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

2008 ◽  
Vol 19 (11) ◽  
pp. 4900-4908 ◽  
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.

scholarly journals Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

2005 ◽  
Vol 170 (7) ◽  
pp. 1047-1055 ◽  
Author(s):  
Kazuhisa Kinoshita ◽  
Tim L. Noetzel ◽  
Laurence Pelletier ◽  
Karl Mechtler ◽  
David N. Drechsel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Microtubule Assembly ◽  
Aurora A ◽  
Specific Mechanism ◽  
Microtubule Polymerization ◽  
Egg Extracts ◽  
Microtubule Growth

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3–XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A–dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

Functional midbody assembly in the absence of a central spindle

2022 ◽  
Vol 221 (3) ◽  
Author(s):  
Sophia M. Hirsch ◽  
Frances Edwards ◽  
Mimi Shirasu-Hiza ◽  
Julien Dumont ◽  
Julie C. Canman
Keyword(s):  
Microtubule Assembly ◽  
Primary Role ◽  
Intercellular Bridge ◽  
Contractile Ring ◽  
Spindle Microtubule ◽  
Central Spindle ◽  
Family Protein ◽  
Spindle Midzone ◽  
Ring Constriction ◽  
Spindle Microtubules

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F–like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2–dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle–independent midbodies appeared to form via contractile ring constriction–driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

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

1995 ◽  
Vol 131 (5) ◽  
pp. 1125-1131 ◽  
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.

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

2015 ◽  
Vol 35 (15) ◽  
pp. 2626-2640 ◽  
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.

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

2016 ◽  
Vol 27 (11) ◽  
pp. 1753-1763 ◽  
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.

scholarly journals Mitotic Effects of a Constitutively Active Mutant of the Xenopus Polo-Like Kinase Plx1

1999 ◽  
Vol 19 (12) ◽  
pp. 8625-8632 ◽  
Author(s):  
Yue-Wei Qian ◽  
Eleanor Erikson ◽  
James L. Maller
Keyword(s):  
Xenopus Laevis ◽  
Aspartic Acid ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Interphase Nuclei ◽  
Aspartic Acid Residue ◽  
Early Embryos ◽  
M Phase ◽  
Egg Extracts

ABSTRACT During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a “trigger” kinase for the activation of Cdc25C during the G2/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.

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

2013 ◽  
Vol 24 (12) ◽  
pp. 1933-1946 ◽  
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.

scholarly journals Spastin Interacts with CRMP5 to Promote  Spindle Organization of Mouse Oocytes by  Severing Microtubules.

2020 ◽  
Author(s):  
Hua-Feng Shou ◽  
Lei-lei Gao ◽  
Zhen Jin ◽  
Jin-Wei Liu ◽  
Shan-Shan Jiang ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Spindle Microtubule ◽  
Microtubule Severing ◽  
Abnormal Phenotype ◽  
Mammalian Oocyte ◽  
Spindle Microtubules ◽  
Normal Meiosis

Abstract Background ： Microtubule-severing protein (MTSP) is highly critical for the survival of both mitotic and post-mitotic cells.However, the study of MTSP in the meiosis of mammalian oocyte has not been reported. Results ：We found that spastin, a member of the MTSP family, was highly expressed in oocyte and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocyte decreased significantly. When the oocyte was cultured in vitro, the oocyte lacking spastin showed obvious maturation obstacles. Combining with the microtubule severing activity of spastin, we speculate that spastin on spindle may increase the microtubule broken ends by severing microtubules, thus playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found that there was co-localization and interaction between CRMP5 and spastin in oocyte. The knockdown of CRMP5 may also lead to spindle abnormalities and developmental disorders in oocyte. Overexpression of spastin may save the abnormal phenotype caused by deletion of CRMP5. Conclusions ：To sum up, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocyte by controlling microtubule dynamics, thus ensuring normal meiosis.

scholarly journals Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

1985 ◽  
Vol 101 (5) ◽  
pp. 1966-1976 ◽  
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.

scholarly journals Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly

2019 ◽  
Vol 218 (10) ◽  
pp. 3237-3257 ◽  
Author(s):  
Mary Kate Bonner ◽  
Julian Haase ◽  
Jason Swinderman ◽  
Hyunmi Halas ◽  
Lisa M. Miller Jenkins ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Central Region ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Kinetochore Assembly ◽  
Egg Extracts ◽  
Chromosome Attachment

Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using Xenopus laevis egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates.

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