scholarly journals Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment

2010 ◽  
Vol 123 (21) ◽  
pp. 3645-3651 ◽  
Author(s):  
W. Fu ◽  
W. Tao ◽  
P. Zheng ◽  
J. Fu ◽  
M. Bian ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Chromosome Alignment

scholarly journals Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

2019 ◽  
Vol 30 (22) ◽  
pp. 2802-2813 ◽  
Author(s):  
Yutaka Shirasugi ◽  
Masamitsu Sato
Keyword(s):  
Fission Yeast ◽  
Motor Proteins ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Double Knockout ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Bipolar Spindles ◽  
Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-­dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

scholarly journals The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

2020 ◽  
Vol 134 (1) ◽  
pp. jcs251025
Author(s):  
Zoë Geraghty ◽  
Christina Barnard ◽  
Pelin Uluocak ◽  
Ulrike Gruneberg
Keyword(s):  
Molecular Mechanisms ◽  
Metaphase Plate ◽  
Direct Interaction ◽  
Mitotic Chromosomes ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

scholarly journals Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.

1996 ◽  
Vol 134 (2) ◽  
pp. 455-464 ◽  
Author(s):  
H J Matthies ◽  
H B McDonald ◽  
L S Goldstein ◽  
W E Theurkauf
Keyword(s):  
Laser Scanning ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Meiotic Spindle ◽  
Bipolar Spindle ◽  
Scanning Confocal Microscopy ◽  
Spindle Poles ◽  
Assembly Kinetics

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.

scholarly journals RanGTP induces an effector gradient of XCTK2 and importin α/β for spindle microtubule cross-linking

2020 ◽  
Vol 219 (2) ◽  
Author(s):  
Stephanie C. Ems-McClung ◽  
Mackenzie Emch ◽  
Stephanie Zhang ◽  
Serena Mahnoor ◽  
Lesley N. Weaver ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Cross Linking ◽  
Tail Domain ◽  
Inhibitory Effects ◽  
Spindle Microtubule ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Assembly Factors ◽  
Importin Α ◽  
Combined Actions

High RanGTP around chromatin is important for governing spindle assembly during meiosis and mitosis by releasing the inhibitory effects of importin α/β. Here we examine how the Ran gradient regulates Kinesin-14 function to control spindle organization. We show that Xenopus Kinesin-14, XCTK2, and importin α/β form an effector gradient that is highest at the poles and diminishes toward the chromatin, which is opposite the RanGTP gradient. Importin α and β preferentially inhibit XCTK2 antiparallel microtubule cross-linking and sliding by decreasing the microtubule affinity of the XCTK2 tail domain. This change in microtubule affinity enables RanGTP to target endogenous XCTK2 to the spindle. We propose that these combined actions of the Ran pathway are critical to promote Kinesin-14 parallel microtubule cross-linking to help focus spindle poles for efficient bipolar spindle assembly. Furthermore, our work illustrates that RanGTP regulation in the spindle is not simply a switch, but rather generates effector gradients where importins α and β gradually tune the activities of spindle assembly factors.

scholarly journals Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Takumi Chinen ◽  
Kaho Yamazaki ◽  
Kaho Hashimoto ◽  
Ken Fujii ◽  
Koki Watanabe ◽  
...  
Keyword(s):  
A549 Cells ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Scaffold Proteins ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Mitotic Cells

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

scholarly journals Interaction of NuMA protein with the kinesin Eg5: its possible role in bipolar spindle assembly and chromosome alignment

2013 ◽  
Vol 451 (2) ◽  
pp. 195-204 ◽  
Author(s):  
Yuko Iwakiri ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Mitotic Apparatus ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Interphase Cells ◽  
Correct Alignment ◽  
Spindle Microtubules

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro. During mitosis Eg5 appears to interact with NuMA in the vicinity of the spindle poles, whereas the interaction does not occur in interphase cells, where Eg5 is distributed throughout the cytoplasm but NuMA exclusively localizes to the nucleus. Slight, but significant, depletion of Eg5 in HeLa cells by RNA interference results in formation of less-focused spindle poles with misaligned chromosomes in metaphase; these phenotypes are similar to those induced by depletion of NuMA. Since NuMA is less accumulated at the spindle poles in Eg5-depleted cells, Eg5 probably contributes to spindle assembly via regulating NuMA localization. Furthermore, depletion of cytoplasmic dynein induces mislocalization of NuMA and phenotypes similar to those observed in NuMA-depleted cells, without affecting Eg5 localization to the spindles. Thus dynein appears to control NuMA function in conjunction with Eg5.

scholarly journals Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Kimberly K. Fong ◽  
Trisha N. Davis ◽  
Charles L. Asbury
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Force Generation ◽  
Initial Contact ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules ◽  
Pole Component

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.

scholarly journals TOGp, the Human Homolog of XMAP215/Dis1, Is Required for Centrosome Integrity, Spindle Pole Organization, and Bipolar Spindle Assembly

2004 ◽  
Vol 15 (4) ◽  
pp. 1580-1590 ◽  
Author(s):  
Lynne Cassimeris ◽  
Justin Morabito
Keyword(s):  
Motor Activity ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Human Homolog ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Multipolar Spindles ◽  
Mitotic Cells

The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336–341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by ∼20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by ∼20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.

scholarly journals A Kinesin Mutant with an Atypical Bipolar Spindle Undergoes Normal Mitosis

2003 ◽  
Vol 14 (4) ◽  
pp. 1717-1726 ◽  
Author(s):  
A. I. Marcus ◽  
W. Li ◽  
H. Ma ◽  
R. J. Cyr
Keyword(s):  
Chromosome Segregation ◽  
Plant Cells ◽  
Spindle Assembly ◽  
Wild Type Allele ◽  
Wild Type ◽  
Bipolar Spindle ◽  
Type Allele ◽  
Spindle Poles ◽  
Normal Mitosis ◽  
Arabidopsis Cells

Motor proteins have been implicated in various aspects of mitosis, including spindle assembly and chromosome segregation. Here, we show that acentrosomal Arabidopsis cells that are mutant for the kinesin, ATK1, lack microtubule accumulation at the predicted spindle poles during prophase and have reduced spindle bipolarity during prometaphase. Nonetheless, all abnormalities are rectified by anaphase and chromosome segregation appears normal. We conclude that ATK1 is required for normal microtubule accumulation at the spindle poles during prophase and possibly functions in spindle assembly during prometaphase. Because aberrant spindle morphology in these mutants is resolved by anaphase, we postulate that mitotic plant cells contain an error-correcting mechanism. Moreover, ATK1 function seems to be dosage-dependent, because cells containing one wild-type allele take significantly longer to proceed to anaphase as compared with cells containing two wild-type alleles.

scholarly journals The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

2004 ◽  
Vol 166 (4) ◽  
pp. 473-478 ◽  
Author(s):  
Neil J. Ganem ◽  
Duane A. Compton
Keyword(s):  
Cell Cycle Progression ◽  
Cultured Cells ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Cycle Progression ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Microtubule Attachment ◽  
Kinesin Eg5 ◽  
Dependent Pathway

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore–microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.

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