The evolution of spindles and their mechanical implications for cancer metastasis
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Abstract:
The mitotic spindle has long been known to play a crucial role in mitosis, orchestrating the segregation of chromosomes into two daughter cells during mitosis with high fidelity. Intracellular forces generated by the mitotic spindle are increasingly well understood, and recent work has revealed that the efficiency and the accuracy of mitosis is ensured by the scaling of mitotic spindle size with cell size. However, the role of the spindle in cancer progression has largely been ignored. Two recent studies point toward the role of mitotic spindle evolution in cancer progression through extracellular force generation. Cancer cells with lengthened spindles exhibit highly increased metastatic potential. Further, interpolar spindle elongation drives protrusive extracellular force generation along the mitotic axis to allow mitotic elongation, a morphological change that is required for cell division. Together, these findings open a new research area studying the role of the mitotic spindle evolution in cancer metastasis.Keywords:
Mitotic exit
Spindle pole body
Multipolar spindles
Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.
Spindle pole body
Kinesin
Mitotic exit
Multipolar spindles
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The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother–bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions.
Mitotic exit
Spindle pole body
Spindle checkpoint
Astral microtubules
Multipolar spindles
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Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.
Spindle checkpoint
Spindle pole body
Mitotic exit
Aurora B kinase
Multipolar spindles
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Spindle pole body
Mitotic exit
Spindle checkpoint
Multipolar spindles
Aurora B kinase
Polo-like kinase
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Spindle pole body
Mitotic exit
Multipolar spindles
Astral microtubules
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The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.
Spindle pole body
Mitotic exit
Spindle checkpoint
Aurora B kinase
Multipolar spindles
Astral microtubules
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Spindle pole body
Multipolar spindles
Mitotic exit
Spindle checkpoint
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The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1–Bub2 guanosine triphosphatase–activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil–like molecule that interacts with the γ-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.
Spindle pole body
Spindle checkpoint
Mitotic exit
Multipolar spindles
Polo-like kinase
Anaphase-promoting complex
Astral microtubules
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Mitotic exit
Polo-like kinase
Spindle checkpoint
Spindle pole body
Multipolar spindles
Microtubule polymerization
Microtubule organizing center
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When a cell divides, it must accurately replicate its genetic material and then faithfully segregate this material into the resulting daughter cells. My research addresses the latter half of this problem, focusing on how the cell regulates the function of the mitotic spindle, an elegant microtubule-based machine that attaches to replicated DNA and pulls it apart during mitosis. Here, I present two studies that investigate how the cell disassembles the mitotic spindle at the end of mitosis and how the cell positions the mitotic spindle prior to mitotic completion.I combined genetic analysis with live-cell fluorescence microscopy to identify the subprocesses driving spindle disassembly as well as the proteins that perform these subprocesses. Our results suggest that mechanistically distinct pathways largely governed by the anaphase-promoting complex, Aurora B kinase, and kinesin-8 cooperate to drive spindle disassembly in budding yeast. We also describe the roles of novel disassembly factors such as the spindle protein She1 and the 7-protein Alternative Replication Factor C complex. Together, these pathways disengage the mitotic spindle halves, inhibit spindle microtubule growth, and promote sustained spindle microtubule depolymerization. Strikingly, combined inhibition of pairs of disassembly pathways yielded cells with hyper-stable spindle remnants, which caused dramatic defects in cell cycle progression, thus establishing that regulated and rapid spindle disassembly is crucial for cell proliferation.To better understand the mechanisms of spindle positioning, I examined how the dynein-driven spindle-positioning pathway in budding yeast is silenced. My work suggests that dynein activity is regulated through interaction with the multi-subunit dynactin complex at anaphase and identifies a new cellular factor, She1, which controls this interaction. Dynactin is a well-known dynein activator, and, in budding yeast, the complete complex is required for dynein-dependent spindle movement. I found that localization of the dynactin complex is cell cycle-regulated, such that dynactin is recruited to astral microtubules, via interaction with dynein, primarily during anaphase. Additionally, we discovered that the protein She1 is a cell cycle-regulated inhibitor of dynein activity. Without She1, dynein activity extends beyond anaphase and, as a result, mis-positions the mitotic spindle. Strikingly, loss of She1 also permits recruitment of the dynactin complex to astral microtubules throughout the cell cycle. These results suggest that in wild-type cells, She1 restricts dynein activity to anaphase by preventing the interaction between dynein and the complete dynactin complex.
Spindle pole body
Multipolar spindles
Spindle checkpoint
Mitotic exit
PLK1
Microtubule organizing center
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