Publications by authors named "Iain M Cheeseman"

82 Publications

Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes.

Elife 2021 08 3;10. Epub 2021 Aug 3.

Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, United States.

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) transition, resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI/MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after MI. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.
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http://dx.doi.org/10.7554/eLife.70588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8370769PMC
August 2021

Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition.

Dev Cell 2021 Aug 30;56(15):2192-2206.e8. Epub 2021 Jul 30.

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.
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http://dx.doi.org/10.1016/j.devcel.2021.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8355204PMC
August 2021

Permitted and restricted steps of human kinetochore assembly in mitotic cell extracts.

Mol Biol Cell 2021 06 6;32(13):1241-1255. Epub 2021 May 6.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

Mitotic kinetochores assemble via the hierarchical recruitment of numerous cytosolic components to the centromere region of each chromosome. However, how these orderly and localized interactions are achieved without spurious macromolecular assemblies forming from soluble kinetochore components in the cell cytosol remains poorly understood. We developed assembly assays to monitor the recruitment of green fluorescent protein-tagged recombinant proteins and native proteins from human cell extracts to inner kinetochore components immobilized on microbeads. In contrast to prior work in yeast and egg extracts, we find that human mitotic cell extracts fail to support de novo assembly of microtubule-binding subcomplexes. A subset of interactions, such as those between CENP-A-containing nucleosomes and CENP-C, are permissive under these conditions. However, the subsequent phospho-dependent binding of the Mis12 complex is less efficient, whereas recruitment of the Ndc80 complex is blocked, leading to weak microtubule-binding activity of assembled particles. Using molecular variants of the Ndc80 complex, we show that auto-inhibition of native Ndc80 complex restricts its ability to bind to the CENP-T/W complex, whereas inhibition of the Ndc80 microtubule binding is driven by a different mechanism. Together, our work reveals regulatory mechanisms that guard against the spurious formation of cytosolic microtubule-binding kinetochore particles.
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http://dx.doi.org/10.1091/mbc.E20-07-0461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8351545PMC
June 2021

Kinetochore assembly throughout the cell cycle.

Semin Cell Dev Biol 2021 Sep 19;117:62-74. Epub 2021 Mar 19.

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

The kinetochore plays an essential role in facilitating chromosome segregation during cell division. This massive protein complex assembles onto the centromere of chromosomes and enables their attachment to spindle microtubules during mitosis. The kinetochore also functions as a signaling hub to regulate cell cycle progression, and is crucial to ensuring the fidelity of chromosome segregation. Despite the fact that kinetochores are large and robust molecular assemblies, they are also highly dynamic structures that undergo structural and organizational changes throughout the cell cycle. This review will highlight our current understanding of kinetochore structure and function, focusing on the dynamic processes that underlie kinetochore assembly.
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http://dx.doi.org/10.1016/j.semcdb.2021.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384650PMC
September 2021

Differential requirements for the CENP-O complex reveal parallel PLK1 kinetochore recruitment pathways.

Mol Biol Cell 2021 04 17;32(8):712-721. Epub 2021 Feb 17.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142.

Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome-wide screens have identified a number of proteins that exhibit cell line-specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the four-subunit centromere-localized CENP-O complex, whose precise function has been difficult to define. Our results support a model in which the CENP-O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell-state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease.
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http://dx.doi.org/10.1091/mbc.E20-11-0751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8108507PMC
April 2021

Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations.

Elife 2020 11 11;9. Epub 2020 Nov 11.

Whitehead Institute for Biomedical Research, Cambridge, United States.

Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere-nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.
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http://dx.doi.org/10.7554/eLife.59770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679138PMC
November 2020

Cellular Mechanisms and Regulation of Quiescence.

Dev Cell 2020 11;55(3):259-271

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

Quiescence is a state of reversible proliferative arrest in which cells are not actively dividing and yet retain the capacity to reenter the cell cycle upon receiving an appropriate stimulus. Quiescent cells are remarkably diverse-they reside in different locations throughout the body, serve distinct roles, and are activated by a variety of signals. Despite this diversity, all quiescent cells must be able to persist in a nondividing state without compromising their proliferative potential, which requires changes to core cellular programs. How drastically different cell types are able to implement extensive changes to their gene-expression programs, metabolism, and cellular structures to induce a common cellular state is a fascinating question in cell and developmental biology. In this review, we explore the diversity of quiescent cells and highlight the unifying characteristics that define the quiescent state.
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http://dx.doi.org/10.1016/j.devcel.2020.09.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665062PMC
November 2020

Chromosome Segregation: Evolving a Plastic Chromosome-Microtubule Interface.

Curr Biol 2020 02;30(4):R174-R177

Whitehead Institute for Biomedical Research, and Department of Biology, Massachusetts Institute of Technology, 455 Main Street, Cambridge, MA 02142, USA. Electronic address:

Despite a conserved requirement in mediating chromosome segregation, kinetochores display remarkable plasticity in their structure and composition. New work in holocentric insect species highlights the molecular rewiring that occurs when key structural components of the kinetochore are lost and centromere structure is changed.
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http://dx.doi.org/10.1016/j.cub.2019.12.058DOI Listing
February 2020

Cohesin Removal Reprograms Gene Expression upon Mitotic Entry.

Mol Cell 2020 04 7;78(1):127-140.e7. Epub 2020 Feb 7.

Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.
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http://dx.doi.org/10.1016/j.molcel.2020.01.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178822PMC
April 2020

Quiescent Cells Actively Replenish CENP-A Nucleosomes to Maintain Centromere Identity and Proliferative Potential.

Dev Cell 2019 10 15;51(1):35-48.e7. Epub 2019 Aug 15.

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.
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http://dx.doi.org/10.1016/j.devcel.2019.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783363PMC
October 2019

The AAA + ATPase TorsinA polymerizes into hollow helical tubes with 8.5 subunits per turn.

Nat Commun 2019 07 22;10(1):3262. Epub 2019 Jul 22.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

TorsinA is an ER-resident AAA + ATPase, whose deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is an unusual AAA + ATPase that needs an external activator. Also, it likely does not thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of its still enigmatic function. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 Å structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner channel of ~ 4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA + ATPases. We discuss the implications of these observations for TorsinA function.
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http://dx.doi.org/10.1038/s41467-019-11194-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646356PMC
July 2019

Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design.

Curr Biol 2019 01 27;29(1):104-119.e10. Epub 2018 Dec 27.

Department of Biophysics, University of Michigan, Ann Arbor, MI 48109, USA; Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address:

Switch-like activation of the spindle assembly checkpoint (SAC) is critical for accurate chromosome segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the "eSAC." The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.
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http://dx.doi.org/10.1016/j.cub.2018.11.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324986PMC
January 2019

Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion.

Curr Biol 2018 11 25;28(21):3422-3429.e5. Epub 2018 Oct 25.

Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. Electronic address:

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments.
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http://dx.doi.org/10.1016/j.cub.2018.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522255PMC
November 2018

Microcephaly Modeling of Kinetochore Mutation Reveals a Brain-Specific Phenotype.

Cell Rep 2018 10;25(2):368-382.e5

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, MIT, 31 Ames Street, Cambridge, MA 02139, USA. Electronic address:

Most genes mutated in microcephaly patients are expressed ubiquitously, and yet the brain is the only major organ compromised in most patients. Why the phenotype remains brain specific is poorly understood. In this study, we used in vitro differentiation of human embryonic stem cells to monitor the effect of a point mutation in kinetochore null protein 1 (KNL1; CASC5), identified in microcephaly patients, during in vitro brain development. We found that neural progenitors bearing a patient mutation showed reduced KNL1 levels, aneuploidy, and an abrogated spindle assembly checkpoint. By contrast, no reduction of KNL1 levels or abnormalities was observed in fibroblasts and neural crest cells. We established that the KNL1 patient mutation generates an exonic splicing silencer site, which mainly affects neural progenitors because of their higher levels of splicing proteins. Our results provide insight into the brain-specific phenomenon, consistent with microcephaly being the only major phenotype of patients bearing KNL1 mutation.
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http://dx.doi.org/10.1016/j.celrep.2018.09.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392048PMC
October 2018

Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes.

Open Biol 2018 09 26;8(9). Epub 2018 Sep 26.

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA

Cytoplasmic dynein is a minus-end-directed microtubule-based motor that acts at diverse subcellular sites. During mitosis, dynein localizes simultaneously to the mitotic spindle, spindle poles, kinetochores and the cell cortex. However, it is unclear what controls the relative targeting of dynein to these locations. As dynein is heavily post-translationally modified, we sought to test a role for these modifications in regulating dynein localization. We find that dynein rapidly and strongly accumulates at mitotic spindle poles following treatment with NSC697923, a small molecule that inhibits the ubiquitin E2 enzyme, Ubc13, or treatment with PYR-41, a ubiquitin E1 inhibitor. Subsets of dynein regulators such as Lis1, ZW10 and Spindly accumulate at the spindle poles, whereas others do not, suggesting that NSC697923 differentially affects specific dynein populations. We additionally find that dynein relocalization induced by NSC697923 or PYR-41 can be suppressed by simultaneous treatment with the non-selective deubiquitinase inhibitor, PR-619. However, we did not observe altered dynein localization following treatment with the selective E1 inhibitor, TAK-243. Although it is possible that off-target effects of NSC697923 and PYR-41 are responsible for the observed changes in dynein localization, the rapid relocalization upon drug treatment highlights the highly dynamic nature of dynein regulation during mitosis.
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http://dx.doi.org/10.1098/rsob.180095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170511PMC
September 2018

The kinetochore-microtubule interface at a glance.

J Cell Sci 2018 08 16;131(16). Epub 2018 Aug 16.

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA

Accurate chromosome segregation critically depends on the formation of attachments between microtubule polymers and each sister chromatid. The kinetochore is the macromolecular complex that assembles at the centromere of each chromosome during mitosis and serves as the link between the DNA and the microtubules. In this Cell Science at a Glance article and accompanying poster, we discuss the activities and molecular players that are involved in generating kinetochore-microtubule attachments, including the initial stages of lateral kinetochore-microtubule interactions and maturation to stabilized end-on attachments. We additionally explore the features that contribute to the ability of the kinetochore to track with dynamic microtubules. Finally, we examine the contributions of microtubule-associated proteins to the organization and stabilization of the mitotic spindle and the control of microtubule dynamics.
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http://dx.doi.org/10.1242/jcs.214577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127730PMC
August 2018

CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.

Mol Biol Cell 2018 10 9;29(20):2370-2377. Epub 2018 Aug 9.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142.

A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for protein depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.
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http://dx.doi.org/10.1091/mbc.E18-04-0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233062PMC
October 2018

Nde1 promotes diverse dynein functions through differential interactions and exhibits an isoform-specific proteasome association.

Mol Biol Cell 2018 09 19;29(19):2336-2345. Epub 2018 Jul 19.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142.

Nde1 is a key regulator of cytoplasmic dynein, binding directly to both dynein itself and the dynein adaptor, Lis1. Nde1 and Lis1 are thought to function together to promote dynein function, yet mutations in each result in distinct neurodevelopment phenotypes. To reconcile these phenotypic differences, we sought to dissect the contribution of Nde1 to dynein regulation and explore the cellular functions of Nde1. Here we show that an Nde1-Lis1 interaction is required for spindle pole focusing and Golgi organization but is largely dispensable for centrosome placement, despite Lis1 itself being required. Thus, diverse functions of dynein rely on distinct Nde1- and Lis1-mediated regulatory mechanisms. Additionally, we discovered a robust, isoform-specific interaction between human Nde1 and the 26S proteasome and identify precise mutations in Nde1 that disrupt the proteasome interaction. Together, our work suggests that Nde1 makes unique contributions to human neurodevelopment through its regulation of both dynein and proteasome function.
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http://dx.doi.org/10.1091/mbc.E18-07-0418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249811PMC
September 2018

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces.

Curr Biol 2017 Dec 16;27(23):3666-3675.e6. Epub 2017 Nov 16.

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends in vitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity in vitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation.
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http://dx.doi.org/10.1016/j.cub.2017.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726585PMC
December 2017

Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80.

Elife 2017 08 25;6. Epub 2017 Aug 25.

Whitehead Institute for Biomedical Research, Cambridge, United States.

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.
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http://dx.doi.org/10.7554/eLife.26866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602300PMC
August 2017

Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition.

Nat Commun 2017 06 9;8:15775. Epub 2017 Jun 9.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the 'arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA.
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http://dx.doi.org/10.1038/ncomms15775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472775PMC
June 2017

Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects.

Dev Cell 2017 02 16;40(4):405-420.e2. Epub 2017 Feb 16.

Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

Defining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, we generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. We performed single-cell microscopic analyses to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, we generated knockouts across cell lines of diverse origins. We demonstrate that p53 modulates the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression.
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http://dx.doi.org/10.1016/j.devcel.2017.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345124PMC
February 2017

A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends.

J Cell Biol 2016 05 2;213(3):315-28. Epub 2016 May 2.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning.
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http://dx.doi.org/10.1083/jcb.201510117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862331PMC
May 2016

Structural comparison of the Caenorhabditis elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior.

Mol Biol Cell 2016 Apr 3;27(8):1197-203. Epub 2016 Mar 3.

Laboratory of Structure Cell Biology, Department of Integrative Structure and Computational Biology, Scripps Research Institute, La Jolla, CA 92037.

During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore-microtubule interactions remains poorly understood. The conserved four-subunit Ndc80 complex plays an essential and direct role in generating dynamic kinetochore-microtubule attachments. Here we compare the binding of theCaenorhabditis elegansand human Ndc80 complexes to microtubules at high resolution using cryo-electron microscopy reconstructions. Despite the conserved roles of the Ndc80 complex in diverse organisms, we find that the attachment mode of these complexes for microtubules is distinct. The human Ndc80 complex binds every tubulin monomer along the microtubule protofilament, whereas theC. elegansNdc80 complex binds more tightly to β-tubulin. In addition, theC. elegansNdc80 complex tilts more toward the adjacent protofilament. These structural differences in the Ndc80 complex between different species may play significant roles in the nature of kinetochore-microtubule interactions.
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http://dx.doi.org/10.1091/mbc.E15-12-0858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4831874PMC
April 2016

The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface.

Mol Cell 2015 Dec 19;60(6):886-98. Epub 2015 Nov 19.

Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.
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http://dx.doi.org/10.1016/j.molcel.2015.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690846PMC
December 2015

The molecular basis for centromere identity and function.

Nat Rev Mol Cell Biol 2016 Jan 25;17(1):16-29. Epub 2015 Nov 25.

Whitehead Institute and Department of Biology, MIT, Nine Cambridge Center, Cambridge, Massachusetts 02142, USA.

The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.
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http://dx.doi.org/10.1038/nrm.2015.5DOI Listing
January 2016

Chromosome Segregation: A Spatial Code to Correct Kinetochore-Microtubule Attachments.

Curr Biol 2015 Jul;25(14):R601-3

Whitehead Institute for Biomedical Research, Cambridge, MA, USA; Department of Biology, MIT, Nine Cambridge Center, Cambridge, MA 02142, USA. Electronic address:

Erroneous kinetochore-microtubule interactions must be detected and corrected before a cell enters anaphase to prevent chromosome mis-segregation. Two new studies describe an Aurora A-mediated error correction mechanism based on the spatial position of a chromosome within the mitotic spindle.
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http://dx.doi.org/10.1016/j.cub.2015.05.056DOI Listing
July 2015

Inferring transient particle transport dynamics in live cells.

Nat Methods 2015 Sep 20;12(9):838-40. Epub 2015 Jul 20.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.
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http://dx.doi.org/10.1038/nmeth.3483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733533PMC
September 2015

Distinct organization and regulation of the outer kinetochore KMN network downstream of CENP-C and CENP-T.

Curr Biol 2015 Mar 5;25(5):671-7. Epub 2015 Feb 5.

Whitehead Institute for Biomedical Research and Department of Biology, MIT, Nine Cambridge Center, Cambridge, MA 02142, USA. Electronic address:

The kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule-binding interface at kinetochores [3] and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5-8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.
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http://dx.doi.org/10.1016/j.cub.2015.01.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348146PMC
March 2015

The outer kinetochore protein KNL-1 contains a defined oligomerization domain in nematodes.

Mol Biol Cell 2015 Jan 19;26(2):229-37. Epub 2014 Nov 19.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

The kinetochore is a large, macromolecular assembly that is essential for connecting chromosomes to microtubules during mitosis. Despite the recent identification of multiple kinetochore components, the nature and organization of the higher-order kinetochore structure remain unknown. The outer kinetochore KNL-1/Mis12 complex/Ndc80 complex (KMN) network plays a key role in generating and sensing microtubule attachments. Here we demonstrate that Caenorhabditis elegans KNL-1 exists as an oligomer, and we identify a specific domain in KNL-1 responsible for this activity. An N-terminal KNL-1 domain from both C. elegans and the related nematode Caenorhabditis remanei oligomerizes into a decameric assembly that appears roughly circular when visualized by electron microscopy. On the basis of sequence and mutational analysis, we identify a small hydrophobic region as responsible for this oligomerization activity. However, mutants that precisely disrupt KNL-1 oligomerization did not alter KNL-1 localization or result in the loss of embryonic viability based on gene replacements in C. elegans. In C. elegans, KNL-1 oligomerization may coordinate with other kinetochore activities to ensure the proper organization, function, and sensory capabilities of the kinetochore-microtubule attachment.
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http://dx.doi.org/10.1091/mbc.E14-06-1125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294671PMC
January 2015
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