiotic chemistry to simple biology, defined as the emergence of chemical assemblies capable of Darwinian evolution. We have proposed that a simple primitive cell, or protocell, would consist of two key components: a protocell membrane that defines a spatially localized compartment, and an informational polymer that allows for the replication and inheritance of functional information. Recent studies of vesicles composed of fatty-acid membranes have shed considerable light on pathways for protocell growth and division, as well as means by which protocells could take up nutrients from their environment. Additional work with genetic polymers has provided insight into the potential for chemical genome replication and compatibility with membrane encapsulation. The integration of a dynamic fatty-acid compartment with robust, generalized genetic polymer replication would yield a laboratory model of a protocell with the potential for classical Darwinian biological evolution, and may help to evaluate potential pathways for the emergence of life on the early Earth. Here we discuss efforts to devise such an integrated protocell model.

Ameres S.L.,Austrian Academy of Sciences | Zamore P.D.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2013

MicroRNAs (miRNAs) regulate the expression of most genes in animals, but we are only now beginning to understand how they are generated, assembled into functional complexes and destroyed. Various mechanisms have now been identified that regulate miRNA stability and that diversify miRNA sequences to create distinct isoforms. The production of different isoforms of individual miRNAs in specific cells and tissues may have broader implications for miRNA-mediated gene expression control. Rigorously testing the many discrepant models for how miRNAs function using quantitative biochemical measurements made in vivo and in vitro remains a major challenge for the future. © 2013 Macmillan Publishers Limited. All rights reserved.

Ramaswami M.,Trinity College Dublin | Taylor J.P.,St Jude Childrens Research Hospital | Parker R.,Howard Hughes Medical Institute
Cell | Year: 2013

The molecular processes that contribute to degenerative diseases are not well understood. Recent observations suggest that some degenerative diseases are promoted by the accumulation of nuclear or cytoplasmic RNA-protein (RNP) aggregates, which can be related to endogenous RNP granules. RNP aggregates arise commonly in degenerative diseases because RNA-binding proteins commonly self-assemble, in part through prion-like domains, which can form self-propagating amyloids. RNP aggregates may be toxic due to multiple perturbations of posttranscriptional control, thereby disrupting the normal "ribostasis" of the cell. This suggests that understanding and modulating RNP assembly or clearance may be effective approaches to developing therapies for these diseases. © 2013 Elsevier Inc.

Marcia M.,Yale University | Pyle A.M.,Yale University | Pyle A.M.,Howard Hughes Medical Institute
Cell | Year: 2012

Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report 14 crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, and they adopt a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome. © 2012 Elsevier Inc.

Irvine D.J.,Massachusetts Institute of Technology | Irvine D.J.,Howard Hughes Medical Institute | Swartz M.A.,Ecole Polytechnique Federale de Lausanne | Szeto G.L.,Massachusetts Institute of Technology
Nature Materials | Year: 2013

Vaccines aim to protect against or treat diseases through manipulation of the immune response, promoting either immunity or tolerance. In the former case, vaccines generate antibodies and T cells poised to protect against future pathogen encounter or attack diseased cells such as tumours; in the latter case, which is far less developed, vaccines block pathogenic autoreactive T cells and autoantibodies that target self tissue. Enormous challenges remain, however, as a consequence of our incomplete understanding of human immunity. A rapidly growing field of research is the design of vaccines based on synthetic materials to target organs, tissues, cells or intracellular compartments; to co-deliver immunomodulatory signals that control the quality of the immune response; or to act directly as immune regulators. There exists great potential for well-defined materials to further our understanding of immunity. Here we describe recent advances in the design of synthetic materials to direct immune responses, highlighting successes and challenges in prophylactic, therapeutic and tolerance-inducing vaccines. © 2013 Macmillan Publishers Limited. All rights reserved.

Haynes B.F.,Duke University | Kelsoe G.,Duke University | Harrison S.C.,Howard Hughes Medical Institute | Kepler T.B.,Duke University | Kepler T.B.,Boston University
Nature Biotechnology | Year: 2012

Failure of immunization with the HIV-1 envelope to induce broadly neutralizing antibodies against conserved epitopes is a major barrier to producing a preventive HIV-1 vaccine. Broadly neutralizing monoclonal antibodies (BnAbs) from those subjects who do produce them after years of chronic HIV-1 infection have one or more unusual characteristics, including polyreactivity for host antigens, extensive somatic hypermutation and long, variable heavy-chain third complementarity-determining regions, factors that may limit their expression by host immunoregulatory mechanisms. The isolation of BnAbs from HIV-1g-infected subjects and the use of computationally derived clonal lineages as templates provide a new path for HIV-1 vaccine immunogen design. This approach, which should be applicable to many infectious agents, holds promise for the construction of vaccines that can drive B cells along rare but desirable maturation pathways. © 2012 Nature America, All Right Reserved.

Sheffield V.C.,Howard Hughes Medical Institute
Transactions of the American Clinical and Climatological Association | Year: 2010

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder affecting multiple organ systems and resulting in blindness, obesity, cognitive impairment, and congenital defects. Interest in the etiology of this disorder stems, in part, from the fact that patients with BBS develop common clinical problems, including obesity, diabetes and hypertension. Twelve genes independently causing BBS have been identified. The heterogeneity is explained by the existence of two BBS complexes, the BBSome consisting of seven known BBS proteins, and the BBS chaperone complex consisting of three known BBS proteins. The formation of the BBSome requires the function of the BBS chaperone complex. Both mouse and zebrafish data support a role for BBS genes in cilia function, and in intracellular and intraflagellar trafficking. From the work described here, a common primary function of BBS proteins has emerged, specifically the mediation and regulation of microtubule-based intracellular transport.

Reddien P.W.,Howard Hughes Medical Institute
Cell | Year: 2013

Embryos and juveniles in many organisms repair tissue injuries better than adults. In this issue, Shyh-Chang et al. find that postnatal activation of Lin28a, a gene typically active in embryonic development, promotes better than normal tissue repair in mice, including following ear and digit injuries. © 2013 Elsevier Inc.

Schinzel R.,University of California at Berkeley | Dillin A.,University of California at Berkeley | Dillin A.,Howard Hughes Medical Institute
Current Opinion in Cell Biology | Year: 2015

Organisms have to cope with an unpredictable and dynamic environment. It is crucial for any living being to respond to these changes by buffering the effects on cellular homeostasis. Failure to appropriately respond to stress can have severe consequences for health and survival. Eukaryotic cells possess several organelle-specific stress responses to cope with this challenge. Besides their central role in stress resistance, these pathways have also been shown to be important in the regulation of proteome maintenance, development and longevity. Intriguingly, many of these effects seem to be controlled by only a subset of cells implying a systemic regulation in a cell non-autonomous manner. The understanding of the nature of this stress communication across tissues, its mechanisms and impact, will be paramount in understanding disease etiology and the development of therapeutic strategies. © 2015.

Laplante M.,Laval University | Sabatini D.M.,Whitehead Institute For Biomedical Research | Sabatini D.M.,Howard Hughes Medical Institute | Sabatini D.M.,Massachusetts Institute of Technology
Journal of Cell Science | Year: 2013

The mechanistic (or mammalian) target of rapamycin (mTOR) is a kinase that regulates key cellular functions linked to the promotion of cell growth and metabolism. This kinase, which is part of two protein complexes termed mTOR complex 1 (mTORC1) and 2 (mTORC2), has a fundamental role in coordinating anabolic and catabolic processes in response to growth factors and nutrients. Of the two mTOR complexes, mTORC1 is by far the best characterized. When active, mTORC1 triggers cell growth and proliferation by promoting protein synthesis, lipid biogenesis, and metabolism, and by reducing autophagy. The fact that mTORC1 deregulation is associated with several human diseases, such as type 2 diabetes, cancer, obesity and neurodegeneration, highlights its importance in the maintenance of cellular homeostasis. Over the last years, several groups observed that mTORC1 inhibition, in addition to reducing protein synthesis, deeply affects gene transcription. Here, we review the connections between mTORC1 and gene transcription by focusing on its impact in regulating the activation of specific transcription factors including including STAT3, SREBPs, PPARγ, PPARα, HIF1α, YY1-PGC1α and TFEB. We also discuss the importance of these transcription factors in mediating the effects of mTORC1 on various cellular processes in physiological and pathological contexts. © 2013. Published by The Company of Biologists Ltd.

Roychowdhury S.,Ohio State University | Chinnaiyan A.M.,University of Michigan | Chinnaiyan A.M.,Howard Hughes Medical Institute
CA Cancer Journal for Clinicians | Year: 2016

Understanding the molecular landscape of cancer has facilitated the development of diagnostic, prognostic, and predictive biomarkers for clinical oncology. Developments in next-generation DNA sequencing technologies have increased the speed and reduced the cost of sequencing the nucleic acids of cancer cells. This has unlocked opportunities to characterize the genomic and transcriptomic landscapes of cancer for basic science research through projects like The Cancer Genome Atlas. The cancer genome includes DNA-based alterations, such as point mutations or gene duplications. The cancer transcriptome involves RNA-based alterations, including changes in messenger RNAs. Together, the genome and transcriptome can provide a comprehensive view of an individual patient's cancer that is beginning to impact real-time clinical decision-making. The authors discuss several opportunities for translating this basic science knowledge into clinical practice, including a molecular classification of cancer, heritable risk of cancer, eligibility for targeted therapies, and the development of innovative, genomic-based clinical trials. In this review, key applications and new directions are outlined for translating the cancer genome and transcriptome into patient care in the clinic. CA Cancer J Clin 2016;75-88. © 2015 American Cancer Society.

Rudd M.E.,Howard Hughes Medical Institute
Journal of Vision | Year: 2013

To maintain color constancy, the human visual system must distinguish surface reflectance-based variations in wavelength and luminance from variations due to illumination. Edge integration theory proposes that this is accomplished by spatially integrating steps in luminance and color contrast that likely result from reflectance changes. Thus, a neural representation of relative reflectance within the visual scene is constructed. An anchoring rule-the largest reflectance in the neural representation appears white-is then applied to map relative lightness onto an absolute lightness scale. A large body of data on human lightness judgments is here shown to be consistent with an edge integration model in which the visual system performs a weighted sum of steps in log luminance across space. Three hypotheses are proposed regarding how weights are applied to edges. First, weights decline with distance from the target surface whose lightness is being computed. Second, larger weights are given to edges whose dark sides point towards the target. Third, edge integration is carried out along a path leading from a common background field, or surround, to the target location. The theory accounts for simultaneous contrast; quantitative lightness judgments made with classical disk-annulus, Gilchrist dome, and Gelb displays; and perceptual filling-in lightness. A cortical theory of lightness in the ventral stream of visual cortex (areas V1 → V4) is proposed to instantiate the edge integration algorithm. The neural model is shown to be capable of unifying the quantitative laws of edge integration in lightness perception with the laws governing brightness, including Stevens' power law brightness model, and makes novel predictions about the quantitative laws governing induced darkness. © 2013 ARVO.

Recent theories of lightness perception assume that lightness (perceived reectance) is computed by a process that contrasts the target's luminance with that of one or more regions in its spatial surround. A challenge for any such theory is the phenomenon of lightness assimilation, which occurs when increasing the luminance of a surround region increases the target lightness: the opposite of contrast. Here contrast and assimilation are studied quantitatively in lightness matching experiments utilizing concentric disk-and-ring displays. Whether contrast or assimilation is seen depends on a number of factors including: the luminance relations of the target, surround, and background; surround size; and matching instructions. When assimilation occurs, it is always part of a larger pattern in which assimilation and contrast both occur over different ranges of surround luminance. These ndings are quantitatively modeled by a theory that assumes lightness is computed from a weighted sum of responses of edge detector neurons in visual cortex. The magnitude of the neural response to an edge is regulated by a combination of contrast gain control acting between neighboring edge detectors and a top-down attentional gain control that selectively weights the response to stimulus edges according to their task relevance. © ARVO.

Yang Z.,University of California at San Francisco | Sullivan B.M.,Howard Hughes Medical Institute | Sullivan B.M.,University of California at San Francisco | Allen C.D.C.,University of California at San Francisco
Immunity | Year: 2012

IgE antibodies may be protective in parasite immunity, but their aberrant production can lead to allergic disease and life-threatening anaphylaxis. Despite the importance of IgE regulation, few studies have directly examined the B cells that express IgE, because these cells are rare and difficult to detect. Here, we describe fluorescent IgE reporter mice and validate a flow cytometry procedure to allow sensitive and specific identification of IgE-expressing B cells in vivo. Similar to IgG1+ cells, IgE+ B cells differentiated into germinal center (GC) B cells and plasma cells (PCs) during primary immune responses to a T cell-dependent hapten-protein conjugate and the helminth Nippostrongylus brasiliensis. However, the participation of IgE+ B cells in GCs was transient. IgE+ B cells had an atypical propensity to upregulate the transcription factor Blimp-1 and undergo PC differentiation. Most IgE+ PCs were short lived and showed reduced affinity maturation, revealing intrinsic mechanisms that restrict the IgE antibody response. © 2012 Elsevier Inc.

Sudhof T.C.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

Upon entering a presynaptic terminal, an action potential opens Ca2+ channels, and transiently increases the local Ca2+ concentration at the presynaptic active zone. Ca2+ then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca2+. Synaptotagmins bind Ca2+ via two C2-domains, and transduce the Ca2+ signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca2{thorn}-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The conserved function of synaptotagmins and complexins operates generally in most, if not all, Ca2+-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release. © 2011 Cold Spring Harbor Laboratory Press.

Breaker R.R.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

Riboswitches are structured noncoding RNA domains that selectively bind metabolites and control gene expression (Mandal and Breaker 2004a; Coppins et al. 2007; Roth and Breaker 2009). Nearly all examples of the known riboswitches reside in noncoding regions of messenger RNAs where they control transcription or translation. Newfound classes of riboswitches are being reported at a rate of about three per year (Ames and Breaker 2009), and these have been shown to selectively respond to fundamental metabolites including coenzymes, nucleobases or their derivatives, amino acids, and other small molecule ligands. The characteristics of some riboswitches suggest they could be modern descendents of an ancient sensory and regulatory system that likely functioned before the emergence of enzymes and genetic factors made of protein (Nahvi et al. 2002; Vitreschak et al. 2004; Breaker 2006). If true, then some of the riboswitch structures and functions that serve modern cells sowell may accurately reflect the capabilities of RNA sensors and switches that existed in the RNAWorld. This article will address some of the characteristics of modern riboswitches that may be relevant to ancient versions of these metabolite-sensing RNAs. © 2012 Cold Spring Harbor Laboratory Press; all rights reserved.

Pfeffer C.K.,Howard Hughes Medical Institute
Current Biology | Year: 2014

Recent studies on vasoactive intestinal peptide-expressing inhibitory neurons in the barrel and auditory cortices of the mouse brain have shown that they form a disinhibitory circuitry that affects the excitability of pyramidal neurons. © 2014 Elsevier Ltd.

Because the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome small interfering RNA (siRNA) screen in melanoma cells to expand our understanding of the kinases that regulate this pathway. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAF(V600E) mutation, inhibits Wnt/β-catenin signaling in human melanoma cells. Because inhibitors of BRAF(V600E) show promise in ongoing clinical trials, we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAF(V600E) inhibitor PLX4720. We found that endogenous β-catenin was required for PLX4720-induced apoptosis of melanoma cells and that activation of Wnt/β-catenin signaling synergized with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlated with reduced abundance of an endogenous negative regulator of β-catenin, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, siRNA directed against AXIN1 rendered resistant melanoma cell lines susceptible to apoptosis in response to treatment with a BRAF(V600E) inhibitor. Thus, Wnt/β-catenin signaling and AXIN1 may regulate the efficacy of inhibitors of BRAF(V600E), suggesting that manipulation of the Wnt/β-catenin pathway could be combined with BRAF inhibitors to treat melanoma.

Willert K.,University of California at San Diego | Nusse R.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

Wnt proteins comprise a major family of signaling molecules that orchestrate and influence a myriad of cell biological and developmental processes. Although our understanding of the role of Wnt signaling in regulating development and affecting disease, such as cancer, has been ever increasing, the study of the Wnt proteins themselves has been painstaking and slow moving. Despite advances in the biochemical characterization of Wnt proteins, many mysteries remain unsolved. In contrast to other developmental signaling molecules, such as fibroblast growth factors (FGF), transforming growth factors (TGFβ), and Sonic hedgehog (Shh), Wnt proteins have not conformed to many standard methods of protein production, such as bacterial overexpression, and analysis, such as ligand-receptor binding assays. The reasons for their recalcitrant nature are likely a consequence of the complex set of posttranslational modifications involving several highly specialized and poorly characterized processing enzymes. With the recent description of the first Wnt protein structure, the time is ripe to uncover and possibly resolve many of the remaining issues surrounding Wnt proteins and their interactions. Here we describe the process of maturation of Wnt from its initial translation to its eventual release from a cell and interactions in the extracellular environment. © 2012 Cold Spring Harbor Laboratory Press; all rights reserved.

Higley M.J.,Yale University | Sabatini B.L.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

Calcium (Ca2+) is a ubiquitous signaling molecule that accumulates in the cytoplasm in response to diverse classes of stimuli and, in turn, regulates many aspects of cell function. In neurons, Ca2+ influx in response to action potentials or synaptic stimulation triggers neurotransmitter release, modulates ion channels, induces synaptic plasticity, and activates transcription. In this article, we discuss the factors that regulate Ca2+ signaling in mammalian neurons with a particular focus on Ca2+ signaling within dendritic spines. This includes consideration of the routes of entry and exit of Ca2+, the cellular mechanisms that establish the temporal and spatial profile of Ca2+ signaling, and the biophysical criteria that determine which downstream signals are activated when Ca2+ accumulates in a spine. Furthermore, we also briefly discuss the technical advances that made possible the quantitative study of Ca2+ signaling in dendritic spines. © 2012 Cold Spring Harbor Laboratory Press; all rights reserved.

Cech T.R.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

There are two RNAworlds. The first is the primordial RNAworld, a hypothetical era when RNA served as both information and function, both genotype and phenotype. The second RNA world is that of today's biological systems, where RNA plays active roles in catalyzing biochemical reactions, in translating mRNA into proteins, in regulating gene expression, and in the constant battle between infectious agents trying to subvert host defense systems and host cells protecting themselves from infection. This second RNAworld is not at all hypothetical, and althoughwe do not have all the answers about howit works,we have the tools to continue our interrogation of this world and refine our understanding. The fun comes whenwe try to use our secure knowledge of the modern RNAworld to infer what the primordial RNAworld might have looked like. © 2012 Cold Spring Harbor Laboratory Press.

Pool A.-H.,University of California at Berkeley | Scott K.,University of California at Berkeley | Scott K.,Howard Hughes Medical Institute
Current Opinion in Neurobiology | Year: 2014

Neuromodulators play a key role in adjusting animal behavior based on environmental cues and internal needs. Here, we review the regulation of Drosophila feeding behavior to illustrate how neuromodulators achieve behavioral plasticity. Recent studies have made rapid progress in determining molecular and cellular mechanisms that translate the metabolic needs of the fly into changes in neuroendocrine and neuromodulatory states. These neuromodulators in turn promote or inhibit discrete feeding behavioral subprograms. This review highlights the links between physiological needs, neuromodulatory states, and feeding decisions. © 2014 Elsevier Ltd.

Ganem D.,Howard Hughes Medical Institute
Journal of Clinical Investigation | Year: 2010

The linkage of Kaposi sarcoma (KS) to infection by a novel human herpesvirus (Kaposi sarcoma-associated herpesvirus [KSHV]) is one of the great successes of contemporary biomedical research and was achieved by using advanced genomic technologies in a manner informed by a nuanced understanding of epidemiology and clinical investigation. Ongoing efforts to understand the molecular mechanisms by which KSHV infection predisposes to KS continue to be powerfully influenced by insights emanating from the clinic. Here, recent developments in KS pathogenesis are reviewed, with particular emphasis on clinical, pathologic, and molecular observations that highlight the many differences between this process and tumorigenesis by other oncogenic viruses.

Zoghbi H.Y.,Howard Hughes Medical Institute
Cell | Year: 2014

This year, the Lasker∼DeBakey Clinical Medical Research Award will be shared by Mahlon R. DeLong and Alim-Louis Benabid for elucidating the role of the subthalamic nucleus in mediating the motor dysfunction of Parkinson's disease and for pioneering the use of deep-brain stimulation to alleviate symptoms of the disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Sokol C.L.,Massachusetts General Hospital | Medzhitov R.,Howard Hughes Medical Institute
Current Opinion in Immunology | Year: 2010

Long appreciated for their role as Type-2 effector cells, basophils have recently come into the spotlight for their role in the initiation of Type-2 immunity. Via an assortment of different activation pathways, basophils produce cytokines such as IL-4 that promote Th2 differentiation. Furthermore, recent studies using different experimental systems have shown that basophils can act as antigen presenting cells both in vitro and in vivo. In addition, basophils shape the Type-2 immune response by guiding antibody production and the memory response.

Davis F.P.,Howard Hughes Medical Institute | Sali A.,University of California at San Francisco
PLoS Computational Biology | Year: 2010

Protein-protein interactions are challenging targets for modulation by small molecules. Here, we propose an approach that harnesses the increasing structural coverage of protein complexes to identify small molecules that may target protein interactions. Specifically, we identify ligand and protein binding sites that overlap upon alignment of homologous proteins. Of the 2,619 protein structure families observed to bind proteins, 1,028 also bind small molecules (250-1000 Da), and 197 exhibit a statistically significant (p<0.01) overlap between ligand and protein binding positions. These "bi-functional positions", which bind both ligands and proteins, are particularly enriched in tyrosine and tryptophan residues, similar to "energetic hotspots" described previously, and are significantly less conserved than mono-functional and solvent exposed positions. Homology transfer identifies ligands whose binding sites overlap at least 20% of the protein interface for 35% of domain-domain and 45% of domain-peptide mediated interactions. The analysis recovered known small-molecule modulators of protein interactions as well as predicted new interaction targets based on the sequence similarity of ligand binding sites. We illustrate the predictive utility of the method by suggesting structural mechanisms for the effects of sanglifehrin A on HIV virion production, bepridil on the cellular entry of anthrax edema factor, and fusicoccin on vertebrate developmental pathways. The results, available at http://pibase.janelia.org, represent a comprehensive collection of structurally characterized modulators of protein interactions, and suggest that homologous structures are a useful resource for the rational design of interaction modulators. © 2010 Davis, Sali.

Haber D.A.,Harvard University | Haber D.A.,Howard Hughes Medical Institute | Velculescu V.E.,Johns Hopkins University
Cancer Discovery | Year: 2014

The ability to study nonhematologic cancers through noninvasive sampling of blood is one of the most exciting and rapidly advancing fields in cancer diagnostics. This has been driven both by major technologic advances, including the isolation of intact cancer cells and the analysis of cancer cell-derived DNA from blood samples, and by the increasing application of molecularly driven therapeutics, which rely on such accurate and timely measurements of critical biomarkers. Moreover, the dramatic efficacy of these potent cancer therapies drives the selection for additional genetic changes as tumors acquire drug resistance, necessitating repeated sampling of cancer cells to adjust therapy in response to tumor evolution. Together, these advanced noninvasive diagnostic capabilities and their applications in guiding precision cancer therapies are poised to change the ways in which we select and monitor cancer treatments. Significance: Recent advances in technologies to analyze circulating tumor cells and circulating tumor DNA are setting the stage for real-time, noninvasive monitoring of cancer and providing novel insights into cancer evolution, invasion, and metastasis. © 2014 American Association for Cancer Research.

Tao H.,Howard Hughes Medical Institute
Nature medicine | Year: 2014

Type 2 diabetes (T2D) has reached an epidemic level globally. Most current treatments ameliorate the hyperglycemic symptom of the disease but are not effective in correcting its underlying cause. One important causal factor of T2D is ectopic accumulation of lipids in metabolically sensitive organs such as liver and muscle. Mitochondrial uncoupling, which reduces cellular energy efficiency and increases lipid oxidation, is an appealing therapeutic strategy. The challenge, however, is to discover safe mitochondrial uncouplers for practical use. Niclosamide is an anthelmintic drug approved by the US Food and Drug Administration that uncouples the mitochondria of parasitic worms. Here we show that niclosamide ethanolamine salt (NEN) uncouples mammalian mitochondria at upper nanomolar concentrations. Oral NEN increases energy expenditure and lipid metabolism in mice. It is also efficacious in preventing and treating hepatic steatosis and insulin resistance induced by a high-fat diet. Moreover, it improves glycemic control and delays disease progression in db/db mice. Given the well-documented safety profile of NEN, our study provides a potentially new and practical pharmacological approach for treating T2D.

Chu C.,Stanford University | Spitale R.C.,Stanford University | Spitale R.C.,University of California at Irvine | Chang H.Y.,Stanford University | Chang H.Y.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2015

Thousands of long noncoding RNAs (lncRNAs) have been discovered, but their functional characterization has been slowed by a limited set of research tools. Here we review emerging RNA-centric methods to interrogate the intrinsic structure of lncRNAs as well as their genomic localization and biochemical partners. Understanding these technologies, including their advantages and caveats, and developing them in the future will be essential to progress from description to comprehension of the myriad roles of lncRNAs. © 2015 Nature America, Inc. All rights reserved.

Wang L.,California Institute of Technology | Anderson D.J.,California Institute of Technology | Anderson D.J.,Howard Hughes Medical Institute
Nature | Year: 2010

Aggression is regulated by pheromones in many animal species. However, in no system have aggression pheromones, their cognate receptors and corresponding sensory neurons been identified. Here we show that 11-cis-vaccenyl acetate (cVA), a male-specific volatile pheromone, robustly promotes male-male aggression in the vinegar fly Drosophila melanogaster. The aggression-promoting effect of synthetic cVA requires olfactory sensory neurons (OSNs) expressing the receptor Or67d, as well as the receptor itself. Activation of Or67d-expressing OSNs, either by genetic manipulation of their excitability or by exposure to male pheromones in the absence of other classes of OSNs, is sufficient to promote aggression. High densities of male flies can promote aggression by the release of volatile cVA. In turn, cVA-promoted aggression can promote male fly dispersal from a food resource, in a manner dependent on Or67d-expressing OSNs. These data indicate that cVA may mediate negative-feedback control of male population density, through its effect on aggression. Identification of a pheromone-OSN pair controlling aggression in a genetic organism opens the way to unravelling the neurobiology of this evolutionarily conserved behaviour. © 2010 Macmillan Publishers Limited. All rights reserved.

Wang H.,Oregon Health And Science University | Elferich J.,Oregon Health And Science University | Gouaux E.,Oregon Health And Science University | Gouaux E.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2012

Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT-substrate complexes in lipid bicelles and in β-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site. © 2012 Nature America, Inc. All rights reserved.

Capra E.J.,Massachusetts Institute of Technology | Perchuk B.S.,Massachusetts Institute of Technology | Skerker J.M.,University of California at Berkeley | Laub M.T.,Massachusetts Institute of Technology | Laub M.T.,Howard Hughes Medical Institute
Cell | Year: 2012

Orthologous proteins often harbor numerous substitutions, but whether these differences result from neutral or adaptive processes is usually unclear. To tackle this challenge, we examined the divergent evolution of a model bacterial signaling pathway comprising the kinase PhoR and its cognate substrate PhoB. We show that the specificity-determining residues of these proteins are typically under purifying selection but have, in α-proteobacteria, undergone a burst of diversification followed by extended stasis. By reversing mutations that accumulated in an α-proteobacterial PhoR, we demonstrate that these substitutions were adaptive, enabling PhoR to avoid crosstalk with a paralogous pathway that arose specifically in α-proteobacteria. Our findings demonstrate that duplication and the subsequent need to avoid crosstalk strongly influence signaling protein evolution. These results provide a concrete example of how system-wide insulation can be achieved postduplication through a surprisingly limited number of mutations. Our work may help explain the apparent ease with which paralogous protein families expanded in all organisms. © 2012 Elsevier Inc.

Littman D.R.,New York University | Littman D.R.,Howard Hughes Medical Institute | Pamer E.G.,Sloan Kettering Cancer Center
Cell Host and Microbe | Year: 2011

The commensal microbiota that inhabit different parts of the gastrointestinal (GI) tract have been shaped by coevolution with the host species. The symbiotic relationship of the hundreds of microbial species with the host requires a tuned response that prevents host damage, e.g., inflammation, while tolerating the presence of the potentially beneficial microbes. Recent studies have begun to shed light on immunological processes that participate in maintenance of homeostasis with the microbiota and on how disturbance of host immunity or the microbial ecosystem can result in disease-provoking dysbiosis. Our growing appreciation of this delicate host-microbe relationship promises to influence our understanding of inflammatory diseases and infection by microbial pathogens and to provide new therapeutic opportunities. © 2011 Elsevier Inc.

Zhang R.,Lawrence Berkeley National Laboratory | Alushin G.M.,University of California at Berkeley | Alushin G.M.,U.S. National Institutes of Health | Brown A.,University of Cambridge | And 2 more authors.
Cell | Year: 2015

Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics. © 2015 Elsevier Inc.

Lutz J.-F.,Charles Sadron Institute | Ouchi M.,Kyoto University | Liu D.R.,Howard Hughes Medical Institute | Sawamoto M.,Kyoto University
Science | Year: 2013

Sequence-controlled polymers are macromolecules in which monomer units of different chemical nature are arranged in an ordered fashion. The most prominent examples are biological and have been studied and used primarily by molecular biologists and biochemists. However, recent progress in protein- and DNA-based nanotechnologies has shown the relevance of sequence-controlled polymers to nonbiological applications, including data storage, nanoelectronics, and catalysis. In addition, synthetic polymer chemistry has provided interesting routes for preparing nonnatural sequence-controlled polymers. Although these synthetic macromolecules do not yet compare in functional scope with their natural counterparts, they open up opportunities for controlling the structure, self-assembly, and macroscopic properties of polymer materials.

Bar-Peled L.,Massachusetts Institute of Technology | Schweitzer L.D.,Massachusetts Institute of Technology | Zoncu R.,Massachusetts Institute of Technology | Sabatini D.M.,Massachusetts Institute of Technology | Sabatini D.M.,Howard Hughes Medical Institute
Cell | Year: 2012

The mTOR Complex 1 (mTORC1) pathway regulates cell growth in response to numerous cues, including amino acids, which promote mTORC1 translocation to the lysosomal surface, its site of activation. The heterodimeric RagA/B-RagC/D GTPases, the Ragulator complex that tethers the Rags to the lysosome, and the v-ATPase form a signaling system that is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of guanosine triphosphate to RagA and RagB but the factors that regulate Rag nucleotide loading are unknown. Here, we identify HBXIP and C7orf59 as two additional Ragulator components that are required for mTORC1 activation by amino acids. The expanded Ragulator has nucleotide exchange activity toward RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, we provide mechanistic insight into how mTORC1 senses amino acids by identifying Ragulator as a guanine nucleotide exchange factor (GEF) for the Rag GTPases. © 2012 Elsevier Inc.

Jazayeri M.,Massachusetts Institute of Technology | Shadlen M.N.,Howard Hughes Medical Institute
Current Biology | Year: 2015

Timing plays a crucial role in sensorimotor function. However, the neural mechanisms that enable the brain to flexibly measure and reproduce time intervals are not known. We recorded neural activity in parietal cortex of monkeys in a time reproduction task. Monkeys were trained to measure and immediately afterward reproduce different sample intervals. While measuring an interval, neural responses had a nonlinear profile that increased with the duration of the sample interval. Activity was reset during the transition from measurement to production and was followed by a ramping activity whose slope encoded the previously measured sample interval. We found that firing rates at the end of the measurement epoch were correlated with both the slope of the ramp and the monkey's corresponding production interval on a trial-by-trial basis. Analysis of response dynamics further linked the rate of change of firing rates in the measurement epoch to the slope of the ramp in the production epoch. These observations suggest that, during time reproduction, an interval is measured prospectively in relation to the desired motor plan to reproduce that interval. © 2015 Elsevier Ltd.

Irvine K.D.,Howard Hughes Medical Institute
Seminars in Cell and Developmental Biology | Year: 2012

Metazoan cells are exposed to a multitude of signals, which they integrate to determine appropriate developmental or physiological responses. Although the Hippo pathway was only discovered recently, and our knowledge of Hippo signal transduction is far from complete, a wealth of interconnections amongst Hippo and other signaling pathways have already been identified. Hippo signaling is particularly important for growth control, and I describe how integration of Hippo and other pathways contributes to regulation of organ growth. Molecular links between Hippo signaling and other signal transduction pathways are summarized. Different types of mechanisms for signal integration are described, and examples of how the complex interconnections between pathways are used to guide developmental and physiological growth responses are discussed. Features of Hippo signaling appear to make it particularly well suited to signal integration, including its responsiveness to cell-cell contact and the mediation of its transcriptional output by transcriptional co-activator proteins that can interact with transcription factors of other pathways. © 2012 Elsevier Ltd.

Amat F.,Max Planck Institute of Molecular Cell Biology and Genetics | Amat F.,Howard Hughes Medical Institute
Nature methods | Year: 2014

The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min(-1) on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

Nannenga B.L.,Howard Hughes Medical Institute
Nature methods | Year: 2014

MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

Glickman M.S.,Sloan Kettering Cancer Center | Sawyers C.L.,Sloan Kettering Cancer Center | Sawyers C.L.,Howard Hughes Medical Institute
Cell | Year: 2012

During the past decade, cancer drug development has shifted from a focus on cytotoxic chemotherapies to drugs that target specific molecular alterations in tumors. Although these drugs dramatically shrink tumors, the responses are temporary. Research is now focused on overcoming drug resistance, a frequent cause of treatment failure. Here we reflect on analogous challenges faced by researchers in infectious diseases. We compare and contrast the resistance mechanisms arising in cancer and infectious diseases and discuss how approaches for overcoming viral and bacterial infections, such as HIV and tuberculosis, are instructive for developing a more rational approach for cancer therapy. In particular, maximizing the effect of the initial treatment response, which often requires synergistic combination therapy, is foremost among these approaches. A remaining challenge in both fields is identifying drugs that eliminate drug-tolerant "persister" cells (infectious disease) or tumor-initiating/stem cells (cancer) to prevent late relapse and shorten treatment duration. © 2012 Elsevier Inc.

Chan D.C.,Howard Hughes Medical Institute
Annual Review of Genetics | Year: 2012

Mitochondria are dynamic organelles that continually undergo fusion and fission. These opposing processes work in concert to maintain the shape, size, and number of mitochondria and their physiological function. Some of the major molecules mediating mitochondrial fusion and fission in mammals have been discovered, but the underlying molecular mechanisms are only partially unraveled. In particular, the cast of characters involved in mitochondrial fission needs to be clarified. By enabling content mixing between mitochondria, fusion and fission serve to maintain a homogeneous and healthy mitochondrial population. Mitochondrial dynamics has been linked to multiple mitochondrial functions, including mitochondrial DNA stability, respiratory capacity, apoptosis, response to cellular stress, and mitophagy. Because of these important functions, mitochondrial fusion and fission are essential in mammals, and even mild defects in mitochondrial dynamics are associated with disease. A better understanding of these processes likely will ultimately lead to improvements in human health. © 2012 by Annual Reviews.

Clevers H.,University Utrecht | Nusse R.,Howard Hughes Medical Institute
Cell | Year: 2012

The WNT signal transduction cascade controls myriad biological phenomena throughout development and adult life of all animals. In parallel, aberrant Wnt signaling underlies a wide range of pathologies in humans. In this Review, we provide an update of the core Wnt/β-catenin signaling pathway, discuss how its various components contribute to disease, and pose outstanding questions to be addressed in the future. © 2012 Elsevier Inc. © 2012 Elsevier Inc.

Yi R.,University of Colorado at Boulder | Fuchs E.,Howard Hughes Medical Institute
Current Topics in Developmental Biology | Year: 2012

MicroRNAs (miRs) comprise a class of tiny (∼ 19-24 nucleotide), noncoding RNAs that regulate gene expression posttranscriptionally. Since the discovery of the founding members lin-4 and let-7 as key regulators in the developing nematode, miRs have been found throughout the eukaryotic kingdom. Functions for miRs are wide-ranging and encompass embryogenesis, stem cell biology, tissue differentiation, and human diseases including cancers. In this chapter, we begin by acquainting our readers with miRs and introducing them to their biogenesis. Then, we focus on the roles of miRs in stem cells during tissue development and homeostasis. We use mammalian skin as our main paradigm, but we also consider miR functions in several different types of adult stem cells. We conclude by discussing future challenges that will lead to a comprehensive understanding of miR functions in stem cells and their lineages. © 2012 Elsevier Inc.

Kondo S.,Howard Hughes Medical Institute
Science signaling | Year: 2011

The DNA damage checkpoint, the first pathway known to be activated in response to DNA damage, is a mechanism by which the cell cycle is temporarily arrested to allow DNA repair. The checkpoint pathway transmits signals from the sites of DNA damage to the cell cycle machinery through the evolutionarily conserved ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) kinase cascades. We conducted a genome-wide RNAi (RNA interference) screen in Drosophila cells to identify previously unknown genes and pathways required for the G2-M checkpoint induced by DNA double-strand breaks (DSBs). Our large-scale analysis provided a systems-level view of the G2-M checkpoint and revealed the coordinated actions of particular classes of proteins, which include those involved in DNA repair, DNA replication, cell cycle control, chromatin regulation, and RNA processing. Further, from the screen and in vivo analysis, we identified previously unrecognized roles of two DNA damage response genes, mus101 and mus312. Our results suggest that the DNA replication preinitiation complex, which includes MUS101, and the MUS312-containing nuclease complexes, which are important for DSB repair, also function in the G2-M checkpoint. Our results provide insight into the diverse mechanisms that link DNA damage and the checkpoint signaling pathway.

Pikaard C.S.,Howard Hughes Medical Institute | Scheid O.M.,Gregor Mendel Institute of Molecular Plant Biology
Cold Spring Harbor Perspectives in Biology | Year: 2014

The study of epigenetics in plants has a long and rich history, from initial descriptions of non-Mendelian gene behaviors to seminal discoveries of chromatin-modifying proteins and RNAs that mediate gene silencing in most eukaryotes, including humans. Genetic screens in the model plant Arabidopsis have been particularly rewarding, identifying more than 130 epigenetic regulators thus far. The diversity of epigenetic pathways in plants is remarkable, presumably contributing to the phenotypic plasticity of plant postembryonic development and the ability to survive and reproduce in unpredictable environments. © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Freeman J.,Howard Hughes Medical Institute
Nature methods | Year: 2014

Understanding brain function requires monitoring and interpreting the activity of large networks of neurons during behavior. Advances in recording technology are greatly increasing the size and complexity of neural data. Analyzing such data will pose a fundamental bottleneck for neuroscience. We present a library of analytical tools called Thunder built on the open-source Apache Spark platform for large-scale distributed computing. The library implements a variety of univariate and multivariate analyses with a modular, extendable structure well-suited to interactive exploration and analysis development. We demonstrate how these analyses find structure in large-scale neural data, including whole-brain light-sheet imaging data from fictively behaving larval zebrafish, and two-photon imaging data from behaving mouse. The analyses relate neuronal responses to sensory input and behavior, run in minutes or less and can be used on a private cluster or in the cloud. Our open-source framework thus holds promise for turning brain activity mapping efforts into biological insights.

Dehnert K.W.,Howard Hughes Medical Institute
ACS chemical biology | Year: 2011

Many developmental processes depend on proper fucosylation, but this post-translational modification is difficult to monitor in vivo. Here we applied a chemical reporter strategy to visualize fucosylated glycans in developing zebrafish. Using azide-derivatized analogues of fucose, we metabolically labeled cell-surface glycans and then detected the incorporated azides via copper-free click chemistry with a difluorinated cyclooctyne probe. We found that the fucose salvage pathway enzymes are expressed during zebrafish embryogenesis but that they process the azide-modified substrates inefficiently. We were able to bypass the salvage pathway by using an azide-functionalized analogue of GDP-fucose. This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis. We used this technique to image fucosylated glycans in the enveloping layer of zebrafish embryos during the first 5 days of development. This work provides a method to study the biosynthesis of fucosylated glycans in vivo.

Bardwell J.C.A.,Howard Hughes Medical Institute | Bardwell J.C.A.,University of Michigan | Jakob U.,University of Michigan
Trends in Biochemical Sciences | Year: 2012

Protein disorder remains an intrinsically fuzzy concept. Its role in protein function is difficult to conceptualize and its experimental study is challenging. Although a wide variety of roles for protein disorder have been proposed, establishing that disorder is functionally important, particularly in vivo, is not a trivial task. Several molecular chaperones have now been identified as conditionally disordered proteins; fully folded and chaperone-inactive under non-stress conditions, they adopt a partially disordered conformation upon exposure to distinct stress conditions. This disorder appears to be vital for their ability to bind multiple aggregation-sensitive client proteins and to protect cells against the stressors. The study of these conditionally disordered chaperones should prove useful in understanding the functional role for protein disorder in molecular recognition. © 2012 Elsevier Ltd.

Paull T.T.,Howard Hughes Medical Institute
Annual Review of Biochemistry | Year: 2015

The ataxia-telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response, and it coordinates checkpoint activation, DNA repair, and metabolic changes in eukaryotic cells in response to DNA double-strand breaks and oxidative stress. Loss of ATM activity in humans results in the pleiotropic neurodegeneration disorder ataxia-telangiectasia. ATM exists in an inactive state in resting cells but can be activated by the Mre11-Rad50-Nbs1 (MRN) complex and other factors at sites of DNA breaks. In addition, oxidation of ATM activates the kinase independently of the MRN complex. This review discusses these mechanisms of activation, as well as the posttranslational modifications that affect this process and the cellular factors that affect the efficiency and specificity of ATM activation and substrate phosphorylation. I highlight functional similarities between the activation mechanisms of ATM, phosphatidylinositol 3-kinases (PI3Ks), and the other PI3K-like kinases, as well as recent structural insights into their regulation. Copyright © 2015 by Annual Reviews. All rights reserved.

Davis F.P.,Howard Hughes Medical Institute
Molecular BioSystems | Year: 2011

Small molecules that modulate protein-protein interactions are of great interest for chemical biology and therapeutics. Here I present a structure-based approach to predict 'bi-functional' sites able to bind both small molecule ligands and proteins, in proteins of unknown structure. First, I develop a homology-based annotation method that transfers binding sites of known three-dimensional structure onto protein sequences, predicting residues in ligand and protein binding sites with estimated true positive rates of 98% and 88%, respectively, at 1% false positive rates. Applying this method to the human proteome predicts 8463 proteins with bi-functional residues and correctly recovers the targets of known interaction modulators. Proteins with significantly (p < 0.01) more bi-functional residues than expected were found to be enriched in regulatory and depleted in metabolism functions. Finally, I demonstrate the utility of the method by describing examples of predicted overlap and evidence of their biological and therapeutic relevance. The results suggest that combining the structures of known binding sites with established fold detection algorithms can predict regions of protein-protein interfaces that are amenable to small molecule modulation. Open-source software and the results for several complete proteomes are available at http://pibase.janelia.org/ homolobind. © The Royal Society of Chemistry.

Xin X.-F.,Michigan State University | He S.Y.,Michigan State University | He S.Y.,Howard Hughes Medical Institute
Annual Review of Phytopathology | Year: 2013

Since the early 1980s, various strains of the gram-negative bacterial pathogen Pseudomonas syringae have been used as models for understanding plant-bacterial interactions. In 1991, a P. syringae pathovar tomato (Pst) strain, DC3000, was reported to infect not only its natural host tomato but also Arabidopsis in the laboratory, a finding that spurred intensive efforts in the subsequent two decades to characterize the molecular mechanisms by which this strain causes disease in plants. Genomic analysis shows that Pst DC3000 carries a large repertoire of potential virulence factors, including proteinaceous effectors that are secreted through the type III secretion system and a polyketide phytotoxin called coronatine, which structurally mimics the plant hormone jasmonate (JA). Study of Pst DC3000 pathogenesis has not only provided several conceptual advances in understanding how a bacterial pathogen employs type III effectors to suppress plant immune responses and promote disease susceptibility but has also facilitated the discovery of the immune function of stomata and key components of JA signaling in plants. The concepts derived from the study of Pst DC3000 pathogenesis may prove useful in understanding pathogenesis mechanisms of other plant pathogens. © Copyright ©2013 by Annual Reviews. All rights reserved.

Deshaies R.J.,Howard Hughes Medical Institute
Sub-Cellular Biochemistry | Year: 2010

The Cullin-RING ubiquitin ligase (CRL) family, which may number as many as 350 different enzymes, has an enormous impact on cellular regulation. CRL enzymes regulate cell biology by conjugating ubiquitin onto target proteins that are involved in a multitude of processes. In most cases this leads to degradation of the target, but in some cases CRL-dependent ubiquitination acts as a switch to activate or repress target function. The ubiquitin ligase activity of CRLs is controlled by cycles of attachment and removal of the ubiquitin-like protein Nedd8. Conjugation of Nedd8 onto the cullin subunit of CRLs promotes assembly of an intact CRL complex and switches on ubiquitin ligase activity. Conversely, removal of Nedd8 switches off ubiquitin ligase activity and initiates CRL disassembly. Continuous maintenance of CRL function in vivo requires the activities of both the Nedd8-conjugating and deconjugating enzymes, pointing to a critical role of complex dynamics in CRL function. Here, we review how the Nedd8 cycle controls CRL activity and how perturbations of this cycle can lead to disease. © 2010 Landes Bioscience and Springer Science+Business Media

Physical inputs, both internal and external to a cell, can directly alter the spatial organization of cell surface receptors and their associated functions. Here we describe a protocol that combines solid-state nanolithography and supported lipid membrane techniques to trigger and manipulate specific receptors on the surface of living cells and to develop an understanding of the interplay between spatial organization and receptor function. While existing protein-patterning techniques are capable of presenting cells with well-defined clusters of protein, this protocol uniquely allows for the control of the spatial organization of laterally fluid receptor-ligand complex at an intermembrane junction. A combination of immunofluorescence and single-cell microscopy methods and complementary biochemical analyses are used to characterize receptor signaling pathways and cell functions. The protocol requires 2-5 d to complete depending on the parameters to be studied. In principle, this protocol is widely applicable to eukaryotic cells and herein is specifically developed to study the role of physical organization and translocation of the EphA2 receptor tyrosine kinase across a library of model breast cancer cell lines.

Lahn B.T.,Howard Hughes Medical Institute
BioEssays | Year: 2011

A simple model, termed "occlusis", is presented here to account for both cell fate restriction during somatic development and reestablishment of pluripotency during reproduction. The model makes three assertions: (1) A gene's transcriptional potential can assume one of two states: the "competent" state, wherein the gene is responsive to, and can be activated by, trans-acting factors in the cellular milieu, and the "occluded" state, wherein the gene is blocked by cis-acting, chromatin-based mechanisms from responding to trans-acting factors such that it remains silent irrespective of whether transcriptional activators are present in the milieu. (2) As differentiation proceeds in somatic lineages, lineage-inappropriate genes shift progressively and irreversibly from competent to occluded state, thereby leading to the restriction of cell fate. (3) During reproduction, global deocclusion takes place in the germline and/or early zygotic cells to reset the genome to the competent state in order to facilitate a new round of organismal development. Copyright © 2011 WILEY Periodicals, Inc.

Yasuda R.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Biology | Year: 2012

Many forms of synaptic plasticity are triggered by biochemical signaling that occurs in small postsynaptic compartments called dendritic spines, each of which typically houses the postsynaptic terminal associated with a single glutamatergic synapse. Recent advances in optical techniques allow investigators to monitor biochemical signaling in single dendritic spines and thus reveal the signaling mechanisms that link synaptic activity and the induction of synaptic plasticity. This is mostly in the study of Ca2+-dependent forms of synaptic plasticity for which many of the steps between Ca2+ influx and changes to the synapse are now known. This article introduces the new techniques used to investigate signaling in single dendritic spines and the neurobiological insights that they have produced. © 2012 Cold Spring Harbor Laboratory Press.

Karch J.,University of Cincinnati | Molkentin J.D.,University of Cincinnati | Molkentin J.D.,Howard Hughes Medical Institute
Circulation Research | Year: 2015

Although The molecular effectors of apoptotic cell death have been largely annotated over The past 30 years, leading to a strong biological understanding of this process and its importance in cell biology, cell death through necrosis has only recently been accepted as a similarly regulated process with definable molecular effectors. The mitochondria are important and central mediators of both apoptosis and regulated necrosis. In apoptosis, The B-cell leukemia/lymphoma 2 (Bcl-2) family members Bcl-2-associated protein x (Bax) and Bcl-2 homologues antagonist/killer (Bak) undergo oligomerization in The outer mitochondrial membrane resulting in The release of apoptosis inducing substrates and The activation of caspases and nucleases. In contrast, during necrosis The mitochondria become dysfunctional and maladaptive in conjunction with reactive oxygen species production and The loss of ATP production, in part through opening of The mitochondrial permeability transition pore. Although regulated necrosis is caspase-independent, recent evidence has shown that it still requires The apoptotic regulators Bax/Bak, which can regulate The permeability characteristics of The outer mitochondrial membrane in their nonoligomerized state. Here, we review The nonapoptotic side of Bcl-2 family, specifically The role of Bax/Bak in regulated necrotic cell death. We will also discuss how these Bcl-2 family member effectors could be part of a larger integrated network that ultimately decides The fate of a given cell somewhere within a molecular continuum between apoptosis and regulated necrosis. © 2015 American Heart Association, Inc.

Davis R.J.,Howard Hughes Medical Institute
Seminars in Immunology | Year: 2014

The binding of tumour necrosis factor α (TNFα) to cell surface receptors engages multiple signal transduction pathways, including three groups of mitogen-activated protein (MAP) kinases: extracellular-signal-regulated kinases (ERKs); the cJun NH2-terminal kinases (JNKs); and the p38 MAP kinases. These MAP kinase signalling pathways induce a secondary response by increasing the expression of several inflammatory cytokines (including TNFα) that contribute to the biological activity of TNFα. MAP kinases therefore function both upstream and down-stream of signalling by TNFα receptors. Here we review mechanisms that mediate these actions of MAP kinases during the response to TNFα. © 2014 Elsevier Ltd.

Hudak J.E.,University of California at Berkeley | Canham S.M.,University of California at Berkeley | Bertozzi C.R.,University of California at Berkeley | Bertozzi C.R.,Howard Hughes Medical Institute
Nature Chemical Biology | Year: 2014

The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human natural killer (NK) cell activation through the recruitment of sialic acid-binding immunoglobulin-like lectin 7 (Siglec-7). Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing, suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy. © 2014 Nature America, Inc. All rights reserved.

Enchev R.I.,ETH Zurich | Schulman B.A.,Howard Hughes Medical Institute | Peter M.,ETH Zurich
Nature Reviews Molecular Cell Biology | Year: 2015

NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin-RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here, we re-evaluate studies of non-cullin targets of NEDD8 in light of the current understanding of the neddylation pathway, and suggest criteria for identifying genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights. © 2014 Macmillan Publishers Limited.

Salazar M.E.,Massachusetts Institute of Technology | Laub M.T.,Massachusetts Institute of Technology | Laub M.T.,Howard Hughes Medical Institute
Current Opinion in Microbiology | Year: 2015

Bacteria sense and respond to numerous environmental signals through two-component signaling pathways. Typically, a given stimulus will activate a sensor histidine kinase to autophosphorylate and then phosphotransfer to a cognate response regulator, which can mount an appropriate response. Although these signaling pathways often appear to be simple switches, they can also orchestrate surprisingly sophisticated and complex responses. These temporal dynamics arise from several key regulatory features, including the bifunctionality of histidine kinases as well as positive and negative feedback loops. Two-component signaling pathways are also dynamic on evolutionary time-scales, expanding dramatically in many species through gene duplication and divergence. Here, we review recent work probing the temporal and evolutionary dynamics of two-component signaling systems. © 2015.

Richardson J.L.,University of Connecticut | Urban M.C.,University of Connecticut | Bolnick D.I.,Howard Hughes Medical Institute | Skelly D.K.,Yale University
Trends in Ecology and Evolution | Year: 2014

Local adaptation has been a major focus of evolutionary ecologists working across diverse systems for decades. However, little of this research has explored variation at microgeographic scales because it has often been assumed that high rates of gene flow will prevent adaptive divergence at fine spatial scales. Here, we establish a quantitative definition of microgeographic adaptation based on Wright's dispersal neighborhood that standardizes dispersal abilities, enabling this measure to be compared across species. We use this definition to evaluate growing evidence of evolutionary divergence at fine spatial scales. We identify the main mechanisms known to facilitate this adaptation and highlight illustrative examples of microgeographic evolution in nature. Collectively, this evidence requires that we revisit our understanding of the spatial scale of adaptation and consider how microgeographic adaptation and its driving mechanisms can fundamentally alter ecological and evolutionary dynamics in nature. © 2014 Elsevier Ltd.

Peng H.,Howard Hughes Medical Institute
Nature Biotechnology | Year: 2010

The V3D system provides three-dimensional (3D) visualization of gigabyte-sized microscopy image stacks in real time on current laptops and desktops. V3D streamlines the online analysis, measurement and proofreading of complicated image patterns by combining ergonomic functions for selecting a location in an image directly in 3D space and for displaying biological measurements, such as from fluorescent probes, using the overlaid surface objects. V3D runs on all major computer platforms and can be enhanced by software plug-ins to address specific biological problems. To demonstrate this extensibility, we built a V3D-based application, V3D-Neuron, to reconstruct complex 3D neuronal structures from high-resolution brain images. V3D-Neuron can precisely digitize the morphology of a single neuron in a fruitfly brain in minutes, with about a 17-fold improvement in reliability and tenfold savings in time compared with other neuron reconstruction tools. Using V3D-Neuron, we demonstrate the feasibility of building a 3D digital atlas of neurite tracts in the fruitfly brain. © 2010 Nature America, Inc. All rights reserved.

Moazed D.,Howard Hughes Medical Institute
Current Biology | Year: 2013

Genetic evidence on the role of specific histone amino acids or their posttranslational modifications in metazoan development has been lacking. A recent study reports that fruit flies carrying histone H3 lysine 27 (H3K27) mutations have the same homeotic gene expression and developmental defects as mutations in the enzyme that trimethylates H3K27. © 2013 Elsevier Ltd.

Lavis L.D.,Howard Hughes Medical Institute | Raines R.T.,University of Wisconsin - Madison
ACS Chemical Biology | Year: 2014

Small-molecule fluorophores manifest the ability of chemistry to solve problems in biology. As we noted in a previous review (Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142-155), the extant collection of fluorescent probes is built on a modest set of "core" scaffolds that evolved during a century of academic and industrial research. Here, we survey traditional and modern synthetic routes to small-molecule fluorophores and highlight recent biological insights attained with customized fluorescent probes. Our intent is to inspire the design and creation of new high-precision tools that empower chemical biologists. © 2014 American Chemical Society.

Bruner K.M.,Johns Hopkins University | Hosmane N.N.,Johns Hopkins University | Siliciano R.F.,Johns Hopkins University | Siliciano R.F.,Howard Hughes Medical Institute
Trends in Microbiology | Year: 2015

The latent reservoir (LR) of HIV-1 in resting memory CD4+ T cells serves as a major barrier to curing HIV-1 infection. While many PCR- and culture-based assays have been used to measure the size of the LR, correlation between results of different assays is poor and recent studies indicate that no available assay provides an accurate measurement of reservoir size. The discrepancies between assays are a hurdle to clinical trials that aim to measure the efficacy of HIV-1 eradication strategies. Here we describe the advantages and disadvantages of various approaches to measuring the LR. © 2015 Elsevier Ltd.

Deshaies R.J.,Howard Hughes Medical Institute
BMC Biology | Year: 2014

Genomic alterations may make cancer cells more dependent than normal cells on mechanisms of proteostasis, including protein folding and degradation. This proposition is the basis for the clinical use of proteasome inhibitors to treat multiple myeloma and mantle cell lymphoma. However, proteasome inhibitors have not proved effective in treating other cancers, and this has called into question the general applicability of this approach. Here, I consider possible explanations for this apparently limited applicability, and discuss whether inhibiting other broadly acting components of the ubiquitin-proteasome system - including ubiquitin-activating enzyme and the AAA-ATPase p97/VCP - might be more generally effective in cancer therapy.

Freeman M.R.,Howard Hughes Medical Institute
Science | Year: 2010

Astrocytes are the most abundant cell type in the mammalian brain. Interest in astrocyte function has increased dramatically in recent years because of their newly discovered roles in synapse formation, maturation, efficacy, and plasticity. However, our understanding of astrocyte development has lagged behind that of other brain cell types. We do not know the molecular mechanism by which astrocytes are specified, how they grow to assume their complex morphologies, and how they interact with and sculpt developing neuronal circuits. Recent work has provided a basic understanding of how intrinsic and extrinsic mechanisms govern the production of astrocytes from precursor cells and the generation of astrocyte diversity. Moreover, new studies of astrocyte morphology have revealed that mature astrocytes are extraordinarily complex, interact with many thousands of synapses, and tile with other astrocytes to occupy unique spatial domains in the brain. A major challenge for the field is to understand how astrocytes talk to each other, and to neurons, during development to establish appropriate astrocytic and neuronal network architectures.

Zhou Y.,Johns Hopkins University | Nathans J.,Johns Hopkins University | Nathans J.,Howard Hughes Medical Institute
Developmental Cell | Year: 2014

Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a coactivator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp coreceptor and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. Theseexperiments identify Gpr124 as a ligand-specific coactivator of canonical Wnt signaling. © 2014 Elsevier Inc.

Hildebrandt F.,Howard Hughes Medical Institute
The Lancet | Year: 2010

Knowledge of the primary cause of a disease is essential for elucidation of its mechanisms, and for adequate classification, prognosis, and treatment. Recently, the causes of many kidney diseases have been shown to be single-gene defects-eg, steroid-resistant nephrotic syndrome, which is caused by podocin mutations in about 25% of children and nearly 15% of adults with the disease. Knowledge of a disease-causing mutation in a single-gene disorder represents one of the most robust diagnostic examples of personalised medicine because the mutation conveys an almost 100% risk of developing the disease by a defined age. Whereas single-gene diseases are rare disorders, polygenic risk alleles arise in common adult-onset diseases. In this Review, I will discuss prominent renal single-gene kidney disorders, and polygenic risk alleles of common disorders. I delineate how emerging techniques of total exome capture and large-scale sequencing will assist molecular genetic diagnosis, prognosis, and specific treatment, and lead to an improved elucidation of disease mechanisms, thus enabling development of new targeted drugs. © 2010 Elsevier Ltd. All rights reserved.

Subramaniam A.R.,Harvard University | Zid B.M.,Harvard University | O'Shea E.K.,Harvard University | O'Shea E.K.,Howard Hughes Medical Institute
Cell | Year: 2014

Ribosomes elongate at a nonuniform rate during translation. Theoretical models and experiments disagree on the in vivo determinants of elongation rate and the mechanism by which elongation rate affects protein levels. To resolve this conflict, we measured transcriptome-wide ribosome occupancy under multiple conditions and used it to formulate a whole-cell model of translation in E. coli. Our model predicts that elongation rates at most codons during nutrient-rich growth are not limited by the intracellular concentrations of aminoacyl-tRNAs. However, elongation pausing during starvation for single amino acids is highly sensitive to the kinetics of tRNA aminoacylation. We further show that translation abortion upon pausing accounts for the observed ribosome occupancy along mRNAs during starvation. Abortion reduces global protein synthesis, but it enhances the translation of a subset of mRNAs. These results suggest a regulatory role for aminoacylation and abortion during stress, and our study provides an experimentally constrained framework for modeling translation. © 2014 Elsevier Inc. All rights reserved.

Little S.C.,Howard Hughes Medical Institute | Gregor T.,Princeton University
Cell | Year: 2013

In the classic picture of morphogen-mediated patterning, cells acquire the correct spatial arrangement of specified fates by reading a precisely distributed gradient of morphogen. Xiong et al. now provide evidence for an alternate strategy - cells of the zebrafish neural tube actively sort to their correct positions following disordered specification by Sonic hedgehog. © 2013 Elsevier Inc.

Laganowsky A.,Howard Hughes Medical Institute
Protein science : a publication of the Protein Society | Year: 2010

Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC(59-163)) and human alphaB crystallin (ABC(68-162)), both containing the C-terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C-terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C-terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC(59-163) and ABC(68-162). A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.

Gunawardena S.,State University of New York at Buffalo | Yang G.,Carnegie Mellon University | Goldstein L.S.B.,Howard Hughes Medical Institute
Human Molecular Genetics | Year: 2013

Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation ofAPP-vesicle transport. Here,weshow that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease. © The Author 2013.

Price J.V.,University of California at Berkeley | Vance R.E.,University of California at Berkeley | Vance R.E.,Howard Hughes Medical Institute
Immunity | Year: 2014

Macrophages are a diverse population of phagocytic cells that reside in tissues throughout the body. At sites of infection, macrophages encounter and engulf invading microbes. Accordingly, macrophages possess specialized effector functions to kill or coordinate the elimination of their prey. Nevertheless, many intracellular bacterial pathogens preferentially replicate inside macrophages. Here we consider explanations for what we call "the macrophage paradox:" why do so many pathogenic bacteria replicate in the very cells equipped to destroy them? We ask whether replication in macrophages is an unavoidable fate that essentially defines a key requirement to be a pathogen. Conversely, we consider whether fundamental aspects of macrophage biology provide unique cellular or metabolic environments that pathogens can exploit. We conclude that resolution of the macrophage paradox requires acknowledgment of the richness and complexity of macrophages as a replicative niche. © 2014 Elsevier Inc.

Park M.-S.,Howard Hughes Medical Institute
PLoS ONE | Year: 2015

Glucose transporters (GLUTs) provide a pathway for glucose transport across membranes. Human GLUTs are implicated in devastating diseases such as heart disease, hyper- and hypo-glycemia, type 2 diabetes and caner. The human GLUT1 has been recently crystalized in the inward-facing open conformation. However, there is no other structural information for other conformations. The X-ray structures of E. coli Xylose permease (XylE), a glucose transporter homolog, are available in multiple conformations with and without the substrates D-xylose and D-glucose. XylE has high sequence homology to human GLUT1 and key residues in the sugar-binding pocket are conserved. Here we construct a homology model for human GLUT1 based on the available XylE crystal structure in the partially occluded outward-facing conformation. A long unbiased all atom molecular dynamics simulation starting from the model can capture a new fully opened outward-facing conformation. Our investigation of molecular interactions at the interface between the transmembrane (TM) domains and the intracellular helices (ICH) domain in the outward- and inward-facing conformation supports that the ICH domain likely stabilizes the outward-facing conformation in GLUT1. Furthermore, inducing a conformational transition, our simulations manifest a global asymmetric rocker switch motion and detailed molecular interactions between the substrate and residues through the water-filled selective pore along a pathway from the extracellular to the intracellular side. The results presented here are consistent with previously published biochemical, mutagenesis and functional studies. Together, this study shed light on the structure and functional relationships of GLUT1 in multiple conformational states. © 2015 Min-Sun Park.

Sudhof T.C.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2012

Upon entering a presynaptic terminal, an action potential opens Ca(2+) channels, and transiently increases the local Ca(2+) concentration at the presynaptic active zone. Ca(2+) then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca(2+). Synaptotagmins bind Ca(2+) via two C2-domains, and transduce the Ca(2+) signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca(2+)-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The conserved function of synaptotagmins and complexins operates generally in most, if not all, Ca(2+)-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release.

Marella S.,University of California at Berkeley | Mann K.,University of California at Berkeley | Scott K.,Howard Hughes Medical Institute | Scott K.,University of California at Berkeley
Neuron | Year: 2012

For an animal to survive in a constantly changing environment, its behavior must be shaped by the complex milieu of sensory stimuli it detects, its previous experience, and its internal state. Although taste behaviors in the fly are relatively simple, with sugars eliciting acceptance behavior and bitter compounds avoidance, these behaviors are also plastic and are modified by intrinsic and extrinsic cues, such as hunger and sensory stimuli. Here, we show that dopamine modulates a simple taste behavior, proboscis extension to sucrose. Conditional silencing of dopaminergic neurons reduces proboscis extension probability, and increased activation of dopaminergic neurons increases extension to sucrose, but not to bitter compounds or water. One dopaminergic neuron with extensive branching in the primary taste relay, the subesophageal ganglion, triggers proboscis extension, and its activity is altered by satiety state. These studies demonstrate the marked specificity of dopamine signaling and provide a foundation to examine neural mechanisms of feeding modulation in the fly. Marella et al. find that dopamine, via the actions of a single dopaminergic neuron with extensive branching in the primary taste relay, selectively modulates sucrose taste behavior in the fly. This work demonstrates a marked specificity of dopamine signaling for this particular taste behavior. © 2012 Elsevier Inc.

Zhou W.,University of Michigan | Hildebrandt F.,University of Michigan | Hildebrandt F.,Howard Hughes Medical Institute
Journal of the American Society of Nephrology | Year: 2012

Damage or loss of podocytes causes glomerulosclerosis inmurinemodels, andmutations inpodocyte-specific genes cause nephrotic syndrome in humans. Zebrafish provide a valuable model for kidney research, but disruption of pronephroi leads to death within a few days, thereby preventing the study of CKD. In this study,wegenerated an inducible model of podocyte injury in zebrafish (pod::NTR-mCherry) by expressing a bacterial nitroreductase, which converts metronidazole to a cytotoxin, specifically in podocytes under the control of the zebrafish nphs2/podocin promoter. Application of the prodrug metronidazole to the transgenic fish induces acute damage to the podocytes in pronephroi of larval zebrafish and the mesonephroi of adult zebrafish, resulting in foot-process effacement and podocyte loss. We also developed a functional assay of the glomerular filtration barrier by creating transgenic zebrafish expressing green fluorescent protein (GFP)-tagged vitamin D-binding protein (VDBP) as a tracer for proteinuria. In the VDBP-GFP and pod::NTR-mCherry double-transgenic fish, induction of podocyte damage led to whole-body edema, and the proximal tubules reabsorbed and accumulated VDBP-GFP that leaked through the glomeruli, mimicking the phenotype of human nephrotic syndrome. Moreover, expression of wt1b::GFP, a marker for the developing nephron, extended into the Bowman capsule in response to podocyte injury, suggesting that zebrafish have a podocyte-specific repair process known to occur in mammalian metanephros. These data support the use of these transgenic zebrafish as a model system for studies of glomerular pathogenesis and podocyte regeneration. Copyright © 2012 by the American Society of Nephrology.

Grossniklaus U.,University of Zurich | Kelly B.,Emory University | Ferguson-Smith A.C.,University of Cambridge | Pembrey M.,University College London | And 2 more authors.
Nature Reviews Genetics | Year: 2013

Much attention has been given to the idea of transgenerational epigenetic inheritance, but fundamental questions remain regarding how much takes place and the impact that this might have on organisms. We asked five leading researchers in this area-working on a range of model organisms and in human disease-for their views on these topics. Their responses highlight the mixture of excitement and caution that surrounds transgenerational epigenetic inheritance and the wide gulf between species in terms of our knowledge of the mechanisms that may be involved. © 2013 Macmillan Publishers Limited. All rights reserved.

Wysocki L.M.,Howard Hughes Medical Institute
Current opinion in chemical biology | Year: 2011

Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Slutter B.,University of Iowa | Pewe L.L.,University of Iowa | Kaech S.M.,Yale University | Kaech S.M.,Howard Hughes Medical Institute | Harty J.T.,University of Iowa
Immunity | Year: 2013

Inducing memory CD8+ Tcells specific for conserved antigens from influenza A virus (IAV) is a potential strategy for broadly protective vaccines. Here we show that memory CD8+ Tcells in the airways played an important role in early control of IAV. Expression of the chemokine receptor CXCR3 was critical for memory CD8+ Tcells to populate the airways during the steady state and vaccination approaches were designed to favor the establishment of memory CD8+ Tcells in the airways. Specifically, we found that interleukin-12 (IL-12) signaling shortly after immunization limited CXCR3 expression on memory CD8+ Tcells. Neutralization of IL-12 or adjuvants that did not induce high amounts of IL-12 enhanced CXCR3 expression, sustained airway localization of memory CD8+ Tcells, and resulted in superior protection against IAV. © 2013 Elsevier Inc.

Elliott J.M.,University of Washington | Wahle J.A.,University of Washington | Yokoyama W.M.,University of Washington | Yokoyama W.M.,Howard Hughes Medical Institute
Journal of Experimental Medicine | Year: 2010

In MHC class I-deficient hosts, natural killer (NK) cells are hyporesponsive to cross-linking of activation receptors. Functional competence requires engagement of a self-major histocompatability complex (MHC) class I-specific inhibitory receptor, a process referred to as "licensing." We previously suggested that licensing is developmentally determined in the bone marrow. In this study, we find that unlicensed mature MHC class I-deficient splenic NK cells show gain-of-function and acquire a licensed phenotype after adoptive transfer into wild-type (WT) hosts. Transferred NK cells produce WT levels of interferon-γ after engagement of multiple activation receptors, and degranulate at levels equivalent to WT NK cells upon coincubation with target cells. Only NK cells expressing an inhibitory Ly49 receptor specific for a cognate host MHC class I molecule show this gain-of-function. Therefore, these findings, which may be relevant to clinical bone marrow transplantation, suggest that neither exposure to MHC class I ligands during NK development in the BM nor endogenous MHC class I expression by NK cells themselves is absolutely required for licensing. © 2010 Elliott et al.

Murphy T.L.,University of Washington | Murphy K.M.,University of Washington | Murphy K.M.,Howard Hughes Medical Institute
Annual Review of Immunology | Year: 2010

B and T lymphocyte associated (BTLA) is an Ig domain superfamily protein with cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Its ligand, herpesvirus entry mediator (HVEM), is a tumor necrosis factor receptor superfamily member. The unique interaction between BTLA and HVEM allows for a system of bidirectional signaling that must be appropriately regulated to balance the outcome of the immune response. HVEM engagement of BTLA produces inhibitory signals through SH2 domain-containing protein tyrosine phosphatase 1 (Shp-1) and Shp-2 association, whereas BTLA engagement of HVEM produces proinflammatory signals via activation of NF-κB. The BTLA-HVEM interaction is intriguing and quite complex given that HVEM has four other ligands that also influence immune responses, the conventional TNF ligand LIGHT and lymphotoxin α, as well as herpes simplex virus glycoprotein D and the glycosylphosphatidylinositol-linked Ig domain protein CD160. BTLA-HVEM interactions have been shown to regulate responses in several pathogen and autoimmune settings, but our understanding of this complex system of interactions is certainly incomplete. Recent findings of spontaneous inflammation in BTLA-deficient mice may provide an important clue. Copyright © 2010 by Annual Reviews. All rights reserved.

Keller P.J.,Howard Hughes Medical Institute
Science | Year: 2013

Morphogenesis, the development of the shape of an organism, is a dynamic process on a multitude of scales, from fast subcellular rearrangements and cell movements to slow structural changes at the whole-organism level. Live-imaging approaches based on light microscopy reveal the intricate dynamics of this process and are thus indispensable for investigating the underlying mechanisms. This Review discusses emerging imaging techniques that can record morphogenesis at temporal scales from seconds to days and at spatial scales from hundreds of nanometers to several millimeters. To unlock their full potential, these methods need to be matched with new computational approaches and physical models that help convert highly complex image data sets into biological insights.

Haswell E.S.,Washington University in St. Louis | Phillips R.,California Institute of Technology | Rees D.C.,Howard Hughes Medical Institute
Structure | Year: 2011

While mechanobiological processes employ diverse mechanisms, at their heart are force-induced perturbations in the structure and dynamics of molecules capable of triggering subsequent events. Among the best characterized force-sensing systems are bacterial mechanosensitive channels. These channels reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane; the membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal, interpreted for the cell by mechanosensitive channels. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension or other functions. This review focuses on the bacterial mechanosensitive channels of large (MscL) and small (MscS) conductance and their eukaryotic homologs, with an emphasis on the outstanding issues surrounding the function and mechanism of this fascinating class of molecules. © 2011 Elsevier Ltd.

Cohn M.J.,Howard Hughes Medical Institute
Developmental Dynamics | Year: 2011

Over the past decade, the genetics of external genital development have begun to be understood. Male and female external genitalia develop from the genital tubercle. The early tubercle has a superficial resemblance to the limb bud, but an important distinction is that the limb consists of only mesoderm and ectoderm, whereas the genital tubercle also has an endodermal component, the urethral epithelium. Urethral epithelium, which expresses Sonic hedgehog, acts as a signaling region that controls outgrowth and pattern formation, and ultimately differentiates into the urethral tube. While there are intriguing parallels between limb and genital development, recent studies have identified some key differences, including the role of Fgf signaling. Our understanding of the mechanisms of genital development still lags far behind the limb, and major questions remain to be answered, including the molecular nature of the signals that initiate genital budding, sustain outgrowth, induce tissue polarity and orchestrate urethral tubulogenesis. © 2011 Wiley-Liss, Inc.

Dickinson B.C.,University of California at Berkeley | Chang C.J.,University of California at Berkeley | Chang C.J.,Howard Hughes Medical Institute
Nature Chemical Biology | Year: 2011

Reactive oxygen species (ROS) are a family of molecules that are continuously generated, transformed and consumed in all living organisms as a consequence of aerobic life. The traditional view of these reactive oxygen metabolites is one of oxidative stress and damage that leads to decline of tissue and organ systems in aging and disease. However, emerging data show that ROS produced in certain situations can also contribute to physiology and increased fitness. This Perspective provides a focused discussion on what factors lead ROS molecules to become signal and/or stress agents, highlighting how increasing knowledge of the underlying chemistry of ROS can lead to advances in understanding their disparate contributions to biology. An important facet of this emerging area at the chemistry-biology interface is the development of new tools to study these small molecules and their reactivity in complex biological systems. © 2011 Nature America, Inc. All rights reserved.

Reddien P.W.,Howard Hughes Medical Institute
Trends in Genetics | Year: 2011

Planarians are flatworms that constitutively maintain adult tissues through cell turnover and can regenerate entire organisms from tiny body fragments. In addition to requiring new cells (from neoblasts), these feats require mechanisms that specify tissue identity in the adult. Crucial roles for Wnt and BMP signaling in the regeneration and maintenance of the body axes have been uncovered, among other regulatory factors. Available data indicate that genes involved in positional identity regulation at key embryonic stages in other animals display persisting regionalized expression in adult planarians. These expression patterns suggest that a constitutively active gene expression map exists for the maintenance of the planarian body. Planarians thus present a fertile ground for the identification of factors regulating the regionalization of the metazoan body plan and for the study of the attributes of these factors that can lead to the maintenance and regeneration of adult tissues. © 2011 Elsevier Ltd.

Brooks E.R.,University of Texas at Austin | Wallingford J.B.,University of Texas at Austin | Wallingford J.B.,Howard Hughes Medical Institute
Current Biology | Year: 2014

Cilia are microtubule-based projections that serve a wide variety of essential functions in animal cells. Defects in cilia structure or function have recently been found to underlie diverse human diseases. While many eukaryotic cells possess only one or two cilia, some cells, including those of many unicellular organisms, exhibit many cilia. In vertebrates, multiciliated cells are a specialized population of post-mitotic cells decorated with dozens of motile cilia that beat in a polarized and synchronized fashion to drive directed fluid flow across an epithelium. Dysfunction of human multiciliated cells is associated with diseases of the brain, airway and reproductive tracts. Despite their importance, multiciliated cells are relatively poorly studied and we are only beginning to understand the mechanisms underlying their development and function. Here, we review the general phylogeny and physiology of multiciliation and detail our current understanding of the developmental and cellular events underlying the specification, differentiation and function of multiciliated cells in vertebrates. © 2014 Elsevier Ltd All rights reserved.

Wang X.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2011

A major surprise arising from genome-wide analyses has been the observation that the majority of the genome is transcribed, generating noncoding RNAs (ncRNAs). It is still an open question whether some or all of these ncRNAs constitute functional networks regulating gene transcriptional programs. However, in light of recent discoveries and given the diversity and flexibility of long ncRNAs and their abilities to nucleate molecular complexes and to form spatially compact arrays of complexes, it becomes likely that many or most ncRNAs act as sensors and integrators of a wide variety of regulated transcriptional responses and probably epigenetic events. Because many RNA-binding proteins, on binding RNAs, show distinct allosteric conformational alterations, we suggest that a ncRNA/RNA-binding protein-based strategy, perhaps in concert with several other mechanistic strategies, serves to integrate transcriptional, as well as RNA processing, regulatory programs.

Legant W.R.,Howard Hughes Medical Institute
Nature Methods | Year: 2016

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high–localization precision, ultrahigh–labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

Hartman N.C.,University of California at Berkeley | Hartman N.C.,Howard Hughes Medical Institute | Groves J.T.,Lawrence Berkeley National Laboratory
Current Opinion in Cell Biology | Year: 2011

Large-scale molecular assemblies, or signaling clusters, at the cell membrane are emerging as important regulators of cell signaling. Here, we review new findings and describe shared characteristics common to signaling clusters from a diverse set of cellular systems. The well-known T cell receptor cluster serves as our paradigmatic model. Specifically, each cluster initiates recruitment of hundreds of molecules to the membrane, interacts with the actin cytoskeleton, and contains a significant fraction of the entire signaling process. Probed by recent advancements in patterning and microscopy techniques, the signaling clusters display functional outcomes that are not readily predictable from the individual components. © 2011 Elsevier Ltd.

Sehgal A.,Howard Hughes Medical Institute | Mignot E.,Stanford University
Cell | Year: 2011

Sleep remains one of the least understood phenomena in biology - even its role in synaptic plasticity remains debatable. Since sleep was recognized to be regulated genetically, intense research has launched on two fronts: the development of model organisms for deciphering the molecular mechanisms of sleep and attempts to identify genetic underpinnings of human sleep disorders. In this Review, we describe how unbiased, high-throughput screens in model organisms are uncovering sleep regulatory mechanisms and how pathways, such as the circadian clock network and specific neurotransmitter signals, have conserved effects on sleep from Drosophila to humans. At the same time, genome-wide association studies (GWAS) have uncovered ∼14 loci increasing susceptibility to sleep disorders, such as narcolepsy and restless leg syndrome. To conclude, we discuss how these different strategies will be critical to unambiguously defining the function of sleep. © 2011 Elsevier Inc.

Whalen E.J.,Duke University | Rajagopal S.,Duke University | Lefkowitz R.J.,Duke University | Lefkowitz R.J.,Howard Hughes Medical Institute
Trends in Molecular Medicine | Year: 2011

Members of the seven-transmembrane receptor (7TMR), or G protein-coupled receptor (GPCR), superfamily represent some of the most successful targets of modern drug therapy, with proven efficacy in the treatment of a broad range of human conditions and disease processes. It is now appreciated that β-arrestins, once viewed simply as negative regulators of traditional 7TMR-stimulated G protein signaling, act as multifunctional adapter proteins that regulate 7TMR desensitization and trafficking and promote distinct intracellular signals in their own right. Moreover, several 7TMR biased agonists, which selectively activate these divergent signaling pathways, have been identified. Here we highlight the diversity of G protein- and β-arrestin-mediated functions and the therapeutic potential of selective targeting of these in disease states. © 2010 Elsevier Ltd.

Mishra P.,California Institute of Technology | Carelli V.,University of Bologna | Manfredi G.,Cornell University | Chan D.C.,California Institute of Technology | Chan D.C.,Howard Hughes Medical Institute
Cell Metabolism | Year: 2014

Mitochondrial fusion is essential for maintenance of mitochondrial function. The mitofusin GTPases control mitochondrial outer membrane fusion, whereas the dynamin-related GTPase Opa1 mediates inner membrane fusion. We show that mitochondrial inner membrane fusion is tuned by the level of oxidative phosphorylation (OXPHOS), whereas outer membrane fusion is insensitive. Consequently, cells from patients with pathogenic mtDNA mutations show a selective defect in mitochondrial inner membrane fusion. In elucidating the molecular mechanism of OXPHOS-stimulated fusion, we uncover that real-time proteolytic processing of Opa1 stimulates mitochondrial inner membrane fusion. OXPHOS-stimulated mitochondrial fusion operates through Yme1L, which cleaves Opa1 more efficiently under high OXPHOS conditions. Engineered cleavage of Opa1 is sufficient to mediate inner membrane fusion, regardless of respiratory state. Proteolytic cleavage therefore stimulates the membrane fusion activity of Opa1, and this feature is exploited to dynamically couple mitochondrial fusion to cellular metabolism. © 2014 Elsevier Inc.

James D.,New York Medical College | Rafii S.,Howard Hughes Medical Institute
Science Translational Medicine | Year: 2014

Endothelial cells in the neointima undergo a transition to the mesenchymal phenotype, suggesting a therapeutic target for vein graft restenosis (Cooley et al., this issue).

Steitz J.,Howard Hughes Medical Institute
Cold Spring Harbor perspectives in biology | Year: 2011

Like their host cells, many viruses produce noncoding (nc)RNAs. These show diversity with respect to time of expression during viral infection, length and structure, protein-binding partners and relative abundance compared with their host-cell counterparts. Viruses, with their limited genomic capacity, presumably evolve or acquire ncRNAs only if they selectively enhance the viral life cycle or assist the virus in combating the host's response to infection. Despite much effort, identifying the functions of viral ncRNAs has been extremely challenging. Recent technical advances and enhanced understanding of host-cell ncRNAs promise accelerated insights into the RNA warfare mounted by this fascinating class of RNPs.

Ryan D.P.,University of California at San Francisco | Ptacek L.J.,University of California at San Francisco | Ptacek L.J.,Howard Hughes Medical Institute
Neuron | Year: 2010

Inherited episodic neurological disorders are often due to mutations in ion channels or their interacting proteins, termed channelopathies. There are a wide variety of such disorders, from those causing paralysis, to extreme pain, to ataxia. A common theme in these is alteration of action potential properties or synaptic transmission and a resulting increased propensity of the resulting tissue to enter into or stay in an altered excitability state. Manifestations of these disorders are triggered by an array of precipitants, all of which stress the particular affected tissue in some way and aid in propelling its activity into an aberrant state. Study of these disorders has aided in the understanding of disease risk factors and elucidated the cause of clinically related sporadic disorders. The findings from study of these disorders will aid in the diagnosis and efficient targeted treatment of affected patients. © 2010 Elsevier Inc.

Wu J.,University of Texas Southwestern Medical Center | Chen Z.J.,University of Texas Southwestern Medical Center | Chen Z.J.,Howard Hughes Medical Institute
Annual Review of Immunology | Year: 2014

The innate immune system utilizes pattern-recognition receptors (PRRs) to detect the invasion of pathogens and initiate host antimicrobial responses such as the production of type I interferons and proinflammatory cytokines. Nucleic acids, which are essential genetic information carriers for all living organisms including viral, bacterial, and eukaryotic pathogens, are major structures detected by the innate immune system. However, inappropriate detection of self nucleic acids can result in autoimmune diseases. PRRs that recognize nucleic acids in cells include several endosomal members of the Toll-like receptor family and several cytosolic sensors for DNA and RNA. Here, we review the recent advances in understanding the mechanism of nucleic acid sensing and signaling in the cytosol of mammalian cells as well as the emerging role of cytosolic nucleic acids in autoimmunity. © 2014 by Annual Reviews. All rights reserved.

Goldberg D.E.,Howard Hughes Medical Institute
Cell Host and Microbe | Year: 2012

Intraerythrocytic malaria parasites send hundreds of effector proteins into the host cell. Diverse modes of export have been proposed for different proteins. In this issue, Grüring et al. (2012) present findings that bring the models together. © 2012 Elsevier Inc.

High K.A.,Childrens Hospital of Philadelphia | High K.A.,Howard Hughes Medical Institute | High K.A.,University of Pennsylvania
Blood | Year: 2012

Since the isolation and characterization of the genes for FVIII and FIX some 30 years ago, a longstanding goal of the field has been development of successful gene therapy for the hemophilias. In a landmark study published in 2011, Nathwani et al demonstrated successful conversion of severe hemophilia B to mild or moderate disease in 6 adult males who underwent intravenous infusion of an adeno-associated viral (AAV) vector expressing factor IX. These 6 subjects have now exhibited expression of FIX at levels ranging from 1% to 6% of normal for periods of > 2 years. This review discusses obstacles that were overcome to reach this goal and the next steps in clinical investigation. Safety issues that will need to be addressed before more widespread use of this approach are discussed. Efforts to extend AAV-mediated gene therapy to hemophilia A, and alternate approaches that may be useful for persons with severe liver disease, who may not be candidates for gene transfer to liver, are also discussed. © 2012 by The American Society of Hematology.

Dillin A.,Howard Hughes Medical Institute | Cohen E.,Hebrew University of Jerusalem
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2011

Late onset is a common hallmark character of numerous disorders including human neurodegenerative maladies such as Huntington's, Parkinson's and Alzheimer's diseases. Why these diseases manifest in aged individuals and why distinct disorders share strikingly similar emergence patterns were until recently unsolved enigmas. During the past decade, invertebrate-based studies indicated that the insulin/IGF signalling pathway (IIS) mechanistically links neurodegenerative-associated toxic protein aggregation and ageing; yet, until recently it was unclear whether this link is conserved from invertebrates to mammals. Recent studies performed in Alzheimer's mouse models indicated that ageing alteration by IIS reduction slows the progression of Alzheimer's-like disease, protects the brain and mitigates the behavioural, pathological and biochemical impairments associated with the disease. Here, we review these novel studies and discuss the potential of ageing alteration as a therapeutic approach for the treatment of late onset neurodegeneration. © 2011 The Royal Society.

Reddy B.V.,Howard Hughes Medical Institute
Developmental cell | Year: 2013

EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. We find that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrate that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. Our observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways. Copyright © 2013 Elsevier Inc. All rights reserved.

Feng S.,University of California at Los Angeles | Jacobsen S.E.,University of California at Los Angeles | Jacobsen S.E.,Howard Hughes Medical Institute
Current Opinion in Plant Biology | Year: 2011

Plant genomes are modified by an array of epigenetic marks that help regulate plant growth and reproduction. Although plants share many epigenetic features with animals and fungi, some epigenetic marks are unique to plants. In different organisms, the same epigenetic mark can play different roles and/or similar functions can be carried out by different epigenetic marks. Furthermore, while the enzymatic systems responsible for generating or eliminating epigenetic marks are often conserved, there are also cases where they are quite divergent between plants and other organisms. DNA methylation and methylation of histone tails on the lysine 4, 9, and 27 positions are among the best characterized epigenetic marks in both plants and animals. Recent studies have greatly enhanced our knowledge about the pattern of these marks in various genomes and provided insights into how they are established and maintained and how they function. This review focuses on the conservation and divergence of the pathways that mediate these four types of epigenetic marks. © 2010 Elsevier Ltd.

Vey J.L.,Massachusetts Institute of Technology | Drennan C.L.,Massachusetts Institute of Technology | Drennan C.L.,Howard Hughes Medical Institute
Chemical Reviews | Year: 2011

The radical SAM enzymes, also referred to as the AdoMet radical enzymes are a newly identified enzyme superfamily capable of catalyzing radical chemistry similar to, but more extensive than, that performed by the AdoCbl-dependent enzymes. Radical SAM enzymes all catalyze radical chemistry and are united as a superfamily by their common mechanism of radical generation. The radical SAM superfamily has been expanded by the characterization of ThiC. While studying thiamine pyrimidine biosynthesis, Downs et al. found that the protein ThiC carries out radical SAM chemistry but does not contain the conserved motif. One of the two initial radical SAM enzymes to be structurally characterized, HemN is one of the more recently biochemically characterized members of this superfamily. Of all of the radical SAM enzymes, Lysine Aminomutase (LAM) is the best characterized. The radical SAM enzyme HydE from Thermotoga maritima is one of three enzymes recently identified as essential for maturation of the hydrogenase from that organism.

Jin C.,Yale University | Henao-Mejia J.,Yale University | Flavell R.A.,Yale University | Flavell R.A.,Howard Hughes Medical Institute
Cell Metabolism | Year: 2013

The study of the intersection of immunology and metabolism is a growing field fueled by the increased prevalence of obesity-associated pathologies. Importantly, the capacity of the innate immune system to sense metabolic stress induced by nutritional surplus has been linked with the progression of obesity, insulin resistance, type 2 diabetes mellitus, nonalcoholic fatty liver disease, and atherosclerosis. Moreover, it is clear that the innate immune system regulates the composition of the intestinal microbiota, which impacts multiple host metabolic processes. Here we review recent studies in this emerging field with an emphasis on how innate immune receptors determine metabolic disease progression. © 2013 Elsevier Inc.

Sparks E.,Duke University | Wachsman G.,Duke University | Benfey P.N.,Duke University | Benfey P.N.,Howard Hughes Medical Institute
Nature Reviews Genetics | Year: 2013

Plants, being sessile organisms, need to respond to changing environments, and as a result they have evolved unique signalling mechanisms that allow rapid communication between different parts of the plant. The signalling mechanisms that direct plant development include long-range effectors, such as phytohormones, and molecules with a local intra-organ range, such as peptides, transcription factors and some small RNAs. In this Review, we highlight recent advances in understanding plant signalling mechanisms and discuss how different classes of signalling networks can integrate with gene regulatory networks and contribute to plant development. In some cases, we also address the evolutionary context of mechanisms and discuss possible links between the lifestyle of plants and selection for different signalling mechanisms. © 2013 Macmillan Publishers Limited. All rights reserved.

Coulon A.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | Chow C.C.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | Singer R.H.,Yeshiva University | Singer R.H.,Howard Hughes Medical Institute | Larson D.R.,U.S. National Cancer Institute
Nature Reviews Genetics | Year: 2013

Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time- and energy-dependence at the molecular level. © 2013 Macmillan Publishers Limited. All rights reserved.

Meyer H.-J.,University of California at Berkeley | Rape M.,Howard Hughes Medical Institute | Rape M.,University of California at Berkeley
Cell | Year: 2014

Posttranslational modification of cell-cycle regulators with ubiquitin chains is essential for eukaryotic cell division. Such chains can be connected through seven lysine residues or the amino terminus of ubiquitin, thereby allowing the assembly of eight homogenous and multiple mixed or branched conjugates. Although functions of homogenous chain types have been described, physiological roles of branched structures are unknown. Here, we report that the anaphase-promoting complex (APC/C) efficiently synthesizes branched conjugates that contain multiple blocks of K11-linked chains. Compared to homogenous chains, the branched conjugates assembled by the APC/C strongly enhance substrate recognition by the proteasome, thereby driving degradation of cell-cycle regulators during early mitosis. Our work, therefore, identifies an enzyme and substrates for modification with branched ubiquitin chains and points to an important role of these conjugates in providing an improved signal for proteasomal degradation. © 2014 Elsevier Inc.

Radhakrishnan S.K.,California Institute of Technology | den Besten W.,California Institute of Technology | Deshaies R.J.,Howard Hughes Medical Institute
eLife | Year: 2014

Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., 'bounce-back') of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. ©Radhakrishnan et al.

Lim W.A.,University of California at San Francisco | Lim W.A.,Howard Hughes Medical Institute | Lee C.M.,University of California at San Francisco | Tang C.,Tsinghua University
Molecular Cell | Year: 2013

A challenge in biology is to understand how complex molecular networks in the cell execute sophisticated regulatory functions. Here we explore the idea that there are common and general principles that link network structures to biological functions, principles that constrain the design solutions that evolution can converge upon for accomplishing a given cellular task. We describe approaches for classifying networks based on abstract architectures and functions, rather than on the specific molecular components of the networks. For any common regulatory task, can we define the space of all possible molecular solutions? Such inverse approaches might ultimately allow the assembly of a design table of core molecular algorithms that could serve as a guide for building synthetic networks and modulating disease networks. © 2013 Elsevier Inc.

Nguyen Q.T.,University of California at San Diego | Tsien R.Y.,Howard Hughes Medical Institute
Nature Reviews Cancer | Year: 2013

A glowing new era in cancer surgery may be dawning. Using fluorescently labelled markers, surgical molecular navigation means that tumours and nerves can be displayed in real time intra-operatively in contrasting pseudocolours, which allows more complete tumour resection while preserving important structures. These advances can potentially cause a paradigm shift in cancer surgery, improving patient outcome and decreasing overall health-care costs. © 2013 Macmillan Publishers Limited. All rights reserved.

Fields S.,Howard Hughes Medical Institute
Genetics | Year: 2014

Fred Sanger developed technologies that won him two Nobel Prizes and revolutionized biological research. Yet, in spite of this record, the question has been raised as to whether, in the current scientific climate, he might be unsuccessful in obtaining a grant because of a productivity that would be viewed as too limited. In imagining how a National Institutes of Health study section today might treat a proposal from Sanger to sequence DNA, we can ask whether there are lessons from his career that suggest changes to the grant review process. © 2014 by the Genetics Society of America.

Lydeard J.R.,Harvard University | Schulman B.A.,Howard Hughes Medical Institute | Harper J.W.,Harvard University
EMBO Reports | Year: 2013

Cullin-RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re-sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how cullin neddylation cycles are integrated with receptor exchange. © 2013 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION.

Eddy S.R.,Howard Hughes Medical Institute
Annual Review of Biophysics | Year: 2014

Transcriptomics experiments and computational predictions both enable systematic discovery of new functional RNAs. However, many putative noncoding transcripts arise instead from artifacts and biological noise, and current computational prediction methods have high false positive rates. I discuss prospects for improving computational methods for analyzing and identifying functional RNAs, with a focus on detecting signatures of conserved RNA secondary structure. An interesting new front is the application of chemical and enzymatic experiments that probe RNA structure on a transcriptome-wide scale. I review several proposed approaches for incorporating structure probing data into the computational prediction of RNA secondary structure. Using probabilistic inference formalisms, I show how all these approaches can be unified in a well-principled framework, which in turn allows RNA probing data to be easily integrated into a wide range of analyses that depend on RNA secondary structure inference. Such analyses include homology search and genome-wide detection of new structural RNAs. Copyright © 2014 by Annual Reviews. All rights reserved.

Yao N.Y.,Howard Hughes Medical Institute
Sub-cellular biochemistry | Year: 2012

The eukaryotic RFC clamp loader couples the energy of ATP hydrolysis to open and close the circular PCNA sliding clamp onto primed sites for use by DNA polymerases and repair factors. Structural studies reveal clamp loaders to be heteropentamers. Each subunit contains a region of homology to AAA+ proteins that defines two domains. The AAA+ domains form a right-handed spiral upon binding ATP. This spiral arrangement generates a DNA binding site within the center of RFC. DNA enters the central chamber through a gap between the AAA+ domains of two subunits. Specificity for a primed template junction is achieved by a third domain that blocks DNA, forcing it to bend sharply. Thus only DNA with a flexible joint can bind the central chamber. DNA entry also requires a slot in the PCNA clamp, which is opened upon binding the AAA+ domains of the clamp loader. ATP hydrolysis enables clamp closing and ejection of RFC, completing the clamp loading reaction.

Iwasaki A.,Yale University | Medzhitov R.,Yale University | Medzhitov R.,Howard Hughes Medical Institute
Cell | Year: 2011

Highly virulent influenza virus infection results in excessive cytokine production, recruitment of leukocytes, and immune-mediated pulmonary injury. Teijaro et al. (2011) now demonstrate that sphingosine-1-phosphate receptor 1 ligands suppress all features of flu-inflicted pathological inflammation and place the endothelium at the center of this regulatory network. © 2011 Elsevier Inc.

Kim H.,Howard Hughes Medical Institute
Reports on progress in physics. Physical Society (Great Britain) | Year: 2013

Precision measurement is a hallmark of physics but the small length scale (∼nanometer) of elementary biological components and thermal fluctuations surrounding them challenge our ability to visualize their action. Here, we highlight the recent developments in single-molecule nanometry where the position of a single fluorescent molecule can be determined with nanometer precision, reaching the limit imposed by the shot noise, and the relative motion between two molecules can be determined with ∼0.3 nm precision at ∼1 ms time resolution, as well as how these new tools are providing fundamental insights into how motor proteins move on cellular highways. We will also discuss how interactions between three and four fluorescent molecules can be used to measure three and six coordinates, respectively, allowing us to correlate the movements of multiple components. Finally, we will discuss recent progress in combining angstrom-precision optical tweezers with single-molecule fluorescent detection, opening new windows for multi-dimensional single-molecule nanometry for biological physics.

Zuo Y.,Yale University | Wang Y.,Yale University | Steitz T.A.,Yale University | Steitz T.A.,Howard Hughes Medical Institute
Molecular Cell | Year: 2013

Guanosine tetraphosphate (ppGpp) is an alarmone that enables bacteria to adapt to their environment. It has been known for years that ppGpp acts directly on RNA polymerase (RNAP) to alter the rate of transcription, but its exact target site is still under debate. Here we report a crystal structure of Escherichia coli RNAP holoenzyme in complex with ppGpp at 4.5 å resolution. The structure reveals that ppGpp binds at an interface between the shelf and core modules on the outer surface of RNAP, away from the catalytic center and the nucleic acid binding path. Bound ppGpp connects these two pivotal modules that may restrain the opening of the RNAP cleft. A detailed mechanism of action of ppGpp is proposed in which ppGpp prevents the closure of the active center that is induced by the binding of NTP, which could slow down nucleotide addition cycles and destabilize the initial transcription complexes. © 2013 Elsevier Inc.

Hastings K.T.,University of Arizona | Cresswell P.,Howard Hughes Medical Institute
Antioxidants and Redox Signaling | Year: 2011

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is constitutively expressed in most antigen presenting cells and is interferon γ inducible in other cell types via signal transducer and activator of transcription 1. Normally, N-and C-terminal propeptides are cleaved in the early endosome, and the mature protein resides in late endosomes and lysosomes. Correspondingly, GILT has maximal reductase activity at an acidic pH. Monocyte differentiation via Toll-like receptor 4 triggers secretion of a disulfide-linked dimer of the enzymatically active precursor, which may contribute to inflammation. GILT facilitates major histocompatibility complex (MHC) class II-restricted processing through reduction of protein disulfide bonds in the endocytic pathway and is hypothesized to expose buried epitopes for MHC class II binding. GILT can also facilitate the transfer of disulfide-containing antigens into the cytosol, enhancing their cross-presentation by MHC class I. A variety of antigens are strongly influenced by GILT-mediated reduction, including hen egg lysozyme, melanocyte differentiation antigens, and viral envelope glycoproteins. In addition, GILT is conserved among lower eukaryotes and likely has additional functions. For example, GILT expression increases the stability of superoxide dismutase 2 and decreases reactive oxygen species, which correlates with decreased cellular proliferation. It is also a critical host factor for infection with Listeria monocytogenes. © 2011 Mary Ann Liebert, Inc.

Zhang J.,Harvard University | Walter J.C.,Harvard University | Walter J.C.,Howard Hughes Medical Institute
DNA Repair | Year: 2014

A critical step in DNA interstrand cross-link repair is the programmed collapse of replication forks that have stalled at an ICL. This event is regulated by the Fanconi anemia pathway, which suppresses bone marrow failure and cancer. In this perspective, we focus on the structure of forks that have stalled at ICLs, how these structures might be incised by endonucleases, and how incision is regulated by the Fanconi anemia pathway. © 2014 Elsevier B.V.

Que E.L.,University of California at Berkeley | Chang C.J.,University of California at Berkeley | Chang C.J.,Howard Hughes Medical Institute
Chemical Society Reviews | Year: 2010

This tutorial review highlights progress in the development of responsive magnetic resonance imaging (MRI) contrast agents for detecting and sensing biologically relevant metal ions. Molecular imaging with bioactivatable MRI indicators offers a potentially powerful methodology for studying the physiology and pathology of metals by capturing dynamic three-dimensional images of living systems for research and clinical applications. This emerging area at the interface of inorganic chemistry and the life sciences offers a broad palette of opportunities for researchers with interests ranging from coordination chemistry and spectroscopy to supramolecular chemistry and molecular recognition to metals in biology and medicine. © 2010 The Royal Society of Chemistry.

Grimes W.N.,Howard Hughes Medical Institute
Visual Neuroscience | Year: 2012

Feedback is a ubiquitous feature of neural circuits in the mammalian central nervous system (CNS). Analogous to pure electronic circuits, neuronal feedback provides either a positive or negative influence on the output of upstream components/neurons. Although the particulars (i.e., connectivity, physiological encoding/processing/signaling) of circuits in higher areas of the brain are often unclear, the inner retina proves an excellent model for studying both the anatomy and physiology of feedback circuits within the functional context of visual processing. Inner retinal feedback to bipolar cells is almost entirely mediated by a single class of interneurons, the amacrine cells. Although this might sound like a simple circuit arrangement with an equally simple function, anatomical, molecular, and functional evidence suggest that amacrine cells represent an extremely diverse class of CNS interneurons that contribute to a variety of retinal processes. In this review, I classify the amacrine cells according to their anatomical output synapses and target cell(s) (i.e., bipolar cells, ganglion cells, and/or amacrine cells) and discuss specifically our current understandings of amacrine cell-mediated feedback and output to bipolar cells on the synaptic, cellular, and circuit levels, while drawing connections to visual processing. © Copyright Cambridge University Press 2012.

Mouquet H.,Rockefeller University | Nussenzweig M.C.,Howard Hughes Medical Institute
Cellular and Molecular Life Sciences | Year: 2012

B cells express immunoglobulins on their surface where they serve as antigen receptors. When secreted as antibodies, the same molecules are key elements of the humoral immune response against pathogens such as viruses. Although most antibodies are restricted to binding a specific antigen, some are polyreactive and have the ability to bind to several different ligands, usually with low affinity. Highly polyreactive antibodies are removed from the repertoire during B-cell development by physiologic tolerancemechanisms including deletion and receptor editing. However, a low level of antibody polyreactivity is tolerated and can confer additional binding properties to pathogen-specific antibodies. For example, high-affinity human antibodies toHIVare frequently polyreactive. Here we review the evidence suggesting that in the case of some pathogens like HIV, polyreactivity may confer a selective advantage to pathogen-specific antibodies. © 2011 Springer Basel AG.

Hudalla G.A.,Howard Hughes Medical Institute | Murphy W.L.,University of Wisconsin - Madison
Advanced Functional Materials | Year: 2011

Growth factor activity is localized within the natural extracellular matrix (ECM) by specific noncovalent interactions with core ECM biomolecules, such as proteins and proteoglycans. Recently, these interactions inspired the development of synthetic biomaterials that can noncovalently regulate growth factor activity for tissue-engineering applications. For example, biomaterials covalently or noncovalently modified with heparin glycosaminoglycans can augment growth factor-release strategies. In addition, recent studies demonstrate that biomaterials modified with heparin-binding peptides can sequester cell-secreted heparin proteoglycans and, in turn, sequester growth factors and regulate stem cell behavior. Another set of studies shows that modular versions of growth factor molecules can be designed to interact with specific components of natural and synthetic ECMs, including collagen and hydroxyapatite. In addition, layer-by-layer assemblies of GAGs and other natural polyelectrolytes retain growth factors at a cell/material interface via specific noncovalent interactions. This Feature Article provides a detailed overview of the various bioinspired strategies that are used to noncovalently localize growth factor activity within biomaterials, and will highlight in vivo examples of the efficacy of these materials to promote tissue regeneration. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Shay J.E.S.,University of Pennsylvania | Celeste Simon M.,University of Pennsylvania | Celeste Simon M.,Howard Hughes Medical Institute
Seminars in Cell and Developmental Biology | Year: 2012

Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors that allow adaptation to hypoxic environments. HIFs function in the cellular response to stress: metabolic, hypoxic, or inflammatory. Metabolic changes occur during tumorigenesis that are, in part, under hypoxia and HIF regulation. Additionally, inflammatory signaling and infiltration secondary to hypoxia are clear drivers of tumor progression. HIF-1α and HIF-2α have opposing and occasionally overlapping roles in both tumor cells and inflammatory cells within the tumor microenvironment and crosstalk between these populations has clear effects on tumor metabolism, inflammation, and progression. It is becoming increasingly apparent that HIFs are one common link between hypoxia, chronic inflammation, metabolic adaptation, and tumor progression through its function in macrophages during cancer development. © 2012 Elsevier Ltd.

Massague J.,Sloan Kettering Cancer Center | Massague J.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2012

The basic elements of the transforming growth factor-β (TGFβ) pathway were revealed more than a decade ago. Since then, the concept of how the TGFβ signal travels from the membrane to the nucleus has been enriched with additional findings, and its multifunctional nature and medical relevance have relentlessly come to light. However, an old mystery has endured: how does the context determine the cellular response to TGFβ? Solving this question is key to understanding TGFβ biology and its many malfunctions. Recent progress is pointing at answers. © 2012 Macmillan Publishers Limited. All rights reserved.

Wallingford J.B.,Howard Hughes Medical Institute
Annual Review of Cell and Developmental Biology | Year: 2012

Planar cell polarity (PCP), the orientation and alignment of cells within a sheet, is a ubiquitous cellular property that is commonly governed by the conserved set of proteins encoded by so-called PCP genes. The PCP proteins coordinate developmental signaling cues with individual cell behaviors in a wildly diverse array of tissues. Consequently, disruptions of PCP protein functions are linked to defects in axis elongation, inner ear patterning, neural tube closure, directed ciliary beating, and left/right patterning, to name only a few. This review attempts to synthesize what is known about PCP and the PCP proteins in vertebrate animals, with a particular focus on the mechanisms by which individual cells respond to PCP cues in order to execute specific cellular behaviors. Copyright © 2012 by Annual Reviews. All rights reserved.

Samuel V.T.,Yale University | Samuel V.T.,Veterans Affairs Medical Center | Shulman G.I.,Yale University | Shulman G.I.,Howard Hughes Medical Institute
Cell | Year: 2012

Insulin resistance is a complex metabolic disorder that defies explanation by a single etiological pathway. Accumulation of ectopic lipid metabolites, activation of the unfolded protein response (UPR) pathway, and innate immune pathways have all been implicated in the pathogenesis of insulin resistance. However, these pathways are also closely linked to changes in fatty acid uptake, lipogenesis, and energy expenditure that can impact ectopic lipid deposition. Ultimately, these cellular changes may converge to promote the accumulation of specific lipid metabolites (diacylglycerols and/or ceramides) in liver and skeletal muscle, a common final pathway leading to impaired insulin signaling and insulin resistance. © 2012 Elsevier Inc.

Ferguson S.M.,Yale University | De Camilli P.,Yale University | De Camilli P.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2012

Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged, highlighting specific contributions of this GTPase to the physiology of different tissues. © 2012 Macmillan Publishers Limited. All rights reserved.

Torii K.U.,University of Washington | Torii K.U.,Howard Hughes Medical Institute
Trends in Cell Biology | Year: 2012

Multicellular organisms produce complex tissues with specialized cell types. During animal development, numerous cell-cell interactions shape tissue patterning through mechanisms involving contact-dependent cell migration and ligand-receptor-mediated lateral inhibition. Owing to the presence of cell walls, plant cells neither migrate nor undergo apoptosis as a means to correct for mis-specified cells. How can plants generate functional tissue patterns? This review aims to deduce fundamental principles of pattern formation through examining two-dimensional (2-D) spatial tissue patterning in plants and animals. Turing's mathematical framework will be introduced and applied to classic examples of de novo 2-D patterning in both animal and plant systems. By comparing their regulatory circuits, new insights into the similarities and differences of the basic principles governing tissue patterning will be discussed. © 2012 Elsevier Ltd.

Little S.C.,Howard Hughes Medical Institute | Tikhonov M.,Princeton University | Gregor T.,Princeton University
Cell | Year: 2013

Early embryonic patterning events are strikingly precise, a fact that appears incompatible with the stochastic gene expression observed across phyla. Using single-molecule mRNA quantification in Drosophila embryos, we determine the magnitude of fluctuations in the expression of four critical patterning genes. The accumulation of mRNAs is identical across genes and fluctuates by only ∼8% between neighboring nuclei, generating precise protein distributions. In contrast, transcribing loci exhibit an intrinsic noise of ∼45% independent of specific promoter-enhancer architecture or fluctuating inputs. Precise transcript distribution in the syncytium is recovered via straightforward spatiotemporal averaging, i.e., accumulation and diffusion of transcripts during nuclear cycles, without regulatory feedback. Common expression characteristics shared between genes suggest that fluctuations in mRNA production are context independent and are a fundamental property of transcription. The findings shed light on how the apparent paradox between stochastic transcription and developmental precision is resolved. © 2013 Elsevier Inc.

Keller P.J.,Howard Hughes Medical Institute
Methods | Year: 2013

The zebrafish Danio rerio has emerged as a powerful vertebrate model system that lends itself particularly well to quantitative investigations with live imaging approaches, owing to its exceptionally high optical clarity in embryonic and larval stages. Recent advances in light microscopy technology enable comprehensive analyses of cellular dynamics during zebrafish embryonic development, systematic mapping of gene expression dynamics, quantitative reconstruction of mutant phenotypes and the system-level biophysical study of morphogenesis.Despite these technical breakthroughs, it remains challenging to design and implement experiments for in vivo long-term imaging at high spatio-temporal resolution. This article discusses the fundamental challenges in zebrafish long-term live imaging, provides experimental protocols and highlights key properties and capabilities of advanced fluorescence microscopes. The article focuses in particular on experimental assays based on light sheet-based fluorescence microscopy, an emerging imaging technology that achieves exceptionally high imaging speeds and excellent signal-to-noise ratios, while minimizing light-induced damage to the specimen. This unique combination of capabilities makes light sheet microscopy an indispensable tool for the in vivo long-term imaging of large developing organisms. © 2013 Elsevier Inc.

Owusu-Ansah E.,Harvard University | Song W.,Harvard University | Perrimon N.,Harvard University | Perrimon N.,Howard Hughes Medical Institute
Cell | Year: 2013

Mitochondrial dysfunction is usually associated with aging. To systematically characterize the compensatory stress signaling cascades triggered in response to muscle mitochondrial perturbation, we analyzed a Drosophila model of muscle mitochondrial injury. We find that mild muscle mitochondrial distress preserves mitochondrial function, impedes the age-dependent deterioration of muscle function and architecture, and prolongs lifespan. Strikingly, this effect is mediated by at least two prolongevity compensatory signaling modules: one involving a muscle-restricted redox-dependent induction of genes that regulate the mitochondrial unfolded protein response (UPR mt) and another involving the transcriptional induction of the Drosophila ortholog of insulin-like growth factor-binding protein 7, which systemically antagonizes insulin signaling and facilitates mitophagy. Given that several secreted IGF-binding proteins (IGFBPs) exist in mammals, our work raises the possibility that muscle mitochondrial injury in humans may similarly result in the secretion of IGFBPs, with important ramifications for diseases associated with aberrant insulin signaling. © 2013 Elsevier Inc.

Hobert O.,Howard Hughes Medical Institute
Annual Review of Cell and Developmental Biology | Year: 2011

The generation of individual neuron types in the nervous system is a multistep process whose endpoint is the expression of neuron typespecific batteries of terminal differentiation genes that determine the functional properties of a neuron. This review focuses on the regulatory mechanisms that are involved in controlling the terminally differentiated state of a neuron. I review several case studies from invertebrate and vertebrate nervous systems that reveal that many terminal differentiation features of a neuron are coregulated via terminal selector transcription factors that initiate and maintain terminal differentiation programs. © 2011 by Annual Reviews. All rights reserved.

A large number of degrees of freedom are required to produce a high quality focus through random scattering media. Previous demonstrations based on spatial phase modulations suffer from either a slow speed or a small number of degrees of freedom. In this work, a high speed wavefront determination technique based on spatial frequency domain wavefront modulations is proposed and experimentally demonstrated, which is capable of providing both a high operation speed and a large number of degrees of freedom. The technique was employed to focus light through a strongly scattering medium and the entire wavefront was determined in 400 milliseconds, ∼three orders of magnitude faster than the previous report. © 2011 Optical Society of America.

Vance R.E.,Howard Hughes Medical Institute | Vance R.E.,University of California at Berkeley
Current Opinion in Immunology | Year: 2015

Inflammasomes comprise a family of cytosolic multi-protein complexes that sense infection, or other threats, and initiate inflammation via the recruitment and activation of the Caspase-1 protease. Although the precise molecular mechanism by which most inflammasomes are activated remains a subject of considerable debate, the NAIP/NLRC4 subfamily of inflammasomes is increasingly well understood. A crystal structure of NLRC4 was recently reported, and a domain in NAIPs that recognizes bacterial ligands was identified. In addition, gain-of-function mutations in NLRC4 have been shown to cause an auto-inflammatory syndrome in humans. Lastly, the NAIP/NLRC4 inflammasome has been shown to provide a novel form of cell intrinsic defense against Salmonella infection, involving expulsion of infected cells from the intestinal epithelium. © 2015 Elsevier Ltd.

Hynes R.O.,Howard Hughes Medical Institute
Journal of Cell Biology | Year: 2012

The modular domain structure of extracellular matrix (ECM) proteins and their genes has allowed extensive exon/domain shuffling during evolution to generate hundreds of ECM proteins. Many of these arose early during metazoan evolution and have been highly conserved ever since. Others have undergone duplication and divergence during evolution, and novel combinations of domains have evolved to generate new ECM proteins, particularly in the vertebrate lineage. The recent sequencing of several genomes has revealed many details of this conservation and evolution of ECM proteins to serve diverse functions in metazoa. © 2012 Hynes.

Yeh E.,Stanford University | DeRisi J.L.,University of California at San Francisco | DeRisi J.L.,Howard Hughes Medical Institute
PLoS Biology | Year: 2011

Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development. © 2011 Yeh, DeRisi.

Hu T.T.,Princeton University | Eisen M.B.,Howard Hughes Medical Institute | Thornton K.R.,University of California at Irvine | Andolfatto P.,Princeton University
Genome Research | Year: 2013

We create a new assembly of the Drosophila simulans genome using 142 million paired short-read sequences and previously published data for strain w501. Our assembly represents a higher-quality genomic sequence with greater coverage, fewer misassemblies, and, by several indexes, fewer sequence errors. Evolutionary analysis of this genome reference sequence reveals interesting patterns of lineage-specific divergence that are different from those previously reported. Specifically, we find that Drosophila melanogaster evolves faster than D. simulans at all annotated classes of sites, including putatively neutrally evolving sites found in minimal introns. While this may be partly explained by a higher mutation rate in D. melanogaster, we also find significant heterogeneity in rates of evolution across classes of sites, consistent with historical differences in the effective population size for the two species. Also contrary to previous findings, we find that the X chromosome is evolving significantly faster than autosomes for nonsynonymous and most noncoding DNA sites and significantly slower for synonymous sites. The absence of a X/A difference for putatively neutral sites and the robustness of the pattern to Gene Ontology and sex-biased expression suggest that partly recessive beneficial mutations may comprise a substantial fraction of noncoding DNA divergence observed between species. Our results have more general implications for the interpretation of evolutionary analyses of genomes of different quality.

Rutherford S.T.,Princeton University | Bassler B.L.,Princeton University | Bassler B.L.,Howard Hughes Medical Institute
Cold Spring Harbor Perspectives in Medicine | Year: 2012

Quorum sensing is a process of cell-cell communication that allows bacteria to share information about cell density and adjust gene expression accordingly. This process enables bacteria to express energetically expensive processes as a collective only when the impact of those processes on the environment or on a host will be maximized. Among the many traits controlled by quorum sensing is the expression of virulence factors by pathogenic bacteria. Here we review the quorum-sensing circuits of Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Vibrio cholerae. We outline these canonical quorum-sensing mechanisms and how each uniquely controls virulence factor production. Additionally, we examine recent efforts to inhibit quorum sensing in these pathogens with the goal of designing novel antimicrobial therapeutics. © 2012 Cold Spring Harbor Laboratory Press.

Mekhail K.,Kings College | Moazed D.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2010

Non-random positioning of chromosomal domains relative to each other and to nuclear landmarks is a common feature of eukaryotic genomes. In particular, the distribution of DNA loci relative to the nuclear periphery has been linked to both transcriptional activation and repression. Nuclear pores and other integral membrane protein complexes are key players in the dynamic organization of the genome in the nucleus, and recent advances in our understanding of the molecular networks that organize genomes at the nuclear periphery point to a further role for non-random locus positioning in DNA repair, recombination and stability. © 2010 Macmillan Publishers Limited. All rights reserved.

Luger K.,Colorado State University | Luger K.,Howard Hughes Medical Institute | Dechassa M.L.,Colorado State University | Tremethick D.J.,Australian National University
Nature Reviews Molecular Cell Biology | Year: 2012

The compaction of genomic DNA into chromatin has profound implications for the regulation of key processes such as transcription, replication and DNA repair. Nucleosomes, the repeating building blocks of chromatin, vary in the composition of their histone protein components. This is the result of the incorporation of variant histones and post-translational modifications of histone amino acid side chains. The resulting changes in nucleosome structure, stability and dynamics affect the compaction of nucleosomal arrays into higher-order structures. It is becoming clear that chromatin structures are not nearly as uniform and regular as previously assumed. This implies that chromatin structure must also be viewed in the context of specific biological functions. © 2012 Macmillan Publishers Limited. All rights reserved.

Robbiani D.F.,Rockefeller University | Nussenzweig M.C.,Rockefeller University | Nussenzweig M.C.,Howard Hughes Medical Institute
Annual Review of Pathology: Mechanisms of Disease | Year: 2013

Studies of B cell lymphomas in the early 1980s led to the cloning of genes (c-MYC and IGH) at a chromosome translocation breakpoint. A rush followed to identify recurrently translocated genes in all types of cancer, which led to remarkable advances in our understanding of cancer genetics. B lymphocyte tumors commonly bear chromosome translocations to immunoglobulin genes, which points to a role for antibody gene diversification processes in tumorigenesis. The discovery of activation-induced cytidine deaminase (AID) and the use of murine models to study translocation have led to a new understanding of how these events contribute to the genesis of lymphomas. Here, we review these advances with a focus on AID and insights gained from the study of translocations in primary cells. © 2013 by Annual Reviews. All rights reserved.

Jindra M.,Academy of Sciences of the Czech Republic | Palli S.R.,University of Kentucky | Riddiford L.M.,Howard Hughes Medical Institute
Annual Review of Entomology | Year: 2013

The molecular action of juvenile hormone (JH), a regulator of vital importance to insects, was until recently regarded as a mystery. The past few years have seen an explosion of studies of JH signaling, sparked by a finding that a JH-resistance gene, Methoprene-tolerant (Met), plays a critical role in insect metamorphosis. Here, we summarize the recently acquired knowledge on the capacity of Met to bind JH, which has been mapped to a particular ligand-binding domain, thus establishing this bHLH-PAS protein as a novel type of an intracellular hormone receptor. Next, we consider the significance of JH-dependent interactions of Met with other transcription factors and signaling pathways. We examine the regulation and biological roles of genes acting downstream of JH and Met in insect metamorphosis. Finally, we discuss the current gaps in our understanding of JH action and outline directions for future research. © 2013 by Annual Reviews. All rights reserved.

Szostak J.W.,Howard Hughes Medical Institute
Journal of Systems Chemistry | Year: 2012

The first RNA World models were based on the concept of an RNA replicase - a ribozyme that was a good enough RNA polymerase that it could catalyze its own replication. Although several RNA polymerase ribozymes have been evolved in vitro, the creation of a true replicase remains a great experimental challenge. At first glance the alternative, in which RNA replication is driven purely by chemical and physical processes, seems even more challenging, given that so many unsolved problems appear to stand in the way of repeated cycles of nonenzymatic RNA replication. Nevertheless the idea of non-enzymatic RNA replication is attractive, because it implies that the first heritable functional RNA need not have improved replication, but could have been a metabolic ribozyme or structural RNA that conferred any function that enhanced protocell reproduction or survival. In this review, I discuss recent findings that suggest that chemically driven RNA replication may not be completely impossible. © 2012 Szostak; licensee Chemistry Central Ltd.

Reddien P.W.,Howard Hughes Medical Institute
Development (Cambridge) | Year: 2013

Planarians are flatworms capable of regenerating all body parts. Planarian regeneration requires neoblasts, a population of dividing cells that has been studied for over a century. Neoblast progeny generate new cells of blastemas, which are the regenerative outgrowths at wounds. If the neoblasts comprise a uniform population of cells during regeneration (e.g. they are all uncommitted and pluripotent), then specialization of new cell types should occur in multipotent, non-dividing neoblast progeny cells. By contrast, recent data indicate that some neoblasts express lineage-specific transcription factors during regeneration and in uninjured animals. These observations raise the possibility that an important early step in planarian regeneration is the specialization of neoblasts to produce specified rather than naïve blastema cells. © 2013. Published by The Company of Biologists Ltd.

Urban S.,Howard Hughes Medical Institute
Biochimica et Biophysica Acta - Biomembranes | Year: 2013

The turn of the millennium coincided with the branding of a fundamentally different class of enzyme - proteases that reside immersed inside the membrane. This new field was the convergence of completely separate lines of research focused on cholesterol homeostasis, Alzheimer's disease, and developmental genetics. None intended their ultimate path, but soon became a richly-integrated fabric for an entirely new field: regulated intramembrane proteolysis. Our aim in this Special Issue is to focus on the ancient and nearly ubiquitous enzymes that catalyze this unexpected yet important reaction. The pace of progress has been dramatic, resulting in a rapidly-expanding universe of known cellular functions, and a paradigm shift in the biochemical understanding of these once heretical enzymes. More recently, the first therapeutic successes have been attained by targeting an intramembrane protease. We consider these advances and identify oncoming opportunities in four parts: growing spectra of cellular roles, insights into biochemical mechanisms, therapeutic strategies, and newly-emerging topics. Recent studies also expose challenges for the future, including non-linear relationships between substrate identification and physiological functions, and the need for potent and specific, not broad-class, inhibitors. © 2013 Published by Elsevier B.V.

Marshall J.D.,Stanford University | Schnitzer M.J.,Stanford University | Schnitzer M.J.,Howard Hughes Medical Institute
ACS Nano | Year: 2013

Biophysicists have long sought optical methods capable of reporting the electrophysiological dynamics of large-scale neural networks with millisecond-scale temporal resolution. Existing fluorescent sensors of cell membrane voltage can report action potentials in individual cultured neurons, but limitations in brightness and dynamic range of both synthetic organic and genetically encoded voltage sensors have prevented concurrent monitoring of spiking activity across large populations of individual neurons. Here we propose a novel, inorganic class of fluorescent voltage sensors: semiconductor nanoparticles, such as ultrabright quantum dots (qdots). Our calculations revealed that transmembrane electric fields characteristic of neuronal spiking (∼10 mV/nm) modulate a qdot's electronic structure and can induce ∼5% changes in its fluorescence intensity and ∼1 nm shifts in its emission wavelength, depending on the qdot's size, composition, and dielectric environment. Moreover, tailored qdot sensors composed of two different materials can exhibit substantial (∼30%) changes in fluorescence intensity during neuronal spiking. Using signal detection theory, we show that conventional qdots should be capable of reporting voltage dynamics with millisecond precision across several tens or more individual neurons over a range of optical and neurophysiological conditions. These results unveil promising avenues for imaging spiking dynamics in neural networks and merit in-depth experimental investigation. © 2013 American Chemical Society.

Schulman B.A.,Howard Hughes Medical Institute
Protein Science | Year: 2011

Post-translational modification by ubiquitin-like proteins (UBLs) is a predominant eukaryotic regulatory mechanism. The vast reach of this form of regulation extends to virtually all eukaryotic processes that involve proteins. UBL modifications play critical roles in controlling the cell cycle, transcription, DNA repair, stress responses, signaling, immunity, plant growth, embryogenesis, circadian rhythms, and a plethora of other pathways. UBLs dynamically modulate target protein properties including enzymatic activity, conformation, half-life, subcellular localization, and intermolecular interactions. Moreover, the enzymatic process of UBL ligation to proteins is itself dynamic, with the UBL moving between multiple enzyme active sites and ultimately to a target. This review highlights our work on how the dynamic conformations of selected enzymes catalyzing UBL ligation help mediate this fascinating form of protein regulation. Published by Wiley-Blackwell. © 2011 The Protein Society.

Bargmann C.I.,Howard Hughes Medical Institute | Marder E.,Brandeis University
Nature Methods | Year: 2013

In this Historical Perspective, we ask what information is needed beyond connectivity diagrams to understand the function of nervous systems. Informed by invertebrate circuits whose connectivities are known, we highlight the importance of neuronal dynamics and neuromodulation, and the existence of parallel circuits. The vertebrate retina has these features in common with invertebrate circuits, suggesting that they are general across animals. Comparisons across these systems suggest approaches to study the functional organization of large circuits based on existing knowledge of small circuits. © 2013 Nature America, Inc. All rights reserved.

Frank J.,Howard Hughes Medical Institute | Frank J.,Columbia University
Current Opinion in Structural Biology | Year: 2012

Recent studies support the notion that the pre-translocation (PRE) ribosomal complex functions, at least in part, as a Brownian machine, stochastically fluctuating among multiple conformations and transfer RNA (tRNA) binding configurations. Apart from the relatively more energetically stable conformational states of the PRE complex, termed macrostate I (MS I) and macrostate II (MS II), several additional intermediate states have been recently discovered. Structural and kinetic analyses of these states, made possible by cryogenic-electron microscopy (cryo-EM), X-ray crystallography, and single-molecule fluorescence resonance energy transfer (smFRET), have provided important insights into the translocation process, which is now understood to proceed, at least in the first step of the process, as a Brownian machine that is transiently stabilized in the 'productive' MS II conformation by the binding of the translocase elongation factor G (EF-G). © 2012 Elsevier Ltd.

Clemente J.C.,University of Colorado at Boulder | Ursell L.K.,University of Colorado at Boulder | Parfrey L.W.,University of Colorado at Boulder | Knight R.,University of Colorado at Boulder | Knight R.,Howard Hughes Medical Institute
Cell | Year: 2012

The human gut harbors diverse microbes that play a fundamental role in the well-being of their host. The constituents of the microbiota - bacteria, viruses, and eukaryotes - have been shown to interact with one another and with the host immune system in ways that influence the development of disease. We review these interactions and suggest that a holistic approach to studying the microbiota that goes beyond characterization of community composition and encompasses dynamic interactions between all components of the microbiota and host tissue over time will be crucial for building predictive models for diagnosis and treatment of diseases linked to imbalances in our microbiota. © 2012 Elsevier Inc.

Hughes J.F.,Howard Hughes Medical Institute | Rozen S.,National University of Singapore
Annual Review of Genomics and Human Genetics | Year: 2012

In mammals, the Y chromosome plays the pivotal role in male sex determination and is essential for normal sperm production. Yet only three Y chromosomes have been completely sequenced to date-those of human, chimpanzee, and rhesus macaque. While Y chromosomes are notoriously difficult to sequence owing to their highly repetitive genomic landscapes, these dedicated sequencing efforts have generated tremendous yields in medical, biological, and evolutionary insight. Knowledge of the complex structural organization of the human Y chromosome and a complete catalog of its gene content have provided a deeper understanding of the mechanisms that generate disease-causing mutations and large-scale rearrangements. Variation among human Y-chromosome sequences has been an invaluable tool for understanding relationships among human populations. Comprehensive comparisons of the human Y-chromosome sequence with those of other primates have illuminated aspects of Y-chromosome evolutionary dynamics over much longer timescales (>25 million years compared with 100,000 years). The future sequencing of additional Y chromosomes will provide a basis for a more comprehensive understanding of the evolution of Y chromosomes and their roles in reproductive biology. © 2012 by Annual Reviews. All rights reserved.

Warren D.L.,Macquarie University | Cardillo M.,Australian National University | Rosauer D.F.,Australian National University | Bolnick D.I.,Howard Hughes Medical Institute
Trends in Ecology and Evolution | Year: 2014

Over the past few decades, there has been a rapid proliferation of statistical methods that infer evolutionary and ecological processes from data on species distributions. These methods have led to considerable new insights, but they often fail to account for the effects of historical biogeography on present-day species distributions. Because the geography of speciation can lead to patterns of spatial and temporal autocorrelation in the distributions of species within a clade, this can result in misleading inferences about the importance of deterministic processes in generating spatial patterns of biodiversity. In this opinion article, we discuss ways in which patterns of species distributions driven by historical biogeography are often interpreted as evidence of particular evolutionary or ecological processes. We focus on three areas that are especially prone to such misinterpretations: community phylogenetics, environmental niche modelling, and analyses of beta diversity (compositional turnover of biodiversity). © 2014.

Baron R.,University of Utah | McCammon J.A.,Howard Hughes Medical Institute
Annual Review of Physical Chemistry | Year: 2013

We review recent developments in our understanding of molecular recognition and ligand association, focusing on two major viewpoints: (a) studies that highlight new physical insight into the molecular recognition process and the driving forces determining thermodynamic signatures of binding and (b) recent methodological advances in applications to protein-ligand binding. In particular, we highlight the challenges posed by compensating enthalpic and entropic terms, competing solute and solvent contributions, and the relevance of complex configurational ensembles comprising multiple protein, ligand, and solvent intermediate states. As more complete physics is taken into account, computational approaches increase their ability to complement experimental measurements, by providing a microscopic, dynamic view of ensemble-averaged experimental observables. Physics-based approaches are increasingly expanding their power in pharmacology applications. © 2013 by Annual Reviews. All rights reserved.

Bargmann C.I.,Howard Hughes Medical Institute
BioEssays | Year: 2012

Powerful ultrastructural tools are providing new insights into neuronal circuits, revealing a wealth of anatomically-defined synaptic connections. These wiring diagrams are incomplete, however, because functional connectivity is actively shaped by neuromodulators that modify neuronal dynamics, excitability, and synaptic function. Studies of defined neural circuits in crustaceans, C. elegans, Drosophila, and the vertebrate retina have revealed the ability of modulators and sensory context to reconfigure information processing by changing the composition and activity of functional circuits. Each ultrastructural connectivity map encodes multiple circuits, some of which are active and some of which are latent at any given time. © 2012 WILEY Periodicals, Inc.

Guler A.D.,Howard Hughes Medical Institute
Nature communications | Year: 2012

The ability to control the electrical activity of a neuronal subtype is a valuable tool in deciphering the role of discreet cell populations in complex neural circuits. Recent techniques that allow remote control of neurons are either labor intensive and invasive or indirectly coupled to neural electrical potential with low temporal resolution. Here we show the rapid, reversible and direct activation of genetically identified neuronal subpopulations by generating two inducible transgenic mouse models. Confined expression of the capsaicin receptor, TRPV1, allows cell-specific activation after peripheral or oral delivery of ligand in freely moving mice. Capsaicin-induced activation of dopaminergic or serotonergic neurons reversibly alters both physiological and behavioural responses within minutes, and lasts ~10 min. These models showcase a robust and remotely controllable genetic tool that modulates a distinct cell population without the need for invasive and labour-intensive approaches.

Cho C.,University of California at San Francisco | Vale R.D.,Howard Hughes Medical Institute
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2012

Dynein is a large cytoskeletal motor protein that belongs to the AAA. + (ATPases associated with diverse cellular activities) superfamily. While dynein has had a rich history of cellular research, its molecular mechanism of motility remains poorly understood. Here we describe recent X-ray crystallographic studies that reveal the architecture of dynein's catalytic ring, mechanical linker element, and microtubule binding domain. This structural information has given rise to new hypotheses on how the dynein motor domain might change its conformation in order to produce motility along microtubules. This article is part of a Special Issue entitled: AAA ATPases: structure and function. © 2011 Elsevier B.V.

Rawling D.C.,Yale University | Pyle A.M.,Yale University | Pyle A.M.,Howard Hughes Medical Institute
Current Opinion in Structural Biology | Year: 2014

Host cell invasion is monitored by a series of pattern recognition receptors (PRRs) that activate the innate immune machinery upon detection of a cognate pathogen associated molecular pattern (PAMP). The RIG-I like receptor (RLR) family of PRRs includes three proteins - RIG-I, MDA5, and LGP2 - responsible for the detection of intracellular pathogenic RNA. All RLR proteins are built around an ATPase core homologous to those found in canonical Superfamily 2 (SF2) RNA helicases, which has been modified through the addition of novel accessory domains to recognize duplex RNA. This review focuses on the structural bases for pathogen-specific dsRNA binding and ATPase activation in RLRs, differential RNA recognition by RLR family members, and implications for other duplex RNA activated ATPases, such as Dicer. © 2013.

Deng W.,Howard Hughes Medical Institute | Blobel G.A.,Childrens Hospital of Philadelphia
Current Opinion in Genetics and Development | Year: 2014

The eukaryotic genome is highly organized in the nucleus. Genes can be localized to specific nuclear compartments in a manner reflecting their activity. A plethora of recent reports has described multiple levels of chromosomal folding that can be related to gene-specific expression states. Here we discuss studies designed to probe the causal impact of genome organization on gene expression. The picture that emerges is that of a reciprocal relationship in which nuclear organization is not only shaped by gene expression states but also directly influences them. © 2013 Elsevier Ltd.

Elinav E.,Weizmann Institute of Science | Nowarski R.,Yale University | Thaiss C.A.,Weizmann Institute of Science | Hu B.,Yale University | And 3 more authors.
Nature Reviews Cancer | Year: 2013

Inflammation is a fundamental innate immune response to perturbed tissue homeostasis. Chronic inflammatory processes affect all stages of tumour development as well as therapy. In this Review, we outline the principal cellular and molecular pathways that coordinate the tumour-promoting and tumour-antagonizing effects of inflammation and we discuss the crosstalk between cancer development and inflammatory processes. In addition, we discuss the recently suggested role of commensal microorganisms in inflammationinduced cancer and we propose that understanding this microbial influence will be crucial for targeted therapy in modern cancer treatment. © 2013 Macmillan Publishers Limited. All right