Centrosomal Actin Assembly Is Required for Proper Mitotic Spindle Formation and Chromosome Congression

The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.

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Regulation of Microtubule Assembly: The View From The End

Microscopy and Microanalysis ◽

10.1017/s143192760003289x ◽

2000 ◽

Vol 6 (S2) ◽

pp. 80-81

Author(s):

L. Cassimeris ◽

C. Spittle ◽

M. Kratzer

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Dynamic Instability ◽

Intrinsic Property ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

Chromosome Movement ◽

Spindle Formation ◽

Associated Proteins

The mitotic spindle is responsible for chromosome movement during mitosis. It is composed of a dynamic array of microtubules and associated proteins whose assembly and constant turnover are required for both spindle formation and chromosome movement. Because microtubule assembly and turnover are necessary for chromosome segregation, we are studying how cells regulate microtubule dynamics. Microtubules are polarized polymers composed of tubulin subunits; they assemble by a process of dynamic instability where individual microtubules exist in persistent phases of elongation or rapid shortening with abrupt transitions between these two states. The switch from elongation to shortening is termed catastrophe, and the switch from shortening to elongation, rescue. Although dynamic instability is an intrinsic property of the tubulin subunits, cells use associated proteins to both speed elongation (∼ 10 fold) and regulate transitions.The only protein isolated to date capable of promoting fast polymerization consistent with rates in vivo is XMAP215, a 215 kD protein from Xenopus eggs.

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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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PLK1 regulates spindle association of phosphorylated eukaryotic translation initiation factor 4E-binding protein and spindle function in mouse oocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00075.2017 ◽

2017 ◽

Vol 313 (5) ◽

pp. C501-C515 ◽

Cited By ~ 8

Author(s):

Ashley L. Severance ◽

Keith E. Latham

Keyword(s):

Translation Initiation ◽

Binding Protein ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Spindle Formation ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Chromosome Congression ◽

Eukaryotic Translation Initiation ◽

Meiotic Spindles

Oocyte meiotic spindles are associated with spindle-enriched mRNAs, phosphorylated ribosome protein S6, and phosphorylated variants of the key translational regulator, eukaryotic translation initiation factor 4E-binding protein 1 (eIF4E-BP1), consistent with translational control of localized mRNAs by eIF4E-BP1 in facilitating spindle formation and stability. Using specific kinase inhibitors, we determined which kinases regulate phosphorylation status of eIF4E-BP1 associated with meiotic spindles in mouse oocytes and effects of kinase inhibition on chromosome congression and spindle formation. Neither ataxia telangiectasia-mutated kinase nor mechanistic target of rapamycin inhibition significantly affected phosphorylation status of spindle-associated eIF4E-BP1 at the phosphorylation sites examined. Spindle-associated phospho-eIF4E-BP1, spindle formation, and chromosome congression were strongly disrupted by polo-like kinase I (PLK1) inhibition at both metaphase I (MI) and MII. In addition, direct inhibition of eIF4E-BP1 via 4EGI led to spindle defects at MI, indicating a direct role for eIF4E-BP1 phosphorylation in meiotic spindle formation. PLK1 also regulated microtubule dynamics throughout the ooplasm, indicating likely coordination between spindle dynamics and broader ooplasm cytoskeletal dynamics. Because diverse upstream signaling pathways converge on PLK1, these results implicate PLK1 as a major regulatory nexus coupling endogenous and exogenous signals via eIF4E-BP1 to the regulation of spindle formation and stability.

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The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

MGG Molecular & General Genetics ◽

10.1007/bf00705642 ◽

1995 ◽

Vol 247 (2) ◽

pp. 123-136 ◽

Cited By ~ 27

Author(s):

Nora Vaisman ◽

Andrey Tsouladze ◽

Kenneth Robzyk ◽

Sigal Ben-Yehuda ◽

Martin Kupiec ◽

...

Keyword(s):

Dna Replication ◽

Mitotic Spindle ◽

Spindle Formation

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Challenges In Modeling Chromosome-driven Mitotic Spindle Formation

Biophysical Journal ◽

10.1016/j.bpj.2008.12.2604 ◽

2009 ◽

Vol 96 (3) ◽

pp. 505a

Author(s):

Stuart Schaffner ◽

Jorge V. Jose

Keyword(s):

Mitotic Spindle ◽

Spindle Formation

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Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Open Biology ◽

10.1098/rsob.140047 ◽

2014 ◽

Vol 4 (5) ◽

pp. 140047 ◽

Cited By ~ 9

Author(s):

Matthew S. Savoian ◽

David M. Glover

Keyword(s):

Mitotic Spindle ◽

Cell Types ◽

Male Meiosis ◽

Negative Regulation ◽

Mitotic Spindles ◽

Animal Cells ◽

Spindle Formation ◽

Formation Pathway ◽

Fixed Cell ◽

Mitotic Cells

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

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