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

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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The sequential activation of the mitotic microtubule assembly pathways favors bipolar spindle formation

Molecular Biology of the Cell ◽

10.1091/mbc.e16-05-0322 ◽

2016 ◽

Vol 27 (19) ◽

pp. 2935-2945 ◽

Cited By ~ 5

Author(s):

Tommaso Cavazza ◽

Paolo Malgaretti ◽

Isabelle Vernos

Keyword(s):

Tissue Culture ◽

Spindle Assembly ◽

Microtubule Assembly ◽

Spindle Formation ◽

Bipolar Spindle ◽

Culture Cells ◽

Tissue Culture Cells ◽

Egg Extracts ◽

Sequential Activation ◽

Assembly Pathways

Centrosome maturation is the process by which the duplicated centrosomes recruit pericentriolar components and increase their microtubule nucleation activity before mitosis. The role of this process in cells entering mitosis has been mostly related to the separation of the duplicated centrosomes and thereby to the assembly of a bipolar spindle. However, spindles can form without centrosomes. In fact, all cells, whether they have centrosomes or not, rely on chromatin-driven microtubule assembly to form a spindle. To test whether the sequential activation of these microtubule assembly pathways, defined by centrosome maturation and nuclear envelope breakdown, plays any role in spindle assembly, we combined experiments in tissue culture cells and Xenopus laevis egg extracts with a mathematical model. We found that interfering with the sequential activation of the microtubule assembly pathways compromises bipolar spindle assembly in tissue culture cells but not in X. laevis egg extracts. Our data suggest a novel function for centrosome maturation that determines the contribution of the chromosomal microtubule assembly pathway and favors bipolar spindle formation in most animal cells in which tubulin is in limiting amounts.

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The impact of chromosomes and centrosomes on spindle assembly as observed in living cells.

The Journal of Cell Biology ◽

10.1083/jcb.129.5.1287 ◽

1995 ◽

Vol 129 (5) ◽

pp. 1287-1300 ◽

Cited By ~ 60

Author(s):

D Zhang ◽

R B Nicklas

Keyword(s):

Spindle Assembly ◽

Living Cells ◽

Microtubule Dynamics ◽

Specific Site ◽

Polarization Microscopy ◽

Spindle Microtubule ◽

Single Chromosome ◽

Bipolar Spindle ◽

The Impact

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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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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Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e15-08-0577 ◽

2016 ◽

Vol 27 (11) ◽

pp. 1753-1763 ◽

Cited By ~ 19

Author(s):

Hirohisa Masuda ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Synthetic Lethality ◽

Spindle Assembly ◽

Spindle Pole ◽

Microtubule Assembly ◽

Spindle Microtubule ◽

Spindle Pole Bodies ◽

Mitotic Spindle Pole

In fission yeast, γ-tubulin ring complex (γTuRC)–specific components Gfh1GCP4, Mod21GCP5, and Alp16GCP6 are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1MOZART1, a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16GCP6 promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1GCP4 and Mod21GCP5 are not required for Alp16GCP6-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16GCP6 and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation.

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Hepatoma Up-Regulated Protein Is Required for Chromatin-induced Microtubule Assembly Independently of TPX2

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0624 ◽

2008 ◽

Vol 19 (11) ◽

pp. 4900-4908 ◽

Cited By ~ 14

Author(s):

Claudia M. Casanova ◽

Sofia Rybina ◽

Hideki Yokoyama ◽

Eric Karsenti ◽

Iain W. Mattaj

Keyword(s):

Xenopus Laevis ◽

Detailed Analysis ◽

Spindle Assembly ◽

Microtubule Assembly ◽

Spindle Microtubule ◽

Microtubule Stabilization ◽

Egg Extracts ◽

Spindle Midzone ◽

Microtubule Growth ◽

Spindle Microtubules

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.

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Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1

Molecular Biology of the Cell ◽

10.1091/mbc.e16-05-0291 ◽

2016 ◽

Vol 27 (20) ◽

pp. 3122-3131 ◽

Cited By ~ 17

Author(s):

Ian D. Wolff ◽

Michael V. Tran ◽

Timothy J. Mullen ◽

Anne M. Villeneuve ◽

Sarah M. Wignall

Keyword(s):

Caenorhabditis Elegans ◽

High Resolution ◽

Nuclear Envelope ◽

Spindle Assembly ◽

Cell Types ◽

High Resolution Imaging ◽

Spindle Formation ◽

Mixed Polarity ◽

Resolution Imaging ◽

Meiosis I

Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a “cage-like” structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles.

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Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e12-12-0851 ◽

2013 ◽

Vol 24 (12) ◽

pp. 1933-1946 ◽

Cited By ~ 5

Author(s):

Shohei Iwase ◽

Ryuhei Sato ◽

Pieter-Jan De Bock ◽

Kris Gevaert ◽

Saburo Fujiki ◽

...

Keyword(s):

Xenopus Laevis ◽

Actin Filament ◽

Spindle Assembly ◽

Microtubule Assembly ◽

Meiotic Spindle ◽

Actin Dynamics ◽

Tail Domain ◽

Latrunculin B ◽

Germinal Vesicle Breakdown ◽

Spindle Formation

We identify Xenopus ADF/cofilin (XAC) and its activator, Slingshot phosphatase (XSSH), as key regulators of actin dynamics essential for spindle microtubule assembly during Xenopus oocyte maturation. Phosphorylation of XSSH at multiple sites within the tail domain occurs just after germinal vesicle breakdown (GVBD) and is accompanied by dephosphorylation of XAC, which was mostly phosphorylated in immature oocytes. This XAC dephosphorylation after GVBD is completely suppressed by latrunculin B, an actin monomer–sequestering drug. On the other hand, jasplakinolide, an F-actin–stabilizing drug, induces dephosphorylation of XAC. Effects of latrunculin B and jasplakinolide are reconstituted in cytostatic factor–arrested extracts (CSF extracts), and XAC dephosphorylation is abolished by depletion of XSSH from CSF extracts, suggesting that XSSH functions as an actin filament sensor to facilitate actin filament dynamics via XAC activation. Injection of anti-XSSH antibody, which blocks full phosphorylation of XSSH after GVBD, inhibits both meiotic spindle formation and XAC dephosphorylation. Coinjection of constitutively active XAC with the antibody suppresses this phenotype. Treatment of oocytes with jasplakinolide also impairs spindle formation. These results strongly suggest that elevation of actin dynamics by XAC activation through XSSH phosphorylation is required for meiotic spindle assembly in Xenopus laevis.

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

The Journal of Cell Biology ◽

10.1083/jcb.201906045 ◽

2020 ◽

Vol 219 (2) ◽

Cited By ~ 3

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.202006085 ◽

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.

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Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

The Journal of Cell Biology ◽

10.1083/jcb.101.5.1966 ◽

1985 ◽

Vol 101 (5) ◽

pp. 1966-1976 ◽

Cited By ~ 18

Author(s):

J B Tucker ◽

S A Mathews ◽

K A Hendry ◽

J B Mackie ◽

D L Roche

Keyword(s):

Nuclear Envelope ◽

Early Stage ◽

Microtubule Assembly ◽

Membrane Association ◽

Paramecium Tetraurelia ◽

Spindle Microtubule ◽

Spatial Control ◽

Spindle Microtubules ◽

Spindle Elongation ◽

Anaphase B

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

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