Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

Hoffman, David; Pearson, Chad G.; Yen, Tim J.; Howell, Bonnie J.; Salmon, Edward D.

doi:10.1091/mbc.12.7.1995

Cited by 328 publications

(391 citation statements)

References 54 publications

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“…Distance was determined by drawing a line from the outer kinetochore extending to the outer edge of its sister kinetochore using the z-stacks. The length of the line was converted from pixels to microns, and the distance was calculated using the Pythagorean Theorem to account for the z-step(s) distance (Hoffman et al, 2001). The interkinetochore distances were measured from three to five kinetochores/cell and then averaged per cell.…”

Section: Immunofluorescence Quantification and Data Collectionmentioning

confidence: 99%

MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

Rizk

Bohannon

Wetzel

et al. 2009

MBoC

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Within the mitotic spindle, there are multiple populations of microtubules with different turnover dynamics, but how these different dynamics are maintained is not fully understood. MCAK is a member of the kinesin-13 family of microtubule-destabilizing enzymes that is required for proper establishment and maintenance of the spindle. Using quantitative immunofluorescence and fluorescence recovery after photobleaching, we compared the differences in spindle organization caused by global suppression of microtubule dynamics, by treating cells with low levels of paclitaxel, versus specific perturbation of spindle microtubule subsets by MCAK inhibition. Paclitaxel treatment caused a disruption in spindle microtubule organization marked by a significant increase in microtubules near the poles and a reduction in K-fiber fluorescence intensity. This was correlated with a faster t 1/2 of both spindle and K-fiber microtubules. In contrast, MCAK inhibition caused a dramatic reorganization of spindle microtubules with a significant increase in astral microtubules and reduction in K-fiber fluorescence intensity, which correlated with a slower t 1/2 of K-fibers but no change in the t 1/2 of spindle microtubules. Our data support the model that MCAK perturbs spindle organization by acting preferentially on a subset of microtubules, and they support the overall hypothesis that microtubule dynamics is differentially regulated in the spindle. INTRODUCTIONThe cytoskeleton must undergo dramatic reorganization as cells transition from interphase to mitosis to assemble the mitotic spindle. The spindle is a macromolecular structure composed of a dynamic array of microtubules (MTs) and their associated proteins that is necessary for proper chromosome segregation (Compton, 2000;Walczak and Heald, 2008). At the onset of mitosis, there is an increase in MT turnover that allows for breakdown of the interphase array and assembly of the mitotic spindle (Saxton et al., 1984;. This change is correlated with an increased catastrophe frequency (transition from growth to shrinkage) and a decreased rescue frequency (transition from shrinkage to growth) (Belmont et al., 1990;Gliksman et al., 1992;Verde et al., 1992;Rusan et al., 2001). The changes in MT dynamics are also correlated with a change in MT polymer levels during mitosis (Zhai et al., 1996). At nuclear envelope breakdown, there is a dramatic decrease in MT polymer levels, which may allow the cell to rapidly assemble a mitotic spindle because the released tubulin dimers can polymerize into new spindle MTs. As the cell progresses from mitosis to the next interphase, there is no change in the levels of MT polymer (Zhai et al., 1996), suggesting that MT polymer gets reorganized to reform the interphase MT cytoskeleton, without a significant change in the dynamics of the MTs. These studies highlight the need for the cell to possess a mechanism to temporally regulate MT dynamics.In addition to the temporal changes in dynamics throughout the cell cycle, the dynamics of MTs within mitosis are also...

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Section: Immunofluorescence Quantification and Data Collectionmentioning

confidence: 99%

MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

Rizk

Bohannon

Wetzel

et al. 2009

MBoC

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“…In prometaphase cells, most kinetochores rapidly become attached to microtubules and align their chromosomes near the spindle equator, while a few unattached kinetochores delay anaphase [reviewed in Rieder and Salmon, 1998]. In contrast, after nocodazole treatment, all spindle microtubules disappear, tension on the kinetochores is lost, and the kinetochores enlarge [Hoffman et al, 2001]. The kinetochores in nocodazole-treated cells also acquire threefold or higher concentrations of most spindle checkpoint proteins in comparison to kinetochores on aligned chromosomes that are fully attached to microtubules [Hoffman et al, 2001].…”

Section: Comments and Conclusionmentioning

confidence: 99%

“…In contrast, after nocodazole treatment, all spindle microtubules disappear, tension on the kinetochores is lost, and the kinetochores enlarge [Hoffman et al, 2001]. The kinetochores in nocodazole-treated cells also acquire threefold or higher concentrations of most spindle checkpoint proteins in comparison to kinetochores on aligned chromosomes that are fully attached to microtubules [Hoffman et al, 2001]. Perhaps the increase in kinetochore protein levels and kinetochore size that comes with nocodazole treatment is responsible for the delay in the timing of precocious anaphase.…”

Section: Comments and Conclusionmentioning

confidence: 99%

Inducing precocious anaphase in cultured mammalian cells

Canman

Salmon

Fang

2002

Cell Motil. Cytoskeleton

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The spindle checkpoint, which prevents anaphase onset upon spindle damage or incorrect chromosome alignment, presents a problem for experimental analysis of protein function in anaphase and cytokinesis. This is because the functional disruption of many proteins before anaphase onset can activate this checkpoint, preventing anaphase and subsequent cell cycle events. This paper compares new and old methods of overriding the spindle checkpoint in prometaphase mammalian tissue culture cells. Cell Motil. Cytoskeleton 52:61–65, 2002. © 2002 Wiley‐Liss, Inc.

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“…How the inhibitory signal is generated and how the checkpoint monitors microtubule attachment and/or tension at the kinetochore are topics of much debate. Microtubule attachment induces the loss of checkpoint proteins at kinetochores, in particular, a substantial loss of Mad2 (Waters et al, 1998;Hoffman et al, 2001). Mad2 is able to bind Cdc20 and inhibit APC/C activation in vitro (Li et al, 1997;Fang et al, 1998), and Mad2 turns over quickly at unattached kinetochores, with a halflife of 24 -28 s (Howell et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

Shannon

Canman

Salmon

2002

MBoC

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The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23°C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23°C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37°C. The metaphase delay at 23°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°C, and metaphase kinetochores at 23°C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37°C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23°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.

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Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

Cited by 328 publications

References 54 publications

MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

Inducing precocious anaphase in cultured mammalian cells

Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

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