scholarly journals Ablation of PRC1 by Small Interfering RNA Demonstrates that Cytokinetic Abscission Requires a Central Spindle Bundle in Mammalian Cells, whereas Completion of Furrowing Does Not

2005 ◽  
Vol 16 (3) ◽  
pp. 1043-1055 ◽  
Author(s):  
Cristiana Mollinari ◽  
Jean-Philippe Kleman ◽  
Yasmina Saoudi ◽  
Sandra A. Jablonski ◽  
Julien Perard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Cell Cortex ◽  
Central Spindle ◽  
Spatial Regulation ◽  
Daughter Cells ◽  
Cell Cleavage ◽  
Temporal And Spatial ◽  
Interfering Rna ◽  
Passenger Proteins

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

The effects of diazepam on mitosis and the microtubule cytoskeleton. I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta

1994 ◽  
Vol 107 (9) ◽  
pp. 2643-2651
Author(s):  
T.P. Spurck ◽  
J.D. Pickett-Heaps
Keyword(s):  
Time Lapse ◽  
Detectable Effect ◽  
Metaphase Chromosomes ◽  
Central Spindle ◽  
Cell Cleavage ◽  
Spindle Poles ◽  
Transmission Electron ◽  
Live Diatoms ◽  
Interphase Cells ◽  
Chromosome Motion

The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy. DZP treatment (250 microM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle. Chromosomes remaining attached to one pole continued to display slight prometaphase oscillations; however, this activity was never observed in metaphase spindles. Following removal of DZP, some chromosomes still bipolarly attached, immediately released elastically from one pole. Within the first 2 minutes of recovery, all chromosomes recommenced spindle attachment, exhibiting normal prometaphase oscillations and proceeded through mitosis. DZP treatment during anaphase had no detectable effect on chromosome motion or cell cleavage. These results suggest that DZP acts as an anti-MT agent, selectively affecting polar MTs at prophase, prometaphase and metaphase, and thereby weakening kinetochore connection to the poles. From these and other results (unpublished), its mode of action is different to that of most anti-MT agents.

scholarly journals Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

2008 ◽  
Vol 181 (4) ◽  
pp. 595-603 ◽  
Author(s):  
Thomas M. Durcan ◽  
Elizabeth S. Halpin ◽  
Trisha Rao ◽  
Nicholas S. Collins ◽  
Emily K. Tribble ◽  
...  
Keyword(s):  
Cell Formation ◽  
Binucleate Cell ◽  
Aurora B ◽  
Microtubule Organization ◽  
Spindle Microtubule ◽  
Central Spindle ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Spindle Midzone ◽  
Interfering Rna

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

scholarly journals Kinesin-like Protein CHO1 Is Required for the Formation of Midbody Matrix and the Completion of Cytokinesis in Mammalian Cells

2002 ◽  
Vol 13 (6) ◽  
pp. 1832-1845 ◽  
Author(s):  
Jurgita Matuliene ◽  
Ryoko Kuriyama
Keyword(s):  
Binding Site ◽  
Mammalian Cells ◽  
Cytoplasmic Bridge ◽  
Central Spindle ◽  
Atp Binding Site ◽  
Daughter Cells ◽  
Dividing Cells ◽  
Spindle Midzone ◽  
Segregation Of Chromosomes

CHO1 is a mammalian kinesin-like motor protein of the MKLP1 subfamily. It associates with the spindle midzone during anaphase and concentrates to a midbody matrix during cytokinesis. CHO1 was originally implicated in karyokinesis, but the invertebrate homologues of CHO1 were shown to function in the midzone formation and cytokinesis. To analyze the role of the protein in mammalian cells, we mutated the ATP-binding site of CHO1 and expressed it in CHO cells. Mutant protein (CHO1F′) was able to interact with microtubules via ATP-independent microtubule-binding site(s) but failed to accumulate at the midline of the central spindle and affected the localization of endogenous CHO1. Although the segregation of chromosomes, the bundling of midzone microtubules, and the initiation of cytokinesis proceeded normally in CHO1F′-expressing cells, the completion of cytokinesis was inhibited. Daughter cells were frequently entering interphase while connected by a microtubule-containing cytoplasmic bridge from which the dense midbody matrix was missing. Depletion of endogenous CHO1 via RNA-mediated interference also affected the formation of midbody matrix in dividing cells, caused the disorganization of midzone microtubules, and resulted in abortive cytokinesis. Thus, CHO1 may not be required for karyokinesis, but it is essential for the proper midzone/midbody formation and cytokinesis in mammalian cells.

scholarly journals Mechanisms regulating targeting of recycling endosomes to the cleavage furrow during cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 391-394 ◽  
Author(s):  
Glenn C. Simon ◽  
Rytis Prekeris
Keyword(s):  
Small Interfering Rna ◽  
Cleavage Furrow ◽  
Gtpase Activating Protein ◽  
Successful Completion ◽  
Interacting Protein ◽  
Recycling Endosomes ◽  
Spatial Regulation ◽  
Dividing Cells ◽  
Temporal And Spatial ◽  
Interfering Rna

Recently, recycling endosomes have emerged as a key components required for the successful completion of cytokinesis. Furthermore, FIP3 (family of Rab11-interacting protein 3), a Rab11 GTPase-binding protein, has been implicated in targeting the recycling endosomes to the midbody of dividing cells. Previously, we have shown that FIP3/Rab11-containing endosomes associate with centrosomes until anaphase, at which time they translocate to the cleavage furrow. At telophase, FIP3/Rab11-containing endosomes move from the furrow into the midbody, and this step is required for abscission. While several other proteins were implicated in regulating FIP3 targeting to the cleavage furrow, the mechanisms regulating the dynamics of FIP3-containing endosomes during mitosis have not been defined. To identify the factors regulating FIP3 targeting to the furrow, we used a combination of siRNA (small interfering RNA) screens and proteomic analysis to identify Cyk-4/MgcRacGAP (GTPase-activating protein) and kinesin I as FIP3-binding proteins. Furthermore, kinesin I mediates the transport of FIP3-containing endosomes to the cleavage furrow. Once in the furrow, FIP3 binds to Cyk-4 as part of centralspindlin complex and accumulates at the midbody. Finally, we demonstrated that ECT2 regulates FIP3 association with the centralspindlin complex. Thus we propose that kinesin I, in concert with centralspindlin complex, plays a role in temporal and spatial regulation of endosome transport to the cleavage furrow during cytokinesis.

Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling

Blood ◽  
2008 ◽  
Vol 112 (8) ◽  
pp. 3164-3174 ◽  
Author(s):  
Larissa Lordier ◽  
Abdelali Jalil ◽  
Fréderic Aurade ◽  
Fréderic Larbret ◽  
Jerôme Larghero ◽  
...  
Keyword(s):  
Ploidy Level ◽  
Time Lapse ◽  
Polyploid Cell ◽  
Nonmuscle Myosin ◽  
Contractile Ring ◽  
Central Spindle ◽  
Daughter Cells ◽  
Late Failure ◽  
Pathway Activation ◽  
Spindle Elongation

Abstract Megakaryocyte (MK) is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered to be an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompanied by a backward movement of the 2 daughter cells. No abnormality was observed in the central spindle of endomitotic MKs. A furrow formation was present, but the contractile ring was abnormal because accumulation of nonmuscle myosin IIA was lacking. In addition, a defect in cell elongation was observed in dipolar endomitotic MKs during telophase. RhoA and F-actin were partially concentrated at the site of furrowing. Inhibition of the Rho/Rock pathway caused the disappearance of F-actin at midzone and increased MK ploidy level. This inhibition was associated with a more pronounced defect in furrow formation as well as in spindle elongation. Our results suggest that the late failure of cytokinesis responsible for the endomitotic process is related to a partial defect in the Rho/Rock pathway activation.

scholarly journals Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission

2013 ◽  
Vol 24 (18) ◽  
pp. 2785-2794 ◽  
Author(s):  
Matthew K. Martz ◽  
Elda Grabocka ◽  
Neil Beeharry ◽  
Timothy J. Yen ◽  
Philip B. Wedegaertner
Keyword(s):  
Small Interfering Rna ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Aurora B ◽  
Intercellular Bridge ◽  
Concerted Effort ◽  
Central Spindle ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Interfering Rna

Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA–resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission.

scholarly journals Failure of cell cleavage induces senescence in tetraploid primary cells

2014 ◽  
Vol 25 (20) ◽  
pp. 3105-3118 ◽  
Author(s):  
Andreas Panopoulos ◽  
Cristina Pacios-Bras ◽  
Justin Choi ◽  
Mythili Yenjerla ◽  
Mark A. Sussman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mammalian Cells ◽  
T Antigen ◽  
Primary Cells ◽  
Ki 67 ◽  
Tetraploid Cell ◽  
Cell Cleavage ◽  
Human Foreskin Fibroblasts ◽  
Interfering Rna

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen–transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

scholarly journals Microtubules are the only structural constituent of the spindle apparatus required for induction of cell cleavage

2003 ◽  
Vol 162 (3) ◽  
pp. 383-390 ◽  
Author(s):  
G. Bradley Alsop ◽  
Dahong Zhang
Keyword(s):  
Cell Types ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Central Spindle ◽  
Structural Constituent ◽  
Cell Cleavage ◽  
Spindle Apparatus

Structural constituents of the spindle apparatus essential for cleavage induction remain undefined. Findings from various cell types using different approaches suggest the importance of all structural constituents, including asters, the central spindle, and chromosomes. In this study, we systematically dissected the role of each constituent in cleavage induction in grasshopper spermatocytes and narrowed the essential one down to bundled microtubules. Using micromanipulation, we produced “cells” containing only asters, a truncated central spindle lacking both asters and chromosomes, or microtubules alone. We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present. Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in “metaphase,” disassembling in “anaphase,” and bundling into arrays in “telophase.” Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress. We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Sign in / Sign up

Export Citation Format

Share Document