The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
E D Salmon - One of the best experts on this subject based on the ideXlab platform.
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a kinesin 5 cin8 recruits protein phosphatase 1 to Kinetochores and regulates chromosome segregation
Current Biology, 2018Co-Authors: Sue Biggins, E D Salmon, Aussie Suzuki, Amitabha Gupta, Sarah K Long, Rena K Evans, Benjamin L Badger, Kerry BloomAbstract:Summary Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at Kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to Kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8’s kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at Kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.
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removal of spindly from microtubule attached Kinetochores controls spindle checkpoint silencing in human cells
Genes & Development, 2010Co-Authors: Reto Gassmann, Don W Cleveland, E D Salmon, Andrew J Holland, Dileep Varma, Xiaohu Wan, Filiz Civril, Karen Oegema, Arshad DesaiAbstract:The spindle checkpoint generates a ‘‘wait anaphase’’ signal at unattached Kinetochores to prevent premature anaphase onset. Kinetochore-localized dynein is thought to silence the checkpoint by transporting checkpoint proteins from microtubule-attached Kinetochores to spindle poles. Throughout metazoans, dynein recruitment to Kinetochores requires the protein Spindly. Here, we identify a conserved motif in Spindly that is essential for kinetochore targeting of dynein. Spindly motif mutants, expressed following depletion of endogenous Spindly, target normally to Kinetochores but prevent dynein recruitment. Spindly depletion and Spindly motif mutants, despite their similar effects on kinetochore dynein, have opposite consequences on chromosome alignment and checkpoint silencing. Spindly depletion delays chromosome alignment, but Spindly motif mutants ameliorate this defect, indicating that Spindly has a dynein recruitment-independent role in alignment. In Spindly depletions, the checkpoint is silenced following delayed alignment by a kinetochore dynein-independent mechanism. In contrast, Spindly motif mutants are retained on microtubule-attached Kinetochores along with checkpoint proteins, resulting in persistent checkpoint signaling. Thus, dynein-mediated removal of Spindly from microtubuleattached Kinetochores, rather than poleward transport per se, is the critical reaction in checkpoint silencing. In the absence of Spindly, a second mechanism silences the checkpoint; this mechanism is likely evolutionarily ancient, as fungi and higher plants lack kinetochore dynein.
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tension dependent regulation of microtubule dynamics at Kinetochores can explain metaphase congression in yeast
Molecular Biology of the Cell, 2005Co-Authors: Melissa K Gardner, E D Salmon, Chad G Pearson, Kerry Bloom, Brian L Sprague, Ted R Zarzar, David J OddeAbstract:During metaphase in budding yeast mitosis, sister Kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of Kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome Kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that Kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.
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anaphase spindle mechanics prevent mis segregation of merotelically oriented chromosomes
Current Biology, 2004Co-Authors: Daniela Cimini, Lisa A. Cameron, E D SalmonAbstract:Abstract Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances [1–5], is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase [6, 7]. In mammalian cells, it occurs at high frequency in early mitosis [5], but few anaphase cells show lagging chromosomes [5]. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic Kinetochores from producing lagging chromosomes. We found that merotelic Kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at Kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.
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direct observation of microtubule dynamics at Kinetochores in xenopus extract spindles implications for spindle mechanics
Journal of Cell Biology, 2003Co-Authors: Paul S Maddox, Timothy J. Mitchison, E D Salmon, Aaron F Straight, Peg CoughlinAbstract:Microtubule plus ends dynamically attach to Kinetochores on mitotic chromosomes. We directly imaged this dynamic interface using high resolution fluorescent speckle microscopy and direct labeling of Kinetochores in Xenopus extract spindles. During metaphase, Kinetochores were stationary and under tension while plus end polymerization and poleward microtubule flux (flux) occurred at velocities varying from 1.5–2.5 μm/min. Because kinetochore microtubules polymerize at metaphase Kinetochores, the primary source of kinetochore tension must be the spindle forces that produce flux and not a kinetochore-based mechanism. We infer that the kinetochore resists translocation of kinetochore microtubules through their attachment sites, and that the polymerization state of the kinetochore acts a “slip-clutch” mechanism that prevents detachment at high tension. At anaphase onset, Kinetochores switched to depolymerization of microtubule plus ends, resulting in chromosome-to-pole rates transiently greater than flux. Kinetochores switched from persistent depolymerization to persistent polymerization and back again during anaphase, bistability exhibited by Kinetochores in vertebrate tissue cells. These results provide the most complete description of spindle microtubule poleward flux to date, with important implications for the microtubule–kinetochore interface and for how flux regulates kinetochore function.
Jennifer G Deluca - One of the best experts on this subject based on the ideXlab platform.
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bugz facilitates loading of spindle assembly checkpoint proteins to Kinetochores in early mitosis
Journal of Biological Chemistry, 2020Co-Authors: Hazheen K Shirnekhi, Jacob A Herman, Patrick J Paddison, Jennifer G DelucaAbstract:BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to Kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and Kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to Kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to Kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto Kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.
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aurora b kinase is recruited to multiple discrete kinetochore and centromere regions in human cells
Journal of Cell Biology, 2020Co-Authors: Amanda J Broad, Keith F Deluca, Jennifer G DelucaAbstract:Aurora B kinase has a critical role in regulating attachments between Kinetochores and spindle microtubules during mitosis. Early in mitosis, kinase activity at Kinetochores is high to promote attachment turnover, and in later mitosis, activity decreases to ensure attachment stabilization. Aurora B localizes prominently to inner centromeres, and a population of the kinase is also detected at Kinetochores. How Aurora B is recruited to and evicted from these regions to regulate kinetochore-microtubule attachments remains unclear. Here, we identified and investigated discrete populations of Aurora B at the centromere/kinetochore region. An inner centromere pool is recruited by Haspin phosphorylation of histone H3, and a kinetochore-proximal outer centromere pool is recruited by Bub1 phosphorylation of histone H2A. Finally, a third pool resides ~20 nm outside of the inner kinetochore protein CENP-C in early mitosis and does not require either the Bub1/pH2A/Sgo1 or Haspin/pH3 pathway for localization or activity. Our results suggest that distinct molecular pathways are responsible for Aurora B recruitment to centromeres and Kinetochores.
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knl1 facilitates phosphorylation of outer kinetochore proteins by promoting aurora b kinase activity
Journal of Cell Biology, 2013Co-Authors: Gina V Caldas, Keith F Deluca, Jennifer G DelucaAbstract:Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore–microtubule (MT) turnover and preventing premature stabilization of kinetochore–MT attachments. Phosphorylation of kinetochore proteins during late mitosis is low, promoting attachment stabilization, which is required for anaphase onset. The kinetochore protein KNL1 recruits Aurora B–counteracting phosphatases and the Aurora B–targeting factor Bub1, yet the consequences of KNL1 depletion on Aurora B phospho-regulation remain unknown. Here, we demonstrate that the KNL1 N terminus is essential for Aurora B activity at Kinetochores. This region of KNL1 is also required for Bub1 kinase activity at Kinetochores, suggesting that KNL1 promotes Aurora B activity through Bub1-mediated Aurora B targeting. However, ectopic targeting of Aurora B to Kinetochores does not fully rescue Aurora B activity in KNL1-depleted cells, suggesting KNL1 influences Aurora B activity through an additional pathway. Our findings establish KNL1 as a requirement for Aurora B activity at Kinetochores and for wild-type kinetochore–MT attachment dynamics.
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Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis.
Journal of cell science, 2011Co-Authors: Keith F Deluca, Susanne M.a. Lens, Jennifer G DelucaAbstract:Precise control of the attachment strength between Kinetochores and spindle microtubules is essential to preserve genomic stability. Aurora B kinase has been implicated in regulating the stability of kinetochore-microtubule attachments but its relevant kinetochore targets in cells remain unclear. Here, we identify multiple serine residues within the N-terminus of the kinetochore protein Hec1 that are phosphorylated in an Aurora-B-kinase-dependent manner during mitosis. On all identified target sites, Hec1 phosphorylation at Kinetochores is high in early mitosis and decreases significantly as chromosomes bi-orient. Furthermore, once dephosphorylated, Hec1 is not highly rephosphorylated in response to loss of kinetochore-microtubule attachment or tension. We find that a subpopulation of Aurora B kinase remains localized at the outer kinetochore even upon Hec1 dephosphorylation, suggesting that Hec1 phosphorylation by Aurora B might not be regulated wholly by spatial positioning of the kinase. Our results define a role for Hec1 phosphorylation in kinetochore-microtubule destabilization and error correction in early mitosis and for Hec1 dephosphorylation in maintaining stable attachments in late mitosis.
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sds22 regulates aurora b activity and microtubule kinetochore interactions at mitosis
Journal of Cell Biology, 2010Co-Authors: Markus Posch, Jennifer G Deluca, Guennadi A Khoudoli, Sam Swift, Emma M King, Jason R SwedlowAbstract:We have studied Sds22, a conserved regulator of protein phosphatase 1 (PP1) activity, and determined its role in modulating the activity of aurora B kinase and kinetochore–microtubule interactions. Sds22 is required for proper progression through mitosis and localization of PP1 to mitotic Kinetochores. Depletion of Sds22 increases aurora B T-loop phosphorylation and the rate of recovery from monastrol arrest. Phospho–aurora B accumulates at Kinetochores in Sds22-depleted cells juxtaposed to critical kinetochore substrates. Sds22 modulates sister kinetochore distance and the interaction between Hec1 and the microtubule lattice and, thus, the activation of the spindle assembly checkpoint. These results demonstrate that Sds22 specifically defines PP1 function and localization in mitosis. Sds22 regulates PP1 targeting to the kinetochore, accumulation of phospho–aurora B, and force generation at the kinetochore–microtubule interface.
Sue Biggins - One of the best experts on this subject based on the ideXlab platform.
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autophosphorylation is sufficient to release mps1 kinase from native Kinetochores
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Lori B Koch, Sue Biggins, Nitobe London, Kwaku Opoku, Yi Deng, Adrienne Barber, Aimee J Littleton, Charles L. AsburyAbstract:Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at Kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible "wait" signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached Kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native Kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated Kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.
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a kinesin 5 cin8 recruits protein phosphatase 1 to Kinetochores and regulates chromosome segregation
Current Biology, 2018Co-Authors: Sue Biggins, E D Salmon, Aussie Suzuki, Amitabha Gupta, Sarah K Long, Rena K Evans, Benjamin L Badger, Kerry BloomAbstract:Summary Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at Kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to Kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8’s kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at Kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.
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Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint
Genes & development, 2014Co-Authors: Nitobe London, Sue BigginsAbstract:The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to Kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and Kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint.
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Phosphoregulation promotes release of Kinetochores from dynamic microtubules via multiple mechanisms
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Krishna K Sarangapani, Bungo Akiyoshi, Sue Biggins, Nicole Duggan, Charles L. AsburyAbstract:During mitosis, multiprotein complexes called Kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole Kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases Kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.
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the structure of purified Kinetochores reveals multiple microtubule attachment sites
Nature Structural & Molecular Biology, 2012Co-Authors: Shane Gonen, Bungo Akiyoshi, Sue Biggins, Nicole Duggan, Matthew G Iadanza, Dan Shi, Tamir GonenAbstract:Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that Kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which Kinetochores control chromosome segregation and cell division.
Carlos Sacristan - One of the best experts on this subject based on the ideXlab platform.
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ectopic activation of the spindle assembly checkpoint signaling cascade reveals its biochemical design
Current Biology, 2019Co-Authors: Chu Chen, Carlos Sacristan, Geert J P L Kops, Ian P Whitney, Anand Banerjee, Palak Sekhri, David M Kern, Adrienne Fontan, John J Tyson, Iain M CheesemanAbstract: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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dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis
Nature Cell Biology, 2018Co-Authors: Carlos Sacristan, Eelco Tromer, Misbha Ud Din Ahmad, Jenny Keller, Job Fermie, Vincent Groenewold, Roberto Melero, Alexander Fish, José María Carazo, Judith KlumpermanAbstract:Faithful chromosome segregation depends on the ability of sister Kinetochores to attach to spindle microtubules. The outer layer of Kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD–Zwilch–ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded Kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.
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dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis
Nature Cell Biology, 2018Co-Authors: Carlos Sacristan, Eelco Tromer, Misbha Ud Din Ahmad, Jenny Keller, Job Fermie, Vincent Groenewold, Roberto Melero, Alexander Fish, José María Carazo, Judith KlumpermanAbstract:Faithful chromosome segregation depends on the ability of sister Kinetochores to attach to spindle microtubules. The outer layer of Kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD–Zwilch–ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded Kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments. Sacristan et al. show that the dynein adaptor Spindly facilitates oligomerisation of the RZZ complex to expand the kinetochore, after which Spindly-associated dynein compacts the kinetochore to allow for faithful chromosome segregation.
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Joined at the hip: Kinetochores, microtubules, and spindle assembly checkpoint signaling.
Trends in cell biology, 2014Co-Authors: Carlos Sacristan, Geert J P L KopsAbstract:Error-free chromosome segregation relies on stable connections between Kinetochores and spindle microtubules. The spindle assembly checkpoint (SAC) monitors such connections and relays their absence to the cell cycle machinery to delay cell division. The molecular network at Kinetochores that is responsible for microtubule binding is integrated with the core components of the SAC signaling system. Molecular-mechanistic understanding of how the SAC is coupled to the kinetochore-microtubule interface has advanced significantly in recent years. The latest insights not only provide a striking view of the dynamics and regulation of SAC signaling events at the outer kinetochore but also create a framework for understanding how that signaling may be terminated when Kinetochores and microtubules connect.
Michael A Lampson - One of the best experts on this subject based on the ideXlab platform.
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minimal model for collective kinetochore microtubule dynamics
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Edward J Banigan, Edward R Ballister, Michael A Lampson, Kevin Chiou, Alyssa M Mayo, Andrea J LiuAbstract:Chromosome segregation during cell division depends on interactions of Kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore-MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force-velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for Kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase Kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo.
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recruitment of mad1 to metaphase Kinetochores is sufficient to reactivate the mitotic checkpoint
Journal of Cell Biology, 2014Co-Authors: Edward R Ballister, Michelle Riegman, Michael A LampsonAbstract:The mitotic checkpoint monitors kinetochore–microtubule attachment and prevents anaphase until all Kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to Kinetochores. Unattached, checkpoint-active Kinetochores accumulate multiple checkpoint proteins, which are depleted from Kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached Kinetochores, it is not known what changes at Kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, we show that recruiting the checkpoint protein Mad1 to metaphase Kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of our checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at Kinetochores.
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Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing.
The Journal of cell biology, 2012Co-Authors: Dan Liu, Olga Davydenko, Michael A LampsonAbstract:Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase Kinetochores and is reduced at metaphase Kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore–microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at Kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore–microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from Kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to Kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at Kinetochores control two critical mitotic processes: initially establishing correct kinetochore–microtubule attachments and subsequently silencing the spindle checkpoint.
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regulated targeting of protein phosphatase 1 to the outer kinetochore by knl1 opposes aurora b kinase
Journal of Cell Biology, 2010Co-Authors: Mathijs Vleugel, Chelsea B Backer, Tatsuo Fukagawa, Tetsuya Hori, Iain M Cheeseman, Michael A LampsonAbstract:Regulated interactions between Kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore–microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at Kinetochores and stabilize microtubule attachments. PP1 levels at Kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1–PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at Kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase.
