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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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Related in: MedlinePlus

The organization of F-actin and myosin II in control (a′  and c′) and Rho inhibited (b′ and d′) cells. Stacks of optical sections were deconvolved and used for reconstruction of the 90°  view. Phalloidin staining of ingressions in C3-injected cells revealed clusters of cortical actin localized above separated chromosomes (b and b′, arrows). No equatorial accumulation of actin  as found in control cells was visible (a and a′). Immunofluorescence of myosin II similarly demonstrated a lack of localization  to the equatorial plane (d and d′) compared to control cells (c  and c′). Myosin II remained diffuse throughout the cell and did not  colocalize with cortical actin over the chromosomes. Bar, 10 μm.
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Figure 5: The organization of F-actin and myosin II in control (a′ and c′) and Rho inhibited (b′ and d′) cells. Stacks of optical sections were deconvolved and used for reconstruction of the 90° view. Phalloidin staining of ingressions in C3-injected cells revealed clusters of cortical actin localized above separated chromosomes (b and b′, arrows). No equatorial accumulation of actin as found in control cells was visible (a and a′). Immunofluorescence of myosin II similarly demonstrated a lack of localization to the equatorial plane (d and d′) compared to control cells (c and c′). Myosin II remained diffuse throughout the cell and did not colocalize with cortical actin over the chromosomes. Bar, 10 μm.

Mentions: The spread morphology of dividing NRK cells facilitated a more detailed investigation of cortical organization and dynamics. Fig. 5 shows the distribution of actin and myosin II in control cells (a and a′, c and c′) and C3-injected cells (b and b′, d and d′). In control cells a band of concentrated actin filaments is visible in the cleavage furrow (Fig. 5 a′). In C3-injected cells, despite the formation of cortical ingressions and elongated furrows, phalloidin staining showed no apparent concentration of actin filaments at the sites of furrowing or ingression. Actin filaments in the equatorial region are oriented predominantly along the spindle axis (Fig. 5 b′). In addition, all injected cells developed a curious local concentration of cortical actin filaments in the vicinity of one or both sets of separated chromosomes and associated spindle poles (Fig. 5 b′, arrows). In most cells these concentrated actin filaments appeared as a cluster of aggregates.


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)

The organization of F-actin and myosin II in control (a′  and c′) and Rho inhibited (b′ and d′) cells. Stacks of optical sections were deconvolved and used for reconstruction of the 90°  view. Phalloidin staining of ingressions in C3-injected cells revealed clusters of cortical actin localized above separated chromosomes (b and b′, arrows). No equatorial accumulation of actin  as found in control cells was visible (a and a′). Immunofluorescence of myosin II similarly demonstrated a lack of localization  to the equatorial plane (d and d′) compared to control cells (c  and c′). Myosin II remained diffuse throughout the cell and did not  colocalize with cortical actin over the chromosomes. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132903&req=5

Figure 5: The organization of F-actin and myosin II in control (a′ and c′) and Rho inhibited (b′ and d′) cells. Stacks of optical sections were deconvolved and used for reconstruction of the 90° view. Phalloidin staining of ingressions in C3-injected cells revealed clusters of cortical actin localized above separated chromosomes (b and b′, arrows). No equatorial accumulation of actin as found in control cells was visible (a and a′). Immunofluorescence of myosin II similarly demonstrated a lack of localization to the equatorial plane (d and d′) compared to control cells (c and c′). Myosin II remained diffuse throughout the cell and did not colocalize with cortical actin over the chromosomes. Bar, 10 μm.
Mentions: The spread morphology of dividing NRK cells facilitated a more detailed investigation of cortical organization and dynamics. Fig. 5 shows the distribution of actin and myosin II in control cells (a and a′, c and c′) and C3-injected cells (b and b′, d and d′). In control cells a band of concentrated actin filaments is visible in the cleavage furrow (Fig. 5 a′). In C3-injected cells, despite the formation of cortical ingressions and elongated furrows, phalloidin staining showed no apparent concentration of actin filaments at the sites of furrowing or ingression. Actin filaments in the equatorial region are oriented predominantly along the spindle axis (Fig. 5 b′). In addition, all injected cells developed a curious local concentration of cortical actin filaments in the vicinity of one or both sets of separated chromosomes and associated spindle poles (Fig. 5 b′, arrows). In most cells these concentrated actin filaments appeared as a cluster of aggregates.

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