scholarly journals Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly

2015 ◽  
Vol 209 (3) ◽  
pp. 387-402 ◽  
Author(s):  
Rafael Lucena ◽  
Noah Dephoure ◽  
Steve P. Gygi ◽  
Douglas R. Kellogg ◽  
Victor A. Tallada ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Nucleocytoplasmic Transport ◽  
Membrane Domain ◽  
Aaa Atpase ◽  
Membrane Compartment ◽  
Spindle Disassembly ◽  
Spindle Midzone ◽  
Anaphase B

During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.

scholarly journals The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

2003 ◽  
Vol 160 (3) ◽  
pp. 329-339 ◽  
Author(s):  
Stéphanie Buvelot ◽  
Sean Y. Tatsutani ◽  
Danielle Vermaak ◽  
Sue Biggins
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Genomic Stability ◽  
Time Lapse ◽  
Time Lapse Microscopy ◽  
Spindle Disassembly ◽  
Spindle Midzone ◽  
Spindle Microtubules

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

scholarly journals Cdc14-regulated midzone assembly controls anaphase B

2007 ◽  
Vol 177 (6) ◽  
pp. 981-993 ◽  
Author(s):  
Anton Khmelinskii ◽  
Clare Lawrence ◽  
Johanna Roostalu ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Cross Linking ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Spindle Midzone ◽  
A Cell ◽  
Spindle Elongation ◽  
And Function ◽  
Anaphase B

Spindle elongation in anaphase of mitosis is a cell cycle–regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase–Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase–Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase–Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase–Slk19 complex.

scholarly journals Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

2002 ◽  
Vol 13 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Brett K. Kaiser ◽  
Zachary A. Zimmerman ◽  
Harry Charbonneau ◽  
Peter K. Jackson
Keyword(s):  
Cell Cycle ◽  
Phosphatase Activity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Genomic Stability ◽  
Mitotic Exit ◽  
Protein Abundance ◽  
Cyclin Dependent Kinase ◽  
Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

scholarly journals Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

1995 ◽  
Vol 129 (6) ◽  
pp. 1617-1628 ◽  
Author(s):  
R M Golsteyn ◽  
K E Mundt ◽  
A M Fry ◽  
E A Nigg
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Subcellular Localization ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Laser Scanning Microscopy ◽  
Human Protein ◽  
Vitro Assay ◽  
Human Protein Kinase ◽  
Spindle Function

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

scholarly journals A Novel Mammalian, Mitotic Spindle–associated Kinase Is Related to Yeast and Fly Chromosome Segregation Regulators

1997 ◽  
Vol 138 (3) ◽  
pp. 643-656 ◽  
Author(s):  
Ganesan Gopalan ◽  
Clarence S.M. Chan ◽  
Peter J. Donovan
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Proximal Part ◽  
Yeast Cells ◽  
Mutant Form ◽  
Wild Type ◽  
Protein Levels ◽  
M Phase

We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases—Ipl1 and aurora, respectively—mutations in which cause disruption of chromosome segregation. We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation.

scholarly journals Astrin is required for the maintenance of sister chromatid cohesion and centrosome integrity

2007 ◽  
Vol 178 (3) ◽  
pp. 345-354 ◽  
Author(s):  
Kerstin H. Thein ◽  
Julia Kleylein-Sohn ◽  
Erich A. Nigg ◽  
Ulrike Gruneberg
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Cysteine Protease ◽  
Mitotic Spindle ◽  
Regulatory Network ◽  
Successful Completion ◽  
Sister Chromatids ◽  
Multipolar Spindles ◽  
Chromatid Cohesion

Faithful chromosome segregation in mitosis requires the formation of a bipolar mitotic spindle with stably attached chromosomes. Once all of the chromosomes are aligned, the connection between the sister chromatids is severed by the cysteine protease separase. Separase also promotes centriole disengagement at the end of mitosis. Temporal coordination of these two activities with the rest of the cell cycle is required for the successful completion of mitosis. In this study, we report that depletion of the microtubule and kinetochore protein astrin results in checkpoint-arrested cells with multipolar spindles and separated sister chromatids, which is consistent with untimely separase activation. Supporting this idea, astrin-depleted cells contain active separase, and separase depletion suppresses the premature sister chromatid separation and centriole disengagement in these cells. We suggest that astrin contributes to the regulatory network that controls separase activity.

scholarly journals Human Survivin Is a Kinetochore-Associated Passenger Protein

2000 ◽  
Vol 151 (7) ◽  
pp. 1575-1582 ◽  
Author(s):  
Dimitrios A. Skoufias ◽  
Cristiana Mollinari ◽  
Françoise B. Lacroix ◽  
Robert L. Margolis
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Mitotic Spindle ◽  
Deletion Mutant ◽  
Microtubule Assembly ◽  
Inhibitor Of Apoptosis ◽  
Nih3t3 Cells ◽  
Cell Cleavage ◽  
Spindle Midzone ◽  
Passenger Protein

Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Δ106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage.

scholarly journals Mechanical design principles of a mitotic spindle

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jonathan J Ward ◽  
Hélio Roque ◽  
Claude Antony ◽  
François Nédélec
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Structural Integrity ◽  
Mechanical Design ◽  
Structural Components ◽  
Cell Type ◽  
And Function ◽  
Anaphase B ◽  
The Relationship ◽  
Spindle Structure

An organised spindle is crucial to the fidelity of chromosome segregation, but the relationship between spindle structure and function is not well understood in any cell type. The anaphase B spindle in fission yeast has a slender morphology and must elongate against compressive forces. This ‘pushing’ mode of chromosome transport renders the spindle susceptible to breakage, as observed in cells with a variety of defects. Here we perform electron tomographic analyses of the spindle, which suggest that it organises a limited supply of structural components to increase its compressive strength. Structural integrity is maintained throughout the spindle's fourfold elongation by organising microtubules into a rigid transverse array, preserving correct microtubule number and dynamically rescaling microtubule length.

scholarly journals Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast

2005 ◽  
Vol 16 (4) ◽  
pp. 1756-1768 ◽  
Author(s):  
Isabelle Loïodice ◽  
Jayme Staub ◽  
Thanuja Gangi Setty ◽  
Nam-Phuong T. Nguyen ◽  
Anne Paoletti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Microtubule Organization ◽  
Cellular Processes ◽  
Spindle Midzone ◽  
Fluorescence Live Cell Imaging

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Δ mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

scholarly journals Separate to operate: control of centrosome positioning and separation

2014 ◽  
Vol 369 (1650) ◽  
pp. 20130461 ◽  
Author(s):  
Fikret G. Agircan ◽  
Elmar Schiebel ◽  
Balca R. Mardin
Keyword(s):  
Cell Cycle ◽  
Cell Motility ◽  
Cell Polarity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
De Novo Assembly ◽  
De Novo ◽  
Animal Cells ◽  
Pericentriolar Material ◽  
Golgi Organization

The centrosome is the main microtubule (MT)-organizing centre of animal cells. It consists of two centrioles and a multi-layered proteinaceous structure that surrounds the centrioles, the so-called pericentriolar material. Centrosomes promote de novo assembly of MTs and thus play important roles in Golgi organization, cell polarity, cell motility and the organization of the mitotic spindle. To execute these functions, centrosomes have to adopt particular cellular positions. Actin and MT networks and the association of the centrosomes to the nuclear envelope define the correct positioning of the centrosomes. Another important feature of centrosomes is the centrosomal linker that connects the two centrosomes. The centrosome linker assembles in late mitosis/G1 simultaneously with centriole disengagement and is dissolved before or at the beginning of mitosis. Linker dissolution is important for mitotic spindle formation, and its cell cycle timing has profound influences on the execution of mitosis and proficiency of chromosome segregation. In this review, we will focus on the mechanisms of centrosome positioning and separation, and describe their functions and mechanisms in the light of recent findings.

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