scholarly journals Taxol-stabilized Microtubules Can Position the Cytokinetic Furrow in Mammalian Cells

2005 ◽  
Vol 16 (9) ◽  
pp. 4423-4436 ◽  
Author(s):  
Katie B. Shannon ◽  
Julie C. Canman ◽  
C. Ben Moree ◽  
Jennifer S. Tirnauer ◽  
E. D. Salmon
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Myosin Ii ◽  
Spindle Checkpoint ◽  
Microtubule Dynamics ◽  
Cell Cortex ◽  
Contractile Ring ◽  
Spindle Microtubule ◽  
Anaphase Onset ◽  
Centromere Protein

How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation.

scholarly journals Adhesion-dependent and Contractile Ring-independent Equatorial Furrowing during Cytokinesis in Mammalian Cells

2005 ◽  
Vol 16 (8) ◽  
pp. 3865-3872 ◽  
Author(s):  
Masamitsu Kanada ◽  
Akira Nagasaki ◽  
Taro Q.P. Uyeda
Keyword(s):  
Mammalian Cells ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Cellular Slime Mold ◽  
Animal Cells ◽  
Contractile Ring ◽  
Normal Rat ◽  
Cellular Slime ◽  
Coated Surfaces

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 μM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 μM), which inhibits Rho-kinase, was similar to 30 μM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 μM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 μM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.

scholarly journals Polar relaxation by dynein-mediated removal of cortical myosin II

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Bernardo Chapa-y-Lazo ◽  
Motonari Hamanaka ◽  
Alexander Wray ◽  
Mohan K. Balasubramanian ◽  
Masanori Mishima
Keyword(s):  
Recent Work ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Myosin Ii ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
C Elegans ◽  
Polar Cell

Nearly six decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction. While this mechanism has remained controversial, recent work has provided evidence for polar relaxation by astral microtubules, although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction, whereas suppression of dynein activity delayed furrowing. We show that dynein-mediated removal of myosin II from the polar cortexes triggers a bidirectional cortical flow toward the cell equator, which induces the assembly of the actomyosin contractile ring. These results provide a molecular mechanism for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis.

scholarly journals Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

1998 ◽  
Vol 9 (8) ◽  
pp. 2173-2184 ◽  
Author(s):  
Sally P. Wheatley ◽  
Christopher B. O’Connell ◽  
Yu-li Wang
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Topoisomerase Ii ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Daughter Cells ◽  
Irregular Shapes ◽  
Anaphase Onset ◽  
Cultured Epithelial Cells ◽  
Normal Rat

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinodermembryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

scholarly journals The Septation Apparatus, an Autonomous System in Budding Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2747-2759 ◽  
Author(s):  
Dong-Hyun Roh ◽  
Blair Bowers ◽  
Martin Schmidt ◽  
Enrico Cabib
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Nuclear Division ◽  
Fluorescent Proteins ◽  
Budding Yeast ◽  
Cell Cortex ◽  
Nonpermissive Temperature ◽  
Contractile Ring ◽  
Primary Septum ◽  
Endocytic Vesicles

Actomyosin ring contraction and chitin primary septum deposition are interdependent processes in cell division of budding yeast. By fusing Myo1p, as representative of the contractile ring, and Chs2p for the primary septum, to different fluorescent proteins we show herein that the two processes proceed essentially at the same location and simultaneously. Chs2p differs from Myo1p in that it reflects the changes in shape of the plasma membrane to which it is attached and in that it is packed after its action into visible endocytic vesicles for its disposal. To ascertain whether this highly coordinated system could function independently of other cell cycle events, we reexamined the septum-like structures made by the septin mutant cdc3 at various sites on the cell cortex at the nonpermissive temperature. With the fluorescent fusion proteins mentioned above, we observed that incdc3 at 37°C both Myo1p and Chs2p colocalize at different spots of the cell cortex. A contraction of the Myo1p patch could also be detected, as well as that of a Chs2p patch, with subsequent appearance of vesicles. Furthermore, the septin Cdc12p, fused with yellow or cyan fluorescent protein, also colocalized with Myo1p and Chs2p at the aberrant locations. The formation of delocalized septa did not require nuclear division. We conclude that the septation apparatus, composed of septins, contractile ring, and the chitin synthase II system, can function at ectopic locations autonomously and independently of cell division, and that it can recruit the other elements necessary for the formation of secondary septa.

scholarly journals Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase

2005 ◽  
Vol 168 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Nasser M. Rusan ◽  
Patricia Wadsworth
Keyword(s):  
Chromosome Segregation ◽  
Equatorial Region ◽  
Cyclin B ◽  
Cell Cortex ◽  
Contractile Ring ◽  
Division Plane ◽  
Anaphase Onset ◽  
Photoactivatable Gfp ◽  
Spinning Disc ◽  
Stimulation Of

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.

scholarly journals Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

2011 ◽  
Vol 195 (1) ◽  
pp. 99-112 ◽  
Author(s):  
Chantal Roubinet ◽  
Barbara Decelle ◽  
Gaëtan Chicanne ◽  
Jonas F. Dorn ◽  
Bernard Payrastre ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Elongation ◽  
Cell Shape ◽  
Mitotic Cell ◽  
Molecular Networks ◽  
Cell Cortex ◽  
Anaphase Onset ◽  
Shape Transformations ◽  
Intracellular Pressure ◽  
Early Mitosis

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. In this paper, we identify two novel networks that collectively control the dynamic reorganization of the mitotic cortex. We demonstrate that Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. We find that the Pp1-87B phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division.

scholarly journals Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anan Chen ◽  
Luisa Ulloa Severino ◽  
Thomas C. Panagiotou ◽  
Trevor F. Moraes ◽  
Darren A. Yuen ◽  
...  
Keyword(s):  
Cell Division ◽  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Actin Network ◽  
Contractile Ring ◽  
Actin Networks ◽  
Membrane Blebbing ◽  
Actin Isoform

AbstractDuring cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a β-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the β- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies β-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a β-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells

1998 ◽  
Vol 111 (9) ◽  
pp. 1227-1240 ◽  
Author(s):  
R. Neujahr ◽  
R. Albrecht ◽  
J. Kohler ◽  
M. Matzner ◽  
J.M. Schwartz ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Myosin Ii ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Mitotic Apparatus ◽  
Alpha Tubulin ◽  
Multinucleate Cells ◽  
Green Fluorescent

To study centrosome motility and the interaction of microtubules with the cell cortex in mitotic, post-mitotic and interphase cells, (alpha)-tubulin was tagged in Dictyostelium discoideum with green fluorescent protein. Multinucleate cells formed by myosin II-null mutants proved to be especially suited for the analysis of the control of cleavage furrow formation by the microtubule system. After docking of the mitotic apparatus onto the cell cortex during anaphase, the cell surface is activated to form ruffles on top of the asters of microtubules that emanate from the centrosomes. Cleavage furrows are initiated at spaces between the asters independently of the positions of spindles. Once initiated, the furrows expand as deep folds without a continued connection to the microtubule system. Occurrence of unilateral furrows indicates that a closed contractile ring is dispensable for cytokinesis in Dictyostelium. The progression of cytokinesis in the multinucleate cells underlines the importance of proteins other than myosin II in specifying a cleavage furrow. The analysis of centrosome motility suggests a major role for a minus-end directed motor protein, probably cytoplasmic dynein, in applying traction forces on guiding microtubules that connect the centrosome with the cell cortex.

scholarly journals Rap1-dependent pathways coordinate cytokinesis in Dictyostelium

2014 ◽  
Vol 25 (25) ◽  
pp. 4195-4204 ◽  
Author(s):  
Katarzyna Plak ◽  
Ineke Keizer-Gunnink ◽  
Peter J. M. van Haastert ◽  
Arjan Kortholt
Keyword(s):  
Cell Division ◽  
Cell Cortex ◽  
Adherent Cells ◽  
Contractile Ring ◽  
Daughter Cells ◽  
Small G Protein ◽  
Rap1 Activation ◽  
Actomyosin Cytoskeleton ◽  
Mutant Cells ◽  
Severe Growth

Cytokinesis is the final step of mitosis when a mother cell is separated into two daughter cells. Major cytoskeletal changes are essential for cytokinesis; it is, however, not well understood how the microtubules and actomyosin cytoskeleton are exactly regulated in time and space. In this paper, we show that during the early stages of cytokinesis, in rounded-up Dictyostelium discoideum cells, the small G-protein Rap1 is activated uniformly at the cell cortex. When cells begin to elongate, active Rap1 becomes restricted from the furrow region, where the myosin contractile ring is subsequently formed. In the final stages of cytokinesis, active Rap1 is only present at the cell poles. Mutant cells with decreased Rap1 activation at the poles showed strongly decreased growth rates. Hyperactivation of Rap1 results in severe growth delays and defective spindle formation in adherent cells and cell death in suspension. Furthermore, Rap mutants show aberrant regulation of the actomyosin cytoskeleton, resulting in extended furrow ingression times and asymmetrical cell division. We propose that Rap1 drives cytokinesis progression by coordinating the three major cytoskeletal components: microtubules, actin, and myosin II. Importantly, mutated forms of Rap also affect cytokinesis in other organisms, suggesting a conserved role for Rap in cell division.

scholarly journals An Essential Role for a Membrane Lipid in Cytokinesis

2000 ◽  
Vol 149 (6) ◽  
pp. 1215-1224 ◽  
Author(s):  
Kazuo Emoto ◽  
Masato Umeda
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Mutant Cell ◽  
Myosin Ii ◽  
Membrane Lipid ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Cytoplasmic Bridge ◽  
Daughter Cells

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is mainly localized in the inner leaflet of the plasma membrane. We previously demonstrated that PE was exposed on the cell surface of the cleavage furrow during cytokinesis. Immobilization of cell surface PE by a PE-binding peptide inhibited disassembly of the contractile ring components, including myosin II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells. This blockade of contractile ring disassembly was reversed by removal of the surface-bound peptide, suggesting that the PE exposure plays a crucial role in cytokinesis. To further examine the role of PE in cytokinesis, we established a mutant cell line with a specific decrease in the cellular PE level. On the culture condition in which the cell surface PE level was significantly reduced, the mutant ceased cell growth in cytokinesis, and the contractile ring remained in the cleavage furrow. Addition of PE or ethanolamine, a precursor of PE synthesis, restored the cell surface PE on the cleavage furrow and normal cytokinesis. These findings provide the first evidence that PE is required for completion of cytokinesis in mammalian cells, and suggest that redistribution of PE on the cleavage furrow may contribute to regulation of contractile ring disassembly.

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