scholarly journals Compartimentalized control of Cdk1 drives mitotic spindle assembly

2021 ◽  
Author(s):  
Angela Flavia Serpico ◽  
Francesco Febbraro ◽  
Caterina Pisauro ◽  
Domenico Grieco
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules ◽  
Growth Promoting ◽  
Fraction Bound

During cell division, dramatic microtubular rearrangements driven by cyclin B-cdk1 (Cdk1) kinase activity mark mitosis onset leading to interphase cytoskeleton dissolution and mitotic spindle assembly. Once activated by Cdc25, that reverses inhibitory phosphorylation operated by Wee1/Myt1, Cdk1 clears the cytoplasm from microtubules by inhibiting microtubule associated proteins (MAPs) with microtubule growth-promoting properties. Nevertheless, some of these MAPs are required for spindle assembly, creating quite a conundrum. We show here that a Cdk1 fraction bound to spindle structures escaped Cdc25 action and remained inhibited by phosphorylation (i-Cdk1) in mitotic human cells. Loss or restoration of i-Cdk1 inhibited or promoted spindle assembly, respectively. Furthermore, polymerizing spindle microtubules fostered i-Cdk1 by aggregating with Wee1 and excluding Cdc25. Our data reveal that spindle assembly relies on compartimentalized control of Cdk1 activity.

scholarly journals Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

2014 ◽  
Vol 204 (7) ◽  
pp. 1111-1121 ◽  
Author(s):  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
Franck Bazile ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Centrosome Separation ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

scholarly journals Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly.

1992 ◽  
Vol 118 (1) ◽  
pp. 109-120 ◽  
Author(s):  
M A Hoyt ◽  
L He ◽  
K K Loo ◽  
W S Saunders
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Viability ◽  
Chromosome Segregation ◽  
Heavy Chain ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Gene Products ◽  
Related Gene ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules

Two Saccharomyces cerevisiae genes, CIN8 and KIP1 (a.k.a. CIN9), were identified by their requirement for normal chromosome segregation. Both genes encode polypeptides related to the heavy chain of the microtubule-based force-generating enzyme kinesin. Cin8p was found to be required for pole separation during mitotic spindle assembly at 37 degrees C, although overproduced Kip1p could substitute. At lower temperatures, the activity of at least one of these proteins was required for cell viability, indicating that they perform an essential but redundant function. Cin8p was observed to be a component of the mitotic spindle, colocalizing with the microtubules that lie between the poles. Taken together, these findings suggest that these proteins interact with spindle microtubules to produce an outwardly directed force acting upon the poles.

scholarly journals Mitotic Acetylation of Microtubules Promotes Centrosomal PLK1 Recruitment and Is Required to Maintain Bipolar Spindle Homeostasis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1859
Author(s):  
Sylvia Fenosoa Rasamizafy ◽  
Claude Delsert ◽  
Gabriel Rabeharivelo ◽  
Julien Cau ◽  
Nathalie Morin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mitotic Spindle ◽  
Mitotic Progression ◽  
Post Translational Modifications ◽  
Microtubule Associated Proteins ◽  
Mitotic Kinase ◽  
Tubulin Acetylation ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
Bipolar Spindles

Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination promotes proper mitotic progression by recruiting specific microtubule-associated proteins motors, tubulin acetylation that occurs on specific microtubule subsets during mitosis is less well understood. Here, we show that siRNA-mediated depletion of the tubulin acetyltransferase ATAT1 in epithelial cells leads to a prolonged prometaphase arrest and the formation of monopolar spindles. This results from collapse of bipolar spindles, as previously described in cells deficient for the mitotic kinase PLK1. ATAT1-depleted mitotic cells have defective recruitment of PLK1 to centrosomes, defects in centrosome maturation and thus microtubule nucleation, as well as labile microtubule-kinetochore attachments. Spindle bipolarity could be restored, in the absence of ATAT1, by stabilizing microtubule plus-ends or by increasing PLK1 activity at centrosomes, demonstrating that the phenotype is not just a consequence of lack of K-fiber stability. We propose that microtubule acetylation of K-fibers is required for a recently evidenced cross talk between centrosomes and kinetochores.

scholarly journals Computer simulations predict that chromosome movements and rotations accelerate mitotic spindle assembly without compromising accuracy

2009 ◽  
Vol 106 (37) ◽  
pp. 15708-15713 ◽  
Author(s):  
Raja Paul ◽  
Roy Wollman ◽  
William T. Silkworth ◽  
Isaac K. Nardi ◽  
Daniela Cimini ◽  
...  
Keyword(s):  
Computer Simulations ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Poles ◽  
Colorectal Cancer Cells ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly ◽  
Chromosome Movements ◽  
Realistic Geometry

The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10–20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.

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 Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

1985 ◽  
Vol 101 (5) ◽  
pp. 1858-1870 ◽  
Author(s):  
N Hirokawa ◽  
R Takemura ◽  
S Hisanaga
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Mitotic Spindle ◽  
Cytoplasmic Dynein ◽  
High Salt ◽  
Mitotic Spindles ◽  
Mitotic Apparatus ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Spindle Microtubules

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The microtubule-associated protein EML3 regulates mitotic spindle assembly by recruiting the Augmin complex to spindle microtubules

2019 ◽  
Vol 294 (14) ◽  
pp. 5643-5656 ◽  
Author(s):  
Jia Luo ◽  
Biying Yang ◽  
Guangwei Xin ◽  
Mengjie Sun ◽  
Boyan Zhang ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules

scholarly journals The contribution of αβ-tubulin curvature to microtubule dynamics

2014 ◽  
Vol 207 (3) ◽  
pp. 323-334 ◽  
Author(s):  
Gary J. Brouhard ◽  
Luke M. Rice
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Fundamental Property ◽  
Microtubule Dynamics ◽  
Cellular Structures ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Microtubule Networks ◽  
And Control

Microtubules are dynamic polymers of αβ-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized αβ-tubulin. The ability of αβ-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. αβ-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks.

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

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