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The type III secretion system (T3SS) is a needle-like molecular machine that gram negative bacteria use to infect cells. Credit: Research Institute of Molecular Pathology Minimalism is an increasingly popular lifestyle choice that encourages individuals to decrease the overall number of possessions owned and live more simply. According to minimalist philosophy, the reduction of unnecessary clutter enables one to live a more functional and purposeful existence. IMP-IMBA Group Leader and Centre for Structural Systems Biology scientist Thomas Marlovits, in collaboration with colleagues from Massachusetts Institute of Technology (MIT), discovered that a minimalist approach can also be applied to complex biological systems, such as the type III secretion system. The findings of this collaborative study have been published in the scientific journal, Nature Communications. The type III secretion system (T3SS) is a needle-like molecular machine found in gram negative bacteria that transports pathogenic proteins from the bacteria to the human host cell thus initiating infection. The proteins in this system are tightly regulated and the regulatory elements of TSS3 vary greatly depending on the surrounding environment of the bacteria. For example, Salmonella, bacteria which cause food poisoning, secretes its pathogenic proteins into human gut cells. "The question we asked ourselves is: Can we remove all of the regulatory elements from this complex biological system and re-build (refactor) the needle complex using basic genetic principles?" explains Marlovits. To accomplish this, scientists from MIT used synthetic biology to recreate the Salmonella needle complex. Using a bottom up approach, coding and non-coding DNA was replaced or altered with synthetic parts and the scientists were able to create an ultra-simplified 'genetic island.' The functionality of this island was then tested in laboratories in both Boston and Vienna using conventional bio-chemistry methods. The Marlovits lab then used electron microscopy to visualize the integrity of the entire system. "Over the course of this three year study, many rounds of debugging were needed to generate a fully functional system," explains Marlovits "this is the first time that synthetic biology has been used successfully on such a complex system. Previous systems that have been refactored contained just three or four proteins; TSS3 is comprised of over 20 proteins." The development of this simplified TSS3 reveals that none of the intrinsic regulatory features of the system are required to generate a functional needle complex and can be exchanged for others. Removing this regulatory "clutter" has not only resulted in the discovery of essential functional roles played by internal start site and small RNA in but has also unveiled key insights regarding the regulatory elements themselves. Regulation, while not directly involved in function, may exist to ensure the efficient utilization of cellular resources and could also increase the number of environmental conditions under which TSS3 can function. The refactored TSS3 could serve as new tool in biotechnology. This simplified needle complex could be inserted into other bacteria and then turned-on via a built in regulatory element that acts as a molecular switch. "TSS3 could be used as a delivery device for novel agents or vaccines," explains Marlovits "future studies will explore the possibility of placing this refactored TSS3 into new environments." This minimalistic approach to understanding complex biological systems could become an essential new tool for scientists at CSSB. "Understanding how the mechanisms of host pathogen interaction impact biological systems is one of the main goals at CSSB. This new approach provides us with a unique way of looking at systems that will help us discover novel elements," stated Marlovits. More information: Miryoung Song et al. Control of type III protein secretion using a minimal genetic system, Nature Communications (2017). DOI: 10.1038/ncomms14737


Wray J.,University College London | Hartmann C.,Research Institute of Molecular Pathology
Trends in Cell Biology | Year: 2012

Embryonic stem cells (ESCs) - undifferentiated cells originating from preimplantation stage embryos - have prolonged self-renewal capacity and are pluripotent. Activation of the canonical Wnt pathway is implicated in maintenance of and exit from the pluripotent state. Recent findings demonstrate that the essential mediator of canonical Wnt signaling, β-catenin, is dispensable for ESC maintenance; however, its activation inhibits differentiation through derepression of T cell factor 3 (Tcf3)-bound genes. Wnt agonists are useful in deriving ESCs from recalcitrant mouse strains and the rat and in nuclear reprogramming of somatic stem cells. We discuss recent advances in our understanding of the role of canonical Wnt signaling in the regulation of ESC self-renewal and how its manipulation can improve pluripotent ESC derivation and maintenance. © 2011 Elsevier Ltd.


Busslinger M.,Research Institute of Molecular Pathology | Tarakhovsky A.,Rockefeller University
Cold Spring Harbor Perspectives in Biology | Year: 2014

Immunity relies on the heterogeneity of immune cells and their ability to respond to pathogen challenges. In the adaptive immune system, lymphocytes display a highly diverse antigen receptor repertoire that matches the vast diversity of pathogens. In the innate immune system, the cell's heterogeneity and phenotypic plasticity enable flexible responses to changes in tissue homeostasis caused by infection or damage. The immune responses are calibrated by the graded activity of immune cells that can vary from yeast-like proliferation to lifetime dormancy. This article describes key epigenetic processes that contribute to the function of immune cells during health and disease. © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.


Schraidt O.,Research Institute of Molecular Pathology
PLoS pathogens | Year: 2010

The correct organization of single subunits of multi-protein machines in a three dimensional context is critical for their functionality. Type III secretion systems (T3SS) are molecular machines with the capacity to deliver bacterial effector proteins into host cells and are fundamental for the biology of many pathogenic or symbiotic bacteria. A central component of T3SSs is the needle complex, a multiprotein structure that mediates the passage of effector proteins through the bacterial envelope. We have used cryo electron microscopy combined with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry to generate an experimentally validated topographic map of a Salmonella typhimurium T3SS needle complex. This study provides insights into the organization of this evolutionary highly conserved nanomachinery and is the basis for further functional analysis.


Pernia-Andrade A.J.,Research Institute of Molecular Pathology
Nature Methods | Year: 2016

Although whole-organism calcium imaging in small and semi-transparent animals has been demonstrated, capturing the functional dynamics of large-scale neuronal circuits in awake behaving mammals at high speed and resolution has remained one of the main frontiers in systems neuroscience. Here we present a method based on light sculpting that enables unbiased single- and dual-plane high-speed (up to 160 Hz) calcium imaging as well as in vivo volumetric calcium imaging of a mouse cortical column (0.5 mm × 0.5 mm × 0.5 mm) at single-cell resolution and fast volume rates (3–6 Hz). We achieved this by tailoring the point-spread function of our microscope to the structures of interest while maximizing the signal-to-noise ratio using a home-built fiber laser amplifier with pulses that are synchronized to the imaging voxel speed. This enabled in vivo recording of calcium dynamics of several thousand neurons across cortical layers and in the hippocampus of awake behaving mice. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.


Dickson B.J.,Research Institute of Molecular Pathology
Cold Spring Harbor perspectives in biology | Year: 2010

In a bilaterally symmetric animal, the midline plays a key role in directing axon growth during wiring of the nervous system. Midline cells provide a variety of guidance cues for growing axons, to which different types of axons respond in different ways and at different times. For some axons, the midline is an intermediate target. They first seek it out, but then move on towards their final targets on the opposite side. For others, the midline is a repulsive barrier that keeps them on their own side of the midline. And for many of these axons the midline provides signals that guide them along specific lateral pathways or up and down the longitudinal axis.


Spitzweck B.,Research Institute of Molecular Pathology | Brankatschk M.,Research Institute of Molecular Pathology | Dickson B.J.,Research Institute of Molecular Pathology
Cell | Year: 2010

The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 "robo swap" alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring. © 2010 Elsevier Inc. All rights reserved.


Grosstessner-Hain K.,Research Institute of Molecular Pathology
Molecular & cellular proteomics : MCP | Year: 2011

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.


Peters J.-M.,Research Institute of Molecular Pathology
EMBO Journal | Year: 2012

It is well known that somatic and germ cells use different cohesin complexes to mediate sister chromatid cohesion, but why different isoforms of cohesin also co-exist within somatic vertebrate cells has remained a mystery. Two papers in this issue of The EMBO Journal have begun to address this question by analysing mouse cells lacking SA1, an isoform of a specific cohesin subunit. © 2012 European Molecular Biology Organization | All Rights Reserved.


Hellerschmied D.,Research Institute of Molecular Pathology | Clausen T.,Research Institute of Molecular Pathology
Current Opinion in Structural Biology | Year: 2014

The folding and assembly of myosin motor proteins is essential for most movement processes at the cellular, but also at the organism level. Importantly, myosins, which represent a very diverse family of proteins, require the activity of general and specialized folding factors to develop their full motor function. The activities of the myosin-specific UCS (UNC-45/Cro1/She4) chaperones range from assisting acto-myosin dependent transport processes to scaffolding multi-subunit chaperone complexes, which are required to assemble myofilaments. Recent structure-function studies revealed the structural organization of TPR (tetratricopeptide repeat)-containing and TPR-less UCS chaperones. The observed structural differences seem to reflect the specialized and remarkably versatile working mechanisms of myosin-directed chaperones, as will be discussed in this review. © 2013.

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