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 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 Defects in nuclear pore assembly lead to activation of an Aurora B–mediated abscission checkpoint

2010 ◽  
Vol 191 (5) ◽  
pp. 923-931 ◽  
Author(s):  
Douglas R. Mackay ◽  
Masaki Makise ◽  
Katharine S. Ullman
Keyword(s):  
Small Interfering Rna ◽  
Nuclear Pore ◽  
Aurora B ◽  
Nuclear Pore Complexes ◽  
Daughter Cells ◽  
Nuclear Environment ◽  
Genomic Regulation ◽  
Pore Complexes ◽  
Interfering Rna ◽  
Nuclear Pore Assembly

Correct assembly of nuclear pore complexes (NPCs), which directly and indirectly control nuclear environment and architecture, is vital to genomic regulation. We previously found that nucleoporin 153 (Nup153) is required for timely progression through late mitosis. In this study, we report that disruption of Nup153 function by either small interfering RNA–mediated depletion or expression of a dominant-interfering Nup153 fragment results in dramatic mistargeting of the pore basket components Tpr and Nup50 in midbody-stage cells. We find a concomitant appearance of aberrantly localized active Aurora B and an Aurora B–dependent delay in abscission. Depletion of Nup50 is also sufficient to increase the number of midbody-stage cells and, likewise, triggers distinctive mislocalization of Aurora B. Together, our results suggest that defects in nuclear pore assembly, and specifically the basket structure, at this time of the cell cycle activate an Aurora B–mediated abscission checkpoint, thereby ensuring that daughter cells are generated only when fully formed NPCs are present.

scholarly journals The Abscission Checkpoint: A Guardian of Chromosomal Stability

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3350
Author(s):  
Eleni Petsalaki ◽  
George Zachos
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Nuclear Pore ◽  
Aurora B ◽  
Intercellular Bridge ◽  
Endosomal Sorting ◽  
Daughter Cells ◽  
Chromosomal Passenger ◽  
Dividing Cells ◽  
Chromatin Bridges

The abscission checkpoint contributes to the fidelity of chromosome segregation by delaying completion of cytokinesis (abscission) when there is chromatin lagging in the intercellular bridge between dividing cells. Although additional triggers of an abscission checkpoint-delay have been described, including nuclear pore defects, replication stress or high intercellular bridge tension, this review will focus only on chromatin bridges. In the presence of such abnormal chromosomal tethers in mammalian cells, the abscission checkpoint requires proper localization and optimal kinase activity of the Chromosomal Passenger Complex (CPC)-catalytic subunit Aurora B at the midbody and culminates in the inhibition of Endosomal Sorting Complex Required for Transport-III (ESCRT-III) components at the abscission site to delay the final cut. Furthermore, cells with an active checkpoint stabilize the narrow cytoplasmic canal that connects the two daughter cells until the chromatin bridges are resolved. Unsuccessful resolution of chromatin bridges in checkpoint-deficient cells or in cells with unstable intercellular canals can lead to chromatin bridge breakage or tetraploidization by regression of the cleavage furrow. In turn, these outcomes can lead to accumulation of DNA damage, chromothripsis, generation of hypermutation clusters and chromosomal instability, which are associated with cancer formation or progression. Recently, many important questions regarding the mechanisms of the abscission checkpoint have been investigated, such as how the presence of chromatin bridges is signaled to the CPC, how Aurora B localization and kinase activity is regulated in late midbodies, the signaling pathways by which Aurora B implements the abscission delay, and how the actin cytoskeleton is remodeled to stabilize intercellular canals with DNA bridges. Here, we review recent progress toward understanding the mechanisms of the abscission checkpoint and its role in guarding genome integrity at the chromosome level, and consider its potential implications for cancer therapy.

scholarly journals Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells

2020 ◽  
Vol 94 (10) ◽  
pp. 3553-3561
Author(s):  
Hauke Reimann ◽  
Helga Stopper ◽  
Henning Hintzsche
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cellular Structures ◽  
Death Rates ◽  
Daughter Cells

Abstract Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.

scholarly journals Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

2014 ◽  
Vol 58 (4) ◽  
pp. 2150-2155 ◽  
Author(s):  
Carmen M. Martín-Navarro ◽  
Jacob Lorenzo-Morales ◽  
Atteneri López-Arencibia ◽  
María Reyes-Batlle ◽  
José E. Piñero ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Drug Target ◽  
Cyst Formation ◽  
Potential Drug Target ◽  
Potential Therapeutic Target ◽  
Content Type ◽  
Daughter Cells ◽  
Sirna Silencing ◽  
And Control ◽  
Interfering Rna

ABSTRACTMembers of the genusAcanthamoebaare facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against theAcanthamoebamyosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as “traction-mediated cytokinesis”. We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited forAcanthamoebatherapy.

scholarly journals Live cell imaging of the hyperthermophilic archaeon Sulfolobus acidocaldarius identifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

2020 ◽  
Author(s):  
Andre Arashiro Pulschen ◽  
Delyan R. Mutavchiev ◽  
Kim Nadine Sebastian ◽  
Jacques Roubinet ◽  
Marc Roubinet ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Halophilic Archaea ◽  
Live Cell ◽  
Tight Coupling ◽  
Dna Compaction ◽  
Cellular Processes ◽  
Daughter Cells

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

scholarly journals Forced apart: a microtubule-based mechanism for equidistant positioning of multiple nuclei in single cells

2021 ◽  
Vol 136 (8) ◽  
Author(s):  
Juliane Teapal ◽  
Leander J. Schuitman ◽  
Bela M. Mulder ◽  
Marcel E. Janson
Keyword(s):  
Molecular Motors ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Single Cells ◽  
Control Cell ◽  
Stochastic Simulations ◽  
Daughter Cells ◽  
Multiple Copies ◽  
Microtubule Growth ◽  
Regular Patterns

AbstractCells can position multiple copies of components like carboxysomes, nucleoids, and nuclei at regular intervals. By controlling positions, cells, for example, ensure equal partitioning of organelles over daughter cells and, in the case of nuclei, control cell sizes during cellularization. Mechanisms that generate regular patterns are as yet poorly understood. We used fission yeast cell cycle mutants to investigate the dispersion of multiple nuclei by microtubule-generated forces in single cells. After removing internuclear attractive forces by microtubule-based molecular motors, we observed the establishment of regular patterns of nuclei. Based on live-cell imaging, we hypothesized that microtubule growth within internuclear spaces pushes neighbouring nuclei apart. In the proposed mechanism, which was validated by stochastic simulations, the repulsive force weakens with increasing separation because stochastic shortening events limit the extent over which microtubules generate forces. Our results, therefore, demonstrate how cells can exploit the dynamics of microtubule growth for the equidistant positioning of organelles.

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.

scholarly journals Annexin 11 is required for midbody formation and completion of the terminal phase of cytokinesis

2004 ◽  
Vol 165 (6) ◽  
pp. 813-822 ◽  
Author(s):  
Alejandra Tomas ◽  
Clare Futter ◽  
Stephen E. Moss
Keyword(s):  
Cell Cycle Progression ◽  
Time Lapse ◽  
Terminal Phase ◽  
Aurora B ◽  
Cycle Progression ◽  
Daughter Cells ◽  
Time Lapse Microscopy ◽  
Spindle Poles ◽  
Spindle Midzone ◽  
Late Telophase

Annexins are Ca2+-binding, membrane-fusogenic proteins with diverse but poorly understood functions. Here, we show that during cell cycle progression annexin 11 translocates from the nucleus to the spindle poles in metaphase and to the spindle midzone in anaphase. Annexin 11 is recruited to the midbody in late telophase, where it forms part of the detergent-resistant matrix that also contains CHO1. To investigate the significance of these observations, we used RNA interference to deplete cells of annexin 11. A combination of confocal and video time-lapse microscopy revealed that cells lacking annexin 11 fail to establish a functional midbody. Instead, daughter cells remain connected by intercellular bridges that contain bundled microtubules and cytoplasmic organelles but exclude normal midbody components such as MKLP1 and Aurora B. Annexin 11–depleted cells failed to complete cytokinesis and died by apoptosis. These findings demonstrate an essential role for annexin 11 in the terminal phase of cytokinesis.

scholarly journals Aurora A promotes chromosome congression by activating the condensin-dependent pool of KIF4A

2019 ◽  
Vol 219 (2) ◽  
Author(s):  
Elena Poser ◽  
Renaud Caous ◽  
Ulrike Gruneberg ◽  
Francis A. Barr
Keyword(s):  
Metaphase Plate ◽  
Aurora Kinase ◽  
Chromosome Condensation ◽  
Aurora B ◽  
Aurora A ◽  
Specific Point ◽  
Aurora Kinases ◽  
Central Spindle ◽  
Spindle Poles ◽  
Chromosome Congression

Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle organization, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1 or are deficient for Aurora kinase regulation. By analyzing these mutants, we show that Aurora A phosphorylates the condensin I–dependent pool of KIF4A and thus actively promotes chromosome congression from the spindle poles to the metaphase plate.

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