scholarly journals Visualization of Mad2 Dynamics at Kinetochores, along Spindle Fibers, and at Spindle Poles in Living Cells

2000 ◽  
Vol 150 (6) ◽  
pp. 1233-1250 ◽  
Author(s):  
B.J. Howell ◽  
D.B. Hoffman ◽  
G. Fang ◽  
A.W. Murray ◽  
E.D. Salmon
Keyword(s):  
Chromosome Segregation ◽  
Binding Sites ◽  
Metaphase Plate ◽  
Atp Depletion ◽  
Anaphase Promoting Complex ◽  
Spindle Fiber ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Kinetochore Function ◽  
Spindle Fibers

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest–deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2–Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t1/2 of ∼24–28 s. Cells entered anaphase ∼10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

scholarly journals The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset.

1992 ◽  
Vol 118 (4) ◽  
pp. 759-773 ◽  
Author(s):  
B C Williams ◽  
T L Karr ◽  
J M Montgomery ◽  
M L Goldberg
Keyword(s):  
Chromosome Segregation ◽  
Metaphase Plate ◽  
Cell Types ◽  
Embryonic Cell ◽  
Larval Brain ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Embryonic Cell Cycle ◽  
Very High

Mutations in the gene l(1)zw10 disrupt the accuracy of chromosome segregation in a variety of cell types during the course of Drosophila development. Cytological analysis of mutant larval brain neuroblasts shows very high levels of aneuploid cells. Many anaphase figures are aberrant, the most frequent abnormality being the presence of lagging chromosomes that remain in the vicinity of the metaphase plate when the other chromosomes have migrated toward the spindle poles. Finally, the centromeric connection between sister chromatids in mutant neuroblasts treated with colchicine often appears to be broken, in contrast with similarly treated control neuroblasts. The 85-kD protein encoded by the l(1)zw10 locus displays a dynamic pattern of localization in the course of the embryonic cell cycle. It is excluded from the nuclei during interphase, but migrates into the nuclear zone during prometaphase. At metaphase, the zw10 antigen is found in a novel filamentous structure that may be specifically associated with kinetochore microtubules. Upon anaphase onset, there is an extremely rapid redistribution of the zw10 protein to a location at or near the kinetochores of the separating chromosomes.

scholarly journals Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics

2003 ◽  
Vol 14 (10) ◽  
pp. 4181-4195 ◽  
Author(s):  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
Ted R. Zarzar ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Regulatory Mechanism ◽  
Metaphase Plate ◽  
Microtubule Dynamics ◽  
Spindle Microtubule ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Kinetochore Function

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.

scholarly journals Merotelic kinetochores in mammalian tissue cells

2005 ◽  
Vol 360 (1455) ◽  
pp. 553-568 ◽  
Author(s):  
E.D Salmon ◽  
D Cimini ◽  
L.A Cameron ◽  
J.G DeLuca
Keyword(s):  
Mitotic Spindle ◽  
Binding Sites ◽  
Metaphase Plate ◽  
Mammalian Tissue ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Pulling Forces ◽  
Tissue Cells ◽  
Early Mitosis

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.

Determinants of Drosophila zw10 protein localization and function

1994 ◽  
Vol 107 (4) ◽  
pp. 785-798 ◽  
Author(s):  
B.C. Williams ◽  
M.L. Goldberg
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Protein Localization ◽  
Metaphase Plate ◽  
Obvious Effect ◽  
Anaphase Onset ◽  
Protein Functions ◽  
Kinetochore Region ◽  
And Function ◽  
Feedback Pathway

We have examined several issues concerning how the Drosophila l(1)zw10 gene product functions to ensure proper chromosome segregation. (a) We have found that in zw10 mutant embryos and larval neuroblasts, absence of the zw10 protein has no obvious effect on either the congression of chromosomes to the metaphase plate or the morphology of the metaphase spindle, although many aberrations are observed subsequently in anaphase. This suggests that activity of the zw10 protein becomes essential at anaphase onset, a time at which the zw10 protein is redistributed to the kinetochore region of the chromosomes. (b) The zw10 protein appears to bind to kinetochores in mitotically arrested cells, eventually accumulating to high levels within the chromosome mass. Our results imply that zw10 may act as part of a novel feedback pathway that normally renders sister chromatid separation dependent upon spindle integrity. (c) The localization of zw10 protein is altered by two mitotic mutations, rough deal and abnormal anaphase resolution, that specifically disrupt anaphase. These findings indicate that the zw10 protein functions as part of a multicomponent mechanism ensuring proper chromosome segregation at the beginning of anaphase.

scholarly journals Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

2008 ◽  
Vol 182 (2) ◽  
pp. 277-288 ◽  
Author(s):  
Ayumu Yamamoto ◽  
Kenji Kitamura ◽  
Daisuke Hihara ◽  
Yukinobu Hirose ◽  
Satoshi Katsuyama ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Related Factor ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Anaphase Onset ◽  
Yeast Meiosis ◽  
Meiosis I

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/CCdc20), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.

scholarly journals Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

2002 ◽  
Vol 158 (5) ◽  
pp. 841-847 ◽  
Author(s):  
Marko J. Kallio ◽  
Victoria A. Beardmore ◽  
Jasminder Weinstein ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Anaphase Promoting Complex ◽  
Late Prophase ◽  
Checkpoint Signaling ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Late Telophase ◽  
Green Fluorescent

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

scholarly journals BUB-1 promotes amphitelic chromosome biorientation via multiple activities at the kinetochore

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Frances Edwards ◽  
Gilliane Maton ◽  
Nelly Gareil ◽  
Julie C Canman ◽  
Julien Dumont
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Underlying Mechanism ◽  
Microtubule Assembly ◽  
Spindle Poles ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Chromatid Segregation ◽  
Associated Proteins ◽  
Chromosome Attachment

Accurate chromosome segregation relies on bioriented amphitelic attachments of chromosomes to microtubules of the mitotic spindle, in which sister chromatids are connected to opposite spindle poles. BUB-1 is a protein of the Spindle Assembly Checkpoint (SAC) that coordinates chromosome attachment with anaphase onset. BUB-1 is also required for accurate sister chromatid segregation independently of its SAC function, but the underlying mechanism remains unclear. Here we show that, in Caenorhabditis elegans embryos, BUB-1 accelerates the establishment of non-merotelic end-on kinetochore-microtubule attachments by recruiting the RZZ complex and its downstream partner dynein-dynactin at the kinetochore. In parallel, BUB-1 limits attachment maturation by the SKA complex. This activity opposes kinetochore-microtubule attachment stabilisation promoted by CLS-2CLASP-dependent kinetochore-microtubule assembly. BUB-1 is therefore a SAC component that coordinates the function of multiple downstream kinetochore-associated proteins to ensure accurate chromosome segregation.

scholarly journals Bipolar spindle attachments affect redistributions of ZW10, a Drosophila centromere/kinetochore component required for accurate chromosome segregation.

1996 ◽  
Vol 134 (5) ◽  
pp. 1127-1140 ◽  
Author(s):  
B C Williams ◽  
M Gatti ◽  
M L Goldberg
Keyword(s):  
Chromosome Segregation ◽  
Male Meiosis ◽  
Chromosome Behavior ◽  
Bipolar Spindle ◽  
Chromosome Missegregation ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Chromosome Separation ◽  
Meiosis I

Previous efforts have shown that mutations in the Drosophila ZW10 gene cause massive chromosome missegregation during mitotic divisions in several tissues. Here we demonstrate that mutations in ZW10 also disrupt chromosome behavior in male meiosis I and meiosis II, indicating that ZW10 function is common to both equational and reductional divisions. Divisions are apparently normal before anaphase onset, but ZW10 mutants exhibit lagging chromosomes and irregular chromosome segregation at anaphase. Chromosome missegregation during meiosis I of these mutants is not caused by precocious separation of sister chromatids, but rather the nondisjunction of homologs. ZW10 is first visible during prometaphase, where it localizes to the kinetochores of the bivalent chromosomes (during meiosis I) or to the sister kinetochores of dyads (during meiosis II). During metaphase of both divisions, ZW10 appears to move from the kinetochores and to spread toward the poles along what appear to be kinetochore microtubules. Redistributions of ZW10 at metaphase require bipolar attachments of individual chromosomes or paired bivalents to the spindle. At the onset of anaphase I or anaphase II, ZW10 rapidly relocalizes to the kinetochore regions of the separating chromosomes. In other mutant backgrounds in which chromosomes lag during anaphase, the presence or absence of ZW10 at a particular kinetochore predicts whether or not the chromosome moves appropriately to the spindle poles. We propose that ZW10 acts as part of, or immediately downstream of, a tension-sensing mechanism that regulates chromosome separation or movement at anaphase onset.

scholarly journals CENP-E Function at Kinetochores Is Essential for Chromosome Alignment

1997 ◽  
Vol 139 (6) ◽  
pp. 1373-1382 ◽  
Author(s):  
B.T. Schaar ◽  
G.K.T. Chan ◽  
P. Maddox ◽  
E.D. Salmon ◽  
T.J. Yen
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Metaphase Plate ◽  
Motor Domain ◽  
Amino Terminal ◽  
Spindle Poles ◽  
Opposite Pole ◽  
Chromosome Alignment ◽  
Combined Data

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate. Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function. The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.

scholarly journals Bub3 promotes Cdc20-dependent activation of the APC/C in S. cerevisiae

2015 ◽  
Vol 209 (4) ◽  
pp. 519-527 ◽  
Author(s):  
Yang Yang ◽  
Dai Tsuchiya ◽  
Soni Lacefield
Keyword(s):  
Chromosome Segregation ◽  
Unknown Function ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Checkpoint Activation ◽  
Anaphase Onset ◽  
Impaired Binding

The spindle checkpoint ensures accurate chromosome segregation by sending a signal from an unattached kinetochore to inhibit anaphase onset. Numerous studies have described the role of Bub3 in checkpoint activation, but less is known about its functions apart from the spindle checkpoint. In this paper, we demonstrate that Bub3 has an unexpected role promoting metaphase progression in budding yeast. Loss of Bub3 resulted in a metaphase delay that was not a consequence of aneuploidy or the activation of a checkpoint. Instead, bub3Δ cells had impaired binding of the anaphase-promoting complex/cyclosome (APC/C) with its activator Cdc20, and the delay could be rescued by Cdc20 overexpression. Kinetochore localization of Bub3 was required for normal mitotic progression, and Bub3 and Cdc20 colocalized at the kinetochore. Although Bub1 binds Bub3 at the kinetochore, bub1Δ cells did not have compromised APC/C and Cdc20 binding. The results demonstrate that Bub3 has a previously unknown function at the kinetochore in activating APC/C-Cdc20 for normal mitotic progression.

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