Faculty Opinions recommendation of Tiam1-Rac signaling counteracts Eg5 during bipolar spindle assembly to facilitate chromosome congression.

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video-enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.

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A role for I B kinase 2 in bipolar spindle assembly

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0706493104 ◽

2007 ◽

Vol 104 (43) ◽

pp. 16940-16945 ◽

Cited By ~ 50

Author(s):

J. T. Irelan ◽

T. J. Murphy ◽

P. D. DeJesus ◽

H. Teo ◽

D. Xu ◽

...

Keyword(s):

Spindle Assembly ◽

Bipolar Spindle

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Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes.

The Journal of Cell Biology ◽

10.1083/jcb.131.5.1125 ◽

1995 ◽

Vol 131 (5) ◽

pp. 1125-1131 ◽

Cited By ~ 46

Author(s):

D Zhang ◽

R B Nicklas

Keyword(s):

Nuclear Envelope ◽

Essential Role ◽

Spindle Assembly ◽

Microtubule Assembly ◽

Chromosomal Protein ◽

Spindle Microtubule ◽

Late Prophase ◽

Spindle Formation ◽

Bipolar Spindle

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.

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Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

Molecular and Cellular Biology ◽

10.1128/mcb.00068-15 ◽

2015 ◽

Vol 35 (15) ◽

pp. 2626-2640 ◽

Cited By ~ 23

Author(s):

Lingjun Meng ◽

Jung-Eun Park ◽

Tae-Sung Kim ◽

Eun Hye Lee ◽

Suk-Youl Park ◽

...

Keyword(s):

Xenopus Laevis ◽

Spindle Assembly ◽

Human Cells ◽

Mitotic Progression ◽

Spindle Formation ◽

Content Type ◽

Bipolar Spindle ◽

Egg Extracts ◽

Cultured Human Cells ◽

Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

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Faculty Opinions recommendation of Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737370364.793576298 ◽

2020 ◽

Author(s):

Alex Mogilner

Keyword(s):

Computational Modeling ◽

Spindle Assembly ◽

Bipolar Spindle

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Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal

mSphere ◽

10.1128/msphere.00610-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Irsa Shoukat ◽

Corey Frazer ◽

John S. Allingham

Keyword(s):

Candida Albicans ◽

Mitotic Spindle ◽

Fungal Pathogens ◽

Spindle Assembly ◽

Spindle Pole ◽

Content Type ◽

Bipolar Spindle ◽

Spindle Elongation ◽

Simultaneous Loss ◽

Bipolar Spindles

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

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A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0497 ◽

2017 ◽

Vol 28 (25) ◽

pp. 3647-3659 ◽

Cited By ~ 23

Author(s):

Masashi Yukawa ◽

Tomoki Kawakami ◽

Masaki Okazaki ◽

Kazunori Kume ◽

Ngang Heok Tang ◽

...

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Spindle Pole ◽

Temperature Sensitive ◽

Bipolar Spindle ◽

Pole Body ◽

Cross Linker ◽

Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e19-07-0366 ◽

2019 ◽

Vol 30 (22) ◽

pp. 2802-2813 ◽

Cited By ~ 4

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.

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

Journal of Cell Science ◽

10.1242/jcs.251025 ◽

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.

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