The Small GTP-binding Protein Rho Regulates Cortical Activities in Cultured Cells during Division

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.

Dissecting the Role of Rho-mediated Signaling in Contractile Ring Formation

“…However, in fluorescence resonance energy transfer (FRET)-based analysis, it has been concluded that Rho activation is not required for contractile ring formation (Yoshizaki et al, 2003). Others reported that Rho is required for cytokinesis in later stages, such as constriction of the contractile ring or severing of the midbody (Drechsel et al, 1997;O'Connell et al, 1999). In this study, we show that Rho acts as a microtubuledependent signal that specifies the position of the contractile ring.…”

“…It is reported that C3-microinjected HeLa cells frequently detach from the dish or fail to initiate anaphase (O'Connell et al, 1999). To avoid these problems, we used the scrape-loading method to deliver C3 to HeLa cells (McNeil et al, 1984).…”

Rho GTPase activity zones and transient contractile arrays

“…Clearly, focusing is not absolutely essential for cytokinesis in situations where either myosin-2 or Rho is inhibited. (71,72) However, when cytokinesis is monitored in living epithelial cells or epithelia in situ, until the terminal phase of the process, the cytokinetic apparatus is tightly focused, cutting through the cell like a knife. Moreover, broad cytokinetic arrays are associated with inefficient furrowing, (20) implying that focusing is important.…”

Myosin II-Independent Cytokinesis in Dictyostelium. Its Mechanism and Implications.

“…O'Connell et al (1999) microinjected C3 ribosyltransferase into mitotic cultured cells to inactivate endogenous Rho, small G proteins which had been implicated in cytokinesis of animal egg cells (Kishi et al, 1993;Mabuchi et al, 1993;Drechel et al, 1996). This treatment enhanced the cortical activities, in that the furrows became wider and ectopic furrows were often formed outside the equatorial region, even when the mitotic spindle was destroyed by nocodazole.…”