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E D Salmon - One of the best experts on this subject based on the ideXlab platform.
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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, Brian L Sprague, Ted R Zarzar, Kerry Bloom, 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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Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to Kinetochores.
Current Biology, 2003Co-Authors: Jennifer G Deluca, Julie C Canman, Bonnie Howell, Jennifer M. Hickey, Guowei Fang, E D SalmonAbstract:Abstract Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable Kinetochore-microtubule attachments and chromosome alignment in mitosis [1–9]. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at Kinetochores that have no or few stable Kinetochore microtubules [5]. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to Kinetochores and that Kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint [6]. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at Kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at Kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from Kinetochores that have not yet formed stable Kinetochore-microtubule attachments.
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hnuf2 inhibition blocks stable Kinetochore microtubule attachment and induces mitotic cell death in hela cells
Journal of Cell Biology, 2002Co-Authors: Jennifer G Deluca, Jennifer M. Hickey, Ben Moree, John V. Kilmartin, E D SalmonAbstract:Identification of proteins that couple Kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at Kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but Kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting Kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached Kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in Kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the Kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.
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hnuf2 inhibition blocks stable Kinetochore microtubule attachment and induces mitotic cell death in hela cells
Journal of Cell Biology, 2002Co-Authors: Jennifer G Deluca, Jennifer M. Hickey, Ben Moree, John V. Kilmartin, E D SalmonAbstract:Identification of proteins that couple Kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at Kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but Kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting Kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached Kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in Kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the Kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.
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mad2 and bubr1 function in a single checkpoint pathway that responds to a loss of tension
Molecular Biology of the Cell, 2002Co-Authors: Katie B Shannon, Julie C Canman, E D SalmonAbstract:The spindle checkpoint monitors microtubule attachment and tension at Kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23 degrees C) were shown to have a pronounced mitotic delay, despite having normal numbers of Kinetochore microtubules. At 23 degrees C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37 degrees C. The metaphase delay at 23 degrees C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that Kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister Kinetochores at metaphase found a 24% decrease in tension at 23 degrees C, and metaphase Kinetochores at 23 degrees C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37 degrees C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23 degrees C, indicating that the higher Kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at Kinetochores with a full complement of Kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of Kinetochore 3F3/2, Bub1, and BubR1 labeling.
Jennifer G Deluca - One of the best experts on this subject based on the ideXlab platform.
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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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Aurora A Kinase Function at Kinetochores.
Cold Spring Harbor symposia on quantitative biology, 2017Co-Authors: Jennifer G DelucaAbstract:One of the most important regulatory aspects of chromosome segregation is the ability of Kinetochores to precisely control their attachment strength to spindle microtubules. Central to this regulation is Aurora B, a mitotic kinase that phosphorylates Kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the Kinetochore protein Ndc80/Hec1, which is a component of the NDC80 complex, the primary force-transducing link between Kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of Kinetochore-microtubule attachment, it is becoming clear that this kinase is not solely responsible for phosphorylating Hec1 and other Kinetochore substrates to facilitate microtubule turnover. In particular, there is growing evidence that Aurora A kinase, whose activities at spindle poles have been extensively described, has additional roles at Kinetochores in regulating the Kinetochore-microtubule interface.
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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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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.
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Merotelic Kinetochores in mammalian tissue cells
Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2005Co-Authors: Edward D. Salmon, Daniela Cimini, Lisa A. Cameron, Jennifer G DelucaAbstract:Merotelic Kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian Kinetochores typically have binding sites for about 20–25 Kinetochore microtubules. In prometaphase, Kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic Kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic Kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the Kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of Kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic Kinetochores based on the ratio of Kinetochore microtubules to the correct versus incorrect pole.
Kozo Tanaka - One of the best experts on this subject based on the ideXlab platform.
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CLIP-170 tethers Kinetochores to microtubule plus ends against poleward force by dynein for stable Kinetochore-microtubule attachment
FEBS Letters, 2015Co-Authors: Mohammed Abdullahel Amin, Kinue Kobayashi, Kozo TanakaAbstract:AbstractThe cytoplasmic linker protein (CLIP)-170 localizes to Kinetochores and is suggested to function in stable attachment of Kinetochores to microtubule ends. Here we show that defects in Kinetochore–microtubule attachment and chromosome alignment in CLIP-170-depleted cells were rescued by co-depletion of p150glued, a dynactin subunit required for Kinetochore localization of CLIP-170. CLIP-170 recruited p150glued to microtubule ends. Kinetochore localization at microtubule ends was perturbed by CLIP-170 depletion, which was rescued by co-depleting p150glued. Our results imply that CLIP-170 tethers Kinetochores to microtubule ends against the dynein-mediated poleward force to slide Kinetochores along microtubules, facilitating the stable Kinetochore attachment to microtubules
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CLIP-170 tethers Kinetochores to microtubule plus ends against poleward force by dynein for stable Kinetochore–microtubule attachment
FEBS Letters, 2015Co-Authors: Mohammed Abdullahel Amin, Kinue Kobayashi, Kozo TanakaAbstract:The cytoplasmic linker protein (CLIP)-170 localizes to Kinetochores and is suggested to function in stable attachment of Kinetochores to microtubule ends. Here we show that defects in Kinetochore–microtubule attachment and chromosome alignment in CLIP-170-depleted cells were rescued by co-depletion of p150glued, a dynactin subunit required for Kinetochore localization of CLIP-170. CLIP-170 recruited p150glued to microtubule ends. Kinetochore localization at microtubule ends was perturbed by CLIP-170 depletion, which was rescued by co-depleting p150glued. Our results imply that CLIP-170 tethers Kinetochores to microtubule ends against the dynein-mediated poleward force to slide Kinetochores along microtubules, facilitating the stable Kinetochore attachment to microtubules.
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Dynamic regulation of Kinetochore-microtubule interaction during mitosis.
Journal of biochemistry, 2012Co-Authors: Kozo TanakaAbstract:Chromosome segregation is carried out by dynamic interplay between Kinetochores and microtubules. First, Kinetochores are efficiently captured by microtubules. Then, flexible interactions between Kinetochores and microtubules allow proper orientation of chromosomes aligned on the centre of the spindle. Finally, microtubules stably attached to Kinetochores drag the chromosomes towards the spindle poles. During these processes, the mode of interaction of Kinetochores with microtubules changes from lateral to end-on attachment, accompanied by changes in Kinetochore structure/composition and microtubule dynamics. The molecular mechanisms for stable Kinetochore-microtubule attachment have been progressively revealed in recent years. On the other hand, the mechanism of dynamic regulation of Kinetochore-microtubule interaction in early mitosis, which is crucial for faithful chromosome segregation, continues to be elusive. Here I focus on this early step of chromosome segregation and discuss how Kinetochores establish proper attachments to microtubules.
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Live-cell analysis of Kinetochore-microtubule interaction in budding yeast.
Methods (San Diego Calif.), 2010Co-Authors: Kozo Tanaka, Etsushi Kitamura, Tomoyuki U. TanakaAbstract:Kinetochore capture and transport by spindle microtubules plays a crucial role in high-fidelity chromosome segregation, although its detailed mechanism has remained elusive. It has been difficult to observe individual Kinetochore–microtubule interactions because multiple Kinetochores are captured by microtubules during a short period within a small space. We have developed a method to visualize individual Kinetochore–microtubule interactions in Saccharomyces cerevisiae, by isolating one of the Kinetochores from others through regulation of the activity of a centromere. We detail this technique, which we call ‘centromere reactivation system’, for dissection of the process of Kinetochore capture and transport on mitotic spindle. Kinetochores are initially captured by the side of microtubules extending from a spindle pole, and subsequently transported poleward along them, which is an evolutionarily conserved process from yeast to vertebrate cells. Our system, in combination with amenable yeast genetics, has proved useful to elucidate the molecular mechanisms of Kinetochore–microtubule interactions. We discuss practical considerations for applying our system to live cell imaging using fluorescence microscopy.
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molecular mechanisms of microtubule dependent Kinetochore transport toward spindle poles
Journal of Cell Biology, 2007Co-Authors: Kozo Tanaka, Etsushi Kitamura, Yoko Kitamura, Tomoyuki U. TanakaAbstract:In mitosis, Kinetochores are initially captured by the lateral sides of single microtubules and are subsequently transported toward spindle poles. Mechanisms for Kinetochore transport are not yet known. We present two mechanisms involved in microtubule-dependent poleward Kinetochore transport in Saccharomyces cerevisiae. First, Kinetochores slide along the microtubule lateral surface, which is mainly and probably exclusively driven by Kar3, a kinesin-14 family member that localizes at Kinetochores. Second, Kinetochores are tethered at the microtubule distal ends and pulled poleward as microtubules shrink (end-on pulling). Kinetochore sliding is often converted to end-on pulling, enabling more processive transport, but the opposite conversion is rare. The establishment of end-on pulling is partly hindered by Kar3, and its progression requires the Dam1 complex. We suggest that the Dam1 complexes, which probably encircle a single microtubule, can convert microtubule depolymerization into the poleward Kinetochore-pulling force. Thus, microtubule-dependent poleward Kinetochore transport is ensured by at least two distinct mechanisms.
Judith Klumperman - One of the best experts on this subject based on the ideXlab platform.
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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.
Tomoyuki U. Tanaka - One of the best experts on this subject based on the ideXlab platform.
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Live-cell analysis of Kinetochore-microtubule interaction in budding yeast.
Methods (San Diego Calif.), 2010Co-Authors: Kozo Tanaka, Etsushi Kitamura, Tomoyuki U. TanakaAbstract:Kinetochore capture and transport by spindle microtubules plays a crucial role in high-fidelity chromosome segregation, although its detailed mechanism has remained elusive. It has been difficult to observe individual Kinetochore–microtubule interactions because multiple Kinetochores are captured by microtubules during a short period within a small space. We have developed a method to visualize individual Kinetochore–microtubule interactions in Saccharomyces cerevisiae, by isolating one of the Kinetochores from others through regulation of the activity of a centromere. We detail this technique, which we call ‘centromere reactivation system’, for dissection of the process of Kinetochore capture and transport on mitotic spindle. Kinetochores are initially captured by the side of microtubules extending from a spindle pole, and subsequently transported poleward along them, which is an evolutionarily conserved process from yeast to vertebrate cells. Our system, in combination with amenable yeast genetics, has proved useful to elucidate the molecular mechanisms of Kinetochore–microtubule interactions. We discuss practical considerations for applying our system to live cell imaging using fluorescence microscopy.
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molecular mechanisms of microtubule dependent Kinetochore transport toward spindle poles
Journal of Cell Biology, 2007Co-Authors: Kozo Tanaka, Etsushi Kitamura, Yoko Kitamura, Tomoyuki U. TanakaAbstract:In mitosis, Kinetochores are initially captured by the lateral sides of single microtubules and are subsequently transported toward spindle poles. Mechanisms for Kinetochore transport are not yet known. We present two mechanisms involved in microtubule-dependent poleward Kinetochore transport in Saccharomyces cerevisiae. First, Kinetochores slide along the microtubule lateral surface, which is mainly and probably exclusively driven by Kar3, a kinesin-14 family member that localizes at Kinetochores. Second, Kinetochores are tethered at the microtubule distal ends and pulled poleward as microtubules shrink (end-on pulling). Kinetochore sliding is often converted to end-on pulling, enabling more processive transport, but the opposite conversion is rare. The establishment of end-on pulling is partly hindered by Kar3, and its progression requires the Dam1 complex. We suggest that the Dam1 complexes, which probably encircle a single microtubule, can convert microtubule depolymerization into the poleward Kinetochore-pulling force. Thus, microtubule-dependent poleward Kinetochore transport is ensured by at least two distinct mechanisms.
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Kinetochore capture and bi-orientation on the mitotic spindle
Nature Reviews Molecular Cell Biology, 2005Co-Authors: Tomoyuki U. Tanaka, Michael J. R. Stark, Kozo TanakaAbstract:To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes accurately to opposite poles during mitosis. For high-fidelity chromosome segregation, Kinetochores must be captured properly on the mitotic spindle before anaphase onset. Correct Kinetochore capture by spindle microtubules is achieved in a stepwise manner. Kinetochores are initially captured by the lateral surface of a single microtubule that extends from either spindle pole. Once captured, Kinetochores are transported poleward along the microtubule. To assure correct Kinetochore capture and transport, microtubules must efficiently locate unattached Kinetochores and, after capture, the Kinetochores must stabilize associated microtubules. Subsequently, microtubules that extend from the other spindle pole also interact with Kinetochores and, eventually, each sister Kinetochore attaches to microtubules that extend from opposite poles (this is known as sister Kinetochore bi-orientation or amphitelic attachment). To achieve this, mal-oriented Kinetochore–spindle-pole connections must be removed, and bi-orientation must be selectively promoted. We discuss how Kinetochores are initially captured by microtubules and how sister Kinetochores subsequently bi-orient on the mitotic spindle. Although we focus mainly on recent research progress in the budding yeast Saccharomyces cerevisiae , we will also discuss findings in other organisms in this context. Kinetochores are large protein complexes that are formed on chromosome regions known as centromeres. For high-fidelity chromosome segregation, Kinetochores must be correctly captured on the mitotic spindle before anaphase onset. During prometaphase, Kinetochores are initially captured by a single microtubule that extends from a spindle pole and are then transported poleward along the microtubule. Subsequently, microtubules that extend from the other spindle pole also interact with Kinetochores and, eventually, each sister Kinetochore attaches to microtubules that extend from opposite poles — this is known as bi-orientation. Here we discuss the molecular mechanisms of these processes, by focusing on budding yeast and drawing comparisons with other organisms.
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molecular mechanisms of Kinetochore capture by spindle microtubules
Nature, 2005Co-Authors: Kozo Tanaka, Hilary Dewar, Naomi Mukae, Mark Van Breugel, Claude Antony, Euan K James, Alan R Prescott, Tomoyuki U. TanakaAbstract:For high-fidelity chromosome segregation, Kinetochores must be properly captured by spindle microtubules, but the mechanisms underlying initial Kinetochore capture have remained elusive. Here we visualized individual Kinetochore–microtubule interactions in Saccharomyces cerevisiae by regulating the activity of a centromere. Kinetochores are captured by the side of microtubules extending from spindle poles, and are subsequently transported poleward along them. The microtubule extension from spindle poles requires microtubule plus-end-tracking proteins and the Ran GDP/GTP exchange factor. Distinct Kinetochore components are used for Kinetochore capture by microtubules and for ensuring subsequent sister Kinetochore bi-orientation on the spindle. Kar3, a kinesin-14 family member, is one of the regulators that promote transport of captured Kinetochores along microtubules. During such transport, Kinetochores ensure that they do not slide off their associated microtubules by facilitating the conversion of microtubule dynamics from shrinkage to growth at the plus ends. This conversion is promoted by the transport of Stu2 from the captured Kinetochores to the plus ends of microtubules.
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Tension between two Kinetochores suffices for their bi-orientation on the mitotic spindle
Nature, 2004Co-Authors: Hilary Dewar, Kozo Tanaka, Kim Nasmyth, Tomoyuki U. TanakaAbstract:The movement of sister chromatids to opposite spindle poles during anaphase depends on the prior capture of sister Kinetochores by microtubules with opposing orientations (amphitelic attachment or bi-orientation)1. In addition to proteins necessary for the Kinetochore–microtubule attachment, bi-orientation requires the Ipl1 (Aurora B in animal cells) protein kinase2,3,4,5,6,7 and tethering of sister chromatids by cohesin8,9. Syntelic attachments, in which sister Kinetochores attach to microtubules with the same orientation, must be either ‘avoided’ or ‘corrected’. Avoidance might be facilitated by the juxtaposition of sister Kinetochores such that they face in opposite directions; Kinetochore geometry is therefore deemed important. Error correction, by contrast, is thought to stem from the stabilization of Kinetochore–spindle pole connections by tension in microtubules, Kinetochores, or the surrounding chromatin arising from amphitelic but not syntelic attachment10,11. The tension model predicts that any type of connection between two Kinetochores suffices for efficient bi-orientation. Here we show that the two Kinetochores of engineered, unreplicated dicentric chromosomes in Saccharomyces cerevisiae bi-orient efficiently, implying that sister Kinetochore geometry is dispensable for bi-orientation. We also show that Ipl1 facilitates bi-orientation by promoting the turnover of Kinetochore–spindle pole connections in a tension-dependent manner.
