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 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 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.

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.

scholarly journals KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle

2019 ◽  
Vol 2 (1) ◽  
pp. e201800169 ◽  
Author(s):  
Heidi LH Malaby ◽  
Dominique V Lessard ◽  
Christopher L Berger ◽  
Jason Stumpff
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Binding Proteins ◽  
Mitotic Chromosome ◽  
Chromosome Movement ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Chromosome Alignment ◽  
Associated Proteins ◽  
Fiber Dynamics

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber–binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

scholarly journals Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo.

1989 ◽  
Vol 109 (6) ◽  
pp. 2977-2991 ◽  
Author(s):  
D R Kellogg ◽  
C M Field ◽  
B M Alberts
Keyword(s):  
Affinity Chromatography ◽  
Mitotic Spindle ◽  
Polyclonal Antibodies ◽  
Drosophila Embryo ◽  
Microtubule Cytoskeleton ◽  
Microtubule Associated Proteins ◽  
Early Drosophila Embryo ◽  
Individual Mouse ◽  
Associated Proteins

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

scholarly journals Two XMAP215/TOG microtubule polymerases, Alp14 and Dis1, play non-exchangeable, distinct roles in microtubule organisation in fission yeast

2019 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Corinne Pinder ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Genome Stability ◽  
Spindle Assembly ◽  
Mitotic Progression ◽  
Spindle Microtubule ◽  
Microtubule Associated Proteins ◽  
Spatial Regulation ◽  
Associated Proteins ◽  
Microtubule Formation

AbstractProper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal “cut” phenotype. By implementing an artificial targetting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Intriguingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.

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.

Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis

1991 ◽  
Vol 98 (4) ◽  
pp. 577-588 ◽  
Author(s):  
D.D. Vandre ◽  
V.E. Centonze ◽  
J. Peloquin ◽  
R.M. Tombes ◽  
G.G. Borisy
Keyword(s):  
Mitotic Spindle ◽  
Immunoblot Analysis ◽  
Entry And Exit ◽  
Immunofluorescence Analysis ◽  
Temporal And Spatial Distribution ◽  
Microtubule Associated Proteins ◽  
Before And After ◽  
Associated Proteins ◽  
Temporal And Spatial ◽  
Monospecific Antibodies

The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 × 10(3) Mr, 350 × 10(3) Mr, a 230–240 X 10(3) Mr doublet, 210 × 10(3) Mr and 120 × 10(3) Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined. The phosphorylated 210 × 10(3) Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 × 10(3) Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies.

scholarly journals Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

1995 ◽  
Vol 128 (5) ◽  
pp. 849-862 ◽  
Author(s):  
K Ookata ◽  
S Hisanaga ◽  
J C Bulinski ◽  
H Murofushi ◽  
H Aizawa ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
Cyclin B ◽  
Microtubule Associated Proteins ◽  
Mitotic Kinase ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Detergent Extraction ◽  
M Phase ◽  
Associated Proteins ◽  
Free Microtubules

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

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