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The small GTP-binding protein rho regulates cortical activities in cultured cells during division.

O'Connell CB, Wheatley SP, Ahmed S, Wang YL - J. Cell Biol. (1999)

Bottom Line: All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully.The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis.Our results indicate that Rho does not simply activate actin-myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

ABSTRACT
We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin-myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

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Time lapse images of control NRK cells and cells microinjected with C3 transferase during prometaphase or metaphase. Approximate time in minutes relative to anaphase onset is located in the upper right corner. Control cells (a–e) developed a narrow cleavage furrow along the equatorial plane (c–e). Cells microinjected with C3 underwent immediate spreading in all directions (f and g, k and  l; f and k were recorded immediately after microinjection). Ingressions were poorly regulated and initiated at several points around the  cell after anaphase onset. An elongated cleavage furrow (j, arrowhead) formed as a result of the broad ingression that developed between separating chromosomes. Another cell initiated an ingression before anaphase onset (l), but did not complete cleavage between  the sets of chromosomes. Ectopic furrows were common in C3-injected cells (i and j and n and o, arrows). Bars, 20 μm.
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Figure 1: Time lapse images of control NRK cells and cells microinjected with C3 transferase during prometaphase or metaphase. Approximate time in minutes relative to anaphase onset is located in the upper right corner. Control cells (a–e) developed a narrow cleavage furrow along the equatorial plane (c–e). Cells microinjected with C3 underwent immediate spreading in all directions (f and g, k and l; f and k were recorded immediately after microinjection). Ingressions were poorly regulated and initiated at several points around the cell after anaphase onset. An elongated cleavage furrow (j, arrowhead) formed as a result of the broad ingression that developed between separating chromosomes. Another cell initiated an ingression before anaphase onset (l), but did not complete cleavage between the sets of chromosomes. Ectopic furrows were common in C3-injected cells (i and j and n and o, arrows). Bars, 20 μm.

Mentions: Both interphase and mitotic NRK cells within a monolayer showed a transient increase in surface area upon microinjection with C3 (Fig. 1, f and g, k and l). Mitotic cells then developed an elongated cleavage furrow between separating chromosomes (95%, n = 20 Fig. 1, h–j and l–o) as if the cleavage activity were spread over a wide area. In addition, while most injected NRK cells completed cytokinesis (55%, n = 20 Fig. 1 j), cleavage became poorly coordinated with mitosis. Ingressions in many cells started at random sites well before the onset of anaphase (25%, n = 16 Fig. 1 l). Moreover, 57% of C3-injected cells developed randomly placed ectopic furrows, which lead to the formation of anuclear fragments (Fig. 1, i and j and n and o, arrows). Cytokinesis failed in 45% of injected cells, not due to the lack of ingression but apparently to the disorganization of cortical activities. As for cleavage in normal cells, ingression in the presence of C3 was inhibited by cytochalasin D.


The small GTP-binding protein rho regulates cortical activities in cultured cells during division.

O'Connell CB, Wheatley SP, Ahmed S, Wang YL - J. Cell Biol. (1999)

Time lapse images of control NRK cells and cells microinjected with C3 transferase during prometaphase or metaphase. Approximate time in minutes relative to anaphase onset is located in the upper right corner. Control cells (a–e) developed a narrow cleavage furrow along the equatorial plane (c–e). Cells microinjected with C3 underwent immediate spreading in all directions (f and g, k and  l; f and k were recorded immediately after microinjection). Ingressions were poorly regulated and initiated at several points around the  cell after anaphase onset. An elongated cleavage furrow (j, arrowhead) formed as a result of the broad ingression that developed between separating chromosomes. Another cell initiated an ingression before anaphase onset (l), but did not complete cleavage between  the sets of chromosomes. Ectopic furrows were common in C3-injected cells (i and j and n and o, arrows). Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132903&req=5

Figure 1: Time lapse images of control NRK cells and cells microinjected with C3 transferase during prometaphase or metaphase. Approximate time in minutes relative to anaphase onset is located in the upper right corner. Control cells (a–e) developed a narrow cleavage furrow along the equatorial plane (c–e). Cells microinjected with C3 underwent immediate spreading in all directions (f and g, k and l; f and k were recorded immediately after microinjection). Ingressions were poorly regulated and initiated at several points around the cell after anaphase onset. An elongated cleavage furrow (j, arrowhead) formed as a result of the broad ingression that developed between separating chromosomes. Another cell initiated an ingression before anaphase onset (l), but did not complete cleavage between the sets of chromosomes. Ectopic furrows were common in C3-injected cells (i and j and n and o, arrows). Bars, 20 μm.
Mentions: Both interphase and mitotic NRK cells within a monolayer showed a transient increase in surface area upon microinjection with C3 (Fig. 1, f and g, k and l). Mitotic cells then developed an elongated cleavage furrow between separating chromosomes (95%, n = 20 Fig. 1, h–j and l–o) as if the cleavage activity were spread over a wide area. In addition, while most injected NRK cells completed cytokinesis (55%, n = 20 Fig. 1 j), cleavage became poorly coordinated with mitosis. Ingressions in many cells started at random sites well before the onset of anaphase (25%, n = 16 Fig. 1 l). Moreover, 57% of C3-injected cells developed randomly placed ectopic furrows, which lead to the formation of anuclear fragments (Fig. 1, i and j and n and o, arrows). Cytokinesis failed in 45% of injected cells, not due to the lack of ingression but apparently to the disorganization of cortical activities. As for cleavage in normal cells, ingression in the presence of C3 was inhibited by cytochalasin D.

Bottom Line: All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully.The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis.Our results indicate that Rho does not simply activate actin-myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

ABSTRACT
We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin-myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

Show MeSH
Related in: MedlinePlus