scholarly journals Microtubule Cytoskeleton Remodeling by Acentriolar Microtubule-organizing Centers at the Entry and Exit from Mitosis in Drosophila Somatic Cells

2009 ◽  
Vol 20 (11) ◽  
pp. 2796-2808 ◽  
Author(s):  
Sara Moutinho-Pereira ◽  
Alain Debec ◽  
Helder Maiato
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
De Novo ◽  
Animal Cell ◽  
Somatic Cells ◽  
Spindle Assembly ◽  
Entry And Exit ◽  
Down Regulation ◽  
Microtubule Organizing Centers ◽  
Cytoskeleton Remodeling

Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain γ-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle–regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.

scholarly journals Coordination of Cell Cycle Progression and Mitotic Spindle Assembly Involves Histone H3 Lysine 4 Methylation by Set1/COMPASS

Genetics ◽  
2016 ◽  
Vol 205 (1) ◽  
pp. 185-199 ◽  
Author(s):  
Traude H. Beilharz ◽  
Paul F. Harrison ◽  
Douglas Maya Miles ◽  
Michael Ming See ◽  
Uyen Minh Merry Le ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Histone H3 ◽  
Spindle Assembly ◽  
Cycle Progression ◽  
Mitotic Spindle Assembly

scholarly journals Aurora Kinase Inhibitor ZM447439 Blocks Chromosome-induced Spindle Assembly, the Completion of Chromosome Condensation, and the Establishment of the Spindle Integrity Checkpoint inXenopusEgg Extracts

2005 ◽  
Vol 16 (3) ◽  
pp. 1305-1318 ◽  
Author(s):  
Bedrick B. Gadea ◽  
Joan V. Ruderman
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Kinase Inhibitor ◽  
Spindle Assembly ◽  
Aurora Kinase ◽  
Chromosome Morphology ◽  
Chromosome Condensation ◽  
Embryonic Cell ◽  
Cell Cycle Regulators ◽  
Cell Cycles

The Aurora family kinases contribute to accurate progression through several mitotic events. ZM447439 (“ZM”), the first Aurora family kinase inhibitor to be developed and characterized, was previously found to interfere with the mitotic spindle integrity checkpoint and chromosome segregation. Here, we have used extracts of Xenopus eggs, which normally proceed through the early embryonic cell cycles in the absence of functional checkpoints, to distinguish between ZM's effects on the basic cell cycle machinery and its effects on checkpoints. ZM clearly had no effect on either the kinetics or amplitude in the oscillations of activity of several key cell cycle regulators. It did, however, have striking effects on chromosome morphology. In the presence of ZM, chromosome condensation began on schedule but then failed to progress properly; instead, the chromosomes underwent premature decondensation during mid-mitosis. ZM strongly interfered with mitotic spindle assembly by inhibiting the formation of microtubules that are nucleated/stabilized by chromatin. By contrast, ZM had little effect on the assembly of microtubules by centrosomes at the spindle poles. Finally, under conditions where the spindle integrity checkpoint was experimentally induced, ZM blocked the establishment, but not the maintenance, of the checkpoint, at a point upstream of the checkpoint protein Mad2. These results show that Aurora kinase activity is required to ensure the maintenance of condensed chromosomes, the generation of chromosome-induced spindle microtubules, and activation of the spindle integrity checkpoint.

scholarly journals The Spindle Checkpoint of Budding Yeast Depends on a Tight Complex between the Mad1 and Mad2 Proteins

1999 ◽  
Vol 10 (8) ◽  
pp. 2607-2618 ◽  
Author(s):  
Rey-Huei Chen ◽  
D. Michelle Brady ◽  
Dana Smith ◽  
Andrew W. Murray ◽  
Kevin G. Hardwick
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Mitotic Spindle ◽  
Mutational Analysis ◽  
Budding Yeast ◽  
Gel Filtration ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Checkpoint Proteins ◽  
Sds Page

The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle. When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.

scholarly journals ProTAME Arrest in Mammalian Oocytes and Embryos Does Not Require Spindle Assembly Checkpoint Activity

2019 ◽  
Vol 20 (18) ◽  
pp. 4537 ◽  
Author(s):  
Lenka Radonova ◽  
Tereza Svobodova ◽  
Michal Skultety ◽  
Ondrej Mrkva ◽  
Lenka Libichova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Spindle Assembly Checkpoint ◽  
Somatic Cells ◽  
Spindle Assembly ◽  
Control Mechanisms ◽  
Anaphase Promoting Complex ◽  
Cycle Arrest ◽  
Molecule Inhibitor ◽  
Early Embryos

In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.

scholarly journals MAP Kinase Is Required for the Spindle Assembly Checkpoint but Is Dispensable for the Normal M Phase Entry and Exit in Xenopus Egg Cell Cycle Extracts

1997 ◽  
Vol 136 (5) ◽  
pp. 1091-1097 ◽  
Author(s):  
Katsuya Takenaka ◽  
Yukiko Gotoh ◽  
Eisuke Nishida
Keyword(s):  
Cell Cycle ◽  
Map Kinase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Egg Cell ◽  
Entry And Exit ◽  
M Phase ◽  
Sperm Nuclei ◽  
Phase Entry

In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti–Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase–depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase– specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase–depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase–depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.

scholarly journals Human Ovarian Granulosa Cells Isolated during an IVF Procedure Exhibit Differential Expression of Genes Regulating Cell Division and Mitotic Spindle Formation

2019 ◽  
Vol 8 (12) ◽  
pp. 2026
Author(s):  
Maciej Brązert ◽  
Wiesława Kranc ◽  
Błażej Chermuła ◽  
Katarzyna Kowalska ◽  
Maurycy Jankowski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Granulosa Cells ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
In Vitro Cultivation ◽  
Cellular Ultrastructure ◽  
Mitotic Spindle Assembly

Granulosa cells (GCs) are a population of somatic cells whose role after ovulation is progesterone production. GCs were collected from patients undergoing controlled ovarian stimulation during an in vitro fertilization procedure, and they were maintained for 1, 7, 15, and 30 days of in vitro primary culture before collection for further gene expression analysis. A study of genes involved in the biological processes of interest was carried out using expression microarrays. To validate the obtained results, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) was performed. The direction of changes in the expression of the selected genes was confirmed in most of the examples. Six ontological groups (“cell cycle arrest”, “cell cycle process”, “mitotic spindle organization”, “mitotic spindle assembly checkpoint”, “mitotic spindle assembly”, and “mitotic spindle checkpoint”) were analyzed in this study. The results of the microarrays obtained by us allowed us to identify two groups of genes whose expressions were the most upregulated (FAM64A, ANLN, TOP2A, CTGF, CEP55, BIRC5, PRC1, DLGAP5, GAS6, and NDRG1) and the most downregulated (EREG, PID1, INHA, RHOU, CXCL8, SEPT6, EPGN, RDX, WNT5A, and EZH2) during the culture. The cellular ultrastructure showed the presence of structures characteristic of mitotic cell division: a centrosome surrounded by a pericentric matrix, a microtubule system, and a mitotic spindle connected to chromosomes. The main goal of the study was to identify the genes involved in mitotic division and to identify the cellular ultrastructure of GCs in a long-term in vitro culture. All of the genes in these groups were subjected to downstream analysis, and their function and relation to the ovarian environment are discussed. The obtained results suggest that long-term in vitro cultivation of GCs may lead to their differentiation toward another cell type, including cells with cancer-like characteristics.

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.

scholarly journals The Xenopus TACC Homologue, Maskin, Functions in Mitotic Spindle Assembly

2005 ◽  
Vol 16 (6) ◽  
pp. 2836-2847 ◽  
Author(s):  
Lori L. O'Brien ◽  
Alison J. Albee ◽  
Lingling Liu ◽  
Wei Tao ◽  
Pawel Dobrzyn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Coiled Coil ◽  
Spindle Assembly ◽  
Cyclin B1 ◽  
Time Points ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Mitotic Spindle Assembly ◽  
Xenopus Egg Extracts

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a ∼200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to “rescue” experiments. Defects in the “early” step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the “late” step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.

scholarly journals Functional Overlap of Microtubule Assembly Factors in Chromatin-Promoted Spindle Assembly

2009 ◽  
Vol 20 (11) ◽  
pp. 2766-2773 ◽  
Author(s):  
Aaron C. Groen ◽  
Thomas J. Maresca ◽  
Jesse C. Gatlin ◽  
Edward D. Salmon ◽  
Timothy J. Mitchison
Keyword(s):  
Mitotic Spindle ◽  
Animal Cell ◽  
Spindle Assembly ◽  
Functional Redundancy ◽  
The Other ◽  
Microtubule Assembly ◽  
Microtubule Polymerization ◽  
Egg Extract ◽  
Assembly Factors ◽  
Mitotic Spindle Assembly

Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, γ-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either γ-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.

scholarly journals Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle.

1990 ◽  
Vol 110 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
D L Gard ◽  
S Hafezi ◽  
T Zhang ◽  
S J Doxsey
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Spindle Assembly ◽  
Centrosome Duplication ◽  
Microtubule Organizing Centers ◽  
Xenopus Embryos ◽  
M Phase ◽  
Pericentriolar Material ◽  
Histone Kinase

Cycloheximide (500 micrograms/ml) rapidly arrests cleavage, spindle assembly, and cycles of an M-phase-specific histone kinase in early Xenopus blastulae. 2 h after cycloheximide addition, most cells contained two microtubule asters radiating from perinuclear microtubule organizing centers (MTOCs). In contrast, blastomeres treated with cycloheximide for longer periods (3-6 h) contained numerous microtubule asters and MTOCs. Immunofluorescence with an anticentrosome serum and EM demonstrated that the MTOCs in cycloheximide-treated cells were typical centrosomes, containing centrioles and pericentriolar material. We conclude that centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. In addition, these observations suggest that Xenopus embryos contain sufficient material to assemble 1,000-2,000 centrosomes in the absence of normal protein synthesis.

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