Sloan Kettering Institute

Sloan, NY, United States

Sloan Kettering Institute

Sloan, NY, United States
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Olsen S.K.,Sloan Kettering Institute | Lima C.D.,Sloan Kettering Institute
Molecular Cell | Year: 2013

Ubiquitin (Ub) conjugation is initiated by an E1 enzyme that catalyzes carboxy-terminal Ub adenylation, thioester bond formation to a catalytic cysteine in the E1 Cys domain, and thioester transfer to a catalytic cysteine in E2 conjugating enzymes. How the E1 and E2 active sites come together during thioester transfer and how Ub E1 interacts with diverse Ub E2s remains unclear. Here we present a crystal structure of a Ub E1-E2(Ubc4)/Ub/ATP{dot operator}Mg complex that was stabilized by induction of a disulfide bond between the E1 and E2 active sites. The structure reveals combinatorial recognition of the E2 by the E1 ubiquitin-fold domain (UFD) and Cys domain and mutational analysis, coupled with thioester transfer assays with E1, Ubc4, and other Ub E2s, show that both interfaces are important for thioester transfer. Comparison to a Ub E1/Ub/ATP{dot operator}Mg structure reveals conformational changes in the E1 that bring the E1 and E2 active sites together. © 2013 Elsevier Inc.

Flynt A.S.,Sloan Kettering Institute
Molecular cell | Year: 2010

microRNAs (miRNAs) are approximately 22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5' end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial "tail" separates its hairpin from the 3'splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RNA exosome-mediated "trimming," followed by conventional dicing and loading into AGO1 to yield a miRNA that can repress seed-matched targets. Analysis of cloned small RNAs yielded six additional candidate 3' tailed mirtrons in D. melanogaster. Altogether, these data reveal an unexpected role for the exosome in the biogenesis of miRNAs from hybrid mirtron substrates. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Sun K.,Sloan Kettering Institute | Lai E.C.,Sloan Kettering Institute
Nature Reviews Genetics | Year: 2013

MicroRNAs (miRNAs) are ~22 nt RNAs that coordinate vast regulatory networks in animals and thereby influence myriad processes. This Review examines evidence that miRNAs have continuous roles in adults in ways that are separable from developmental control. Adult-specific activities for miRNAs have been described in various stem cell populations, in the context of neural function and cardiovascular biology, in metabolism and ageing, and during cancer. In addition to reviewing recent results, we also discuss methods for studying miRNA activities specifically in adults and evaluate their relative strengths and weaknesses. A fuller understanding of continuous functions of miRNAs in adults has bearing on efforts and opportunities to manipulate miRNAs for therapeutic purposes. © 2013 Macmillan Publishers Limited. All rights reserved.

Lee G.,Sloan Kettering Institute
Nature protocols | Year: 2010

Human pluripotent stem cell (hPSC)-derived neural crest (NC) cells present a valuable tool for modeling aspects of human NC development, including cell fate specification, multipotency and cell migration. hPSC-derived NC cells are also suitable for modeling human disease and as a renewable cell source for applications in regenerative medicine. Here we provide protocols for the step-wise differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into neuroectodermal and NC cells using either the MS5 coculture system or a novel defined culture method based on pharmacological inhibition of bone morphogenetic protein and transforming growth factor-beta signaling pathways. Furthermore, we present protocols for the purification and propagation of hPSC-NC cells using flow cytometry and defined in vitro culture conditions. Our protocol has been validated in multiple independent hESC and hiPSC lines. The average time required for generating purified hPSC-NC precursors using this protocol is 2-5 weeks.

Foley E.A.,Sloan Kettering Institute | Kapoor T.M.,Rockefeller University
Nature Reviews Molecular Cell Biology | Year: 2013

In eukaryotes, chromosome segregation during cell division is facilitated by the kinetochore, a multiprotein structure that is assembled on centromeric DNA. The kinetochore attaches chromosomes to spindle microtubules, modulates the stability of these attachments and relays the microtubule-binding status to the spindle assembly checkpoint (SAC), a cell cycle surveillance pathway that delays chromosome segregation in response to unattached kinetochores. Recent studies are shaping current thinking on how each of these kinetochore-centred processes is achieved, and how their integration ensures faithful chromosome segregation, focusing on the essential roles of kinase-phosphatase signalling and the microtubule-binding KMN protein network. © 2012 Macmillan Publishers Limited. All rights reserved.

Chambers S.M.,Sloan Kettering Institute | Studer L.,Sloan Kettering Institute
Cell | Year: 2011

Building on the discovery that MyoD expression reprograms fibroblasts into muscle, three papers (Vierbuchen et al.; 2010; Ieda et al.; 2010; Szabo et al.; 2010) recently reported the reprogramming of fibroblasts into neurons, cardiomyocytes, and blood cell progenitors without first passing the cells through a pluripotent state. Here we discuss the advantages and challenges of harnessing this direct reprogramming method for regenerative medicine. © 2011 Elsevier Inc. All Rights Reserved.

Goetz S.C.,Sloan Kettering Institute | Anderson K.V.,Sloan Kettering Institute
Nature Reviews Genetics | Year: 2010

The primary cilium has recently stepped into the spotlight, as a flood of data show that this organelle has crucial roles in vertebrate development and human genetic diseases. Cilia are required for the response to developmental signals, and evidence is accumulating that the primary cilium is specialized for hedgehog signal transduction. The formation of cilia, in turn, is regulated by other signalling pathways, possibly including the planar cell polarity pathway. The cilium therefore represents a nexus for signalling pathways during development. The connections between cilia and developmental signalling have begun to clarify the basis of human diseases associated with ciliary dysfunction. © 2010 Macmillan Publishers Limited. All rights reserved.

Okamura K.,Sloan Kettering Institute
Wiley Interdisciplinary Reviews: RNA | Year: 2012

Higher eukaryotes employ extensive post-transcriptional gene regulation to accomplish fine control of gene expression. The microRNA (miRNA) family plays important roles in the post-transcriptional gene regulation of broad networks of target mRNA expression. Most miRNAs are generated by a conserved mechanism involving two RNase III enzymes Drosha and Dicer. However, work from the past few years has uncovered diverse noncanonical miRNA pathways, which exploit a variety of other RNA processing enzymes. In addition, the discovery of another abundant small RNA family, endogenous short interfering RNAs (endo-siRNAs), has also broadened the catalogs of short regulatory RNAs. This review highlights recent studies that revealed novel small RNA biogenesis pathways, and discusses their relevance to gene regulatory networks. © 2011 John Wiley & Sons, Ltd.

Cell migration and cell rearrangements are critical for establishment of the body plan of vertebrate embryos. The first step in organization of the body plan of the mouse embryo, specification of the anterior-posterior body axis, depends on migration of the anterior visceral endoderm from the distal tip of the embryo to a more proximal region overlying the future head. The anterior visceral endoderm (AVE) is a cluster of extra-embryonic cells that secretes inhibitors of the Wnt and Nodal pathways to inhibit posterior development. Because Rac proteins are crucial regulators of cell migration and mouse Rac1 mutants die early in development, we tested whether Rac1 plays a role in AVE migration. Here we show that Rac1 mutant embryos fail to specify an anterior-posterior axis and, instead, express posterior markers in a ring around the embryonic circumference. Cells that express the molecular markers of the AVE are properly specified in Rac1 mutants but remain at the distal tip of the embryo at the time when migration should take place. Using tissue specific deletions, we show that Rac1 acts autonomously within the visceral endoderm to promote cell migration. High-resolution imaging shows that the leading wild-type AVE cells extend long lamellar protrusions that span several cell diameters and are polarized in the direction of cell movement. These projections are tipped by filopodia-like structures that appear to sample the environment. Wild-type AVE cells display hallmarks of collective cell migration: they retain tight and adherens junctions as they migrate and exchange neighbors within the plane of the visceral endoderm epithelium. Analysis of mutant embryos shows that Rac1 is not required for intercellular signaling, survival, proliferation, or adhesion in the visceral endoderm but is necessary for the ability of visceral endoderm cells to extend projections, change shape, and exchange neighbors. The data show that Rac1-mediated epithelial migration of the AVE is a crucial step in the establishment of the mammalian body plan and suggest that Rac1 is essential for collective migration in mammalian tissues.

Gareau J.R.,Sloan Kettering Institute | Lima C.D.,Sloan Kettering Institute
Nature Reviews Molecular Cell Biology | Year: 2010

Proteins of the small ubiquitin-related modifier (SUMO) family are conjugated to proteins to regulate such cellular processes as nuclear transport, transcription, chromosome segregation and DNA repair. Recently, numerous insights into regulatory mechanisms of the SUMO modification pathway have emerged. Although SUMO-conjugating enzymes can discriminate between SUMO targets, many substrates possess characteristics that facilitate their modification. Other post-translational modifications also regulate SUMO conjugation, suggesting that SUMO signalling is integrated with other signal transduction pathways. A better understanding of SUMO regulatory mechanisms will lead to improved approaches for analysing the function of SUMO and substrate conjugation in distinct cellular pathways. © 2010 Macmillan Publishers Limited. All rights reserved.

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