Over the last few years, research on the ras-related, low molecular weight G proteins has revealed many new members. Currently this superfamily accounts for ovcr 40 proteins divided into four groups. These proteins all share sequence homology and several common properties; in particular, they become activated upon GTP binding and have intrinsic GTPase activity that hydrolyses the bound GTP, rendering the proteins inactive. The small G proteins appear to be important regulatory or signalling components that have been implicated in many different cellular activities, from control of cell proliferation to membrane trafficking and cytoskcletal organization. Some of the most striking evidence that members of the rho subfamily of small G proteins play a role in the regulation of cytoskeletal activity has come from two recent studies, reported in Cell by Ridley and her colleagues in Hall's group('.2). The first indication that the rho subfamily of proteins affects cytoskeletal organization came from the use of the C3 exotransferase of Clostridium botulinum, which ADP-ribosylates and thereby inactivates rho proteins. This modification does not affect the binding or hydrolysis of CTP but appears to inhibit key interactions of rho with other components. When C3 transferase was introduced into cells it had a pronounced effect on their morphology, causing them to round up and lose their stress fibers (the large bundles of actin filaments that are prominent in many cultured cell^)(^,^). More direct evidence that rho has an effect on the actin microfilament system was obtained two years ago by Paterson and coworkers, using a constitutively activated form of rho that was generated by mutating amino acid 14 in rho from glycine to ~ a l i n e ( ~This ) . change is equivalent to the oncogenic mutation in codon 12 of ras, which leads to activation of thc protein by decreasing its GTPase activity. Microinjection of this activated form of rho into normal fibroblasts growing at low density caused them to contract and display an unusual morphology. When the activated rho was injected into quiescent, serum-starved, confluent cells that displayed few stress fibers, prominent stress fibers reappeared. Ridley and Hall have now extended these results and demonstrated that, not only does microinjection of activated rho into serum-starved, quiescent fibroblasts lead to prominent stress fibers, but also to the appearance of focal adhesions('). Focal adhesions are the regions where stress fibers are anchored to the plasma membrane and where the cell adheres most tightly to the underlying substratum. Since under most conditions focal adhesions are closely correlated with the pres-
ence of stress fibers, this finding in itself is not too surprising. However, the authors go on to show that the effect of microinjecting activated rho into serum-starved, quiescent cells is mimicked by the addition of serum and, furthermore, that this response to serum is mediated by rho. Surprisingly, the factor in serum responsible for this effect is shown not to be a polypeptide growth factor but lysophosphatidic acid. How might rho affect the cytoskeleton? Several lines of evidence suggest that it promotes contractility. Paterson and coworkers observed that fibroblasts at low density injected with rho appcared to ont tract'^). The reappearance of prominent stress fibers in quiescent cells may also be consistent with increased contractility since it has been argued previously that stress fibers are under a state of isometric contraction and that this contraction contributes to their formation(@). Contractility in nonmuscle cells depends on the interaction of actin and myosin. This interaction is under several levels of control, any of which could be influenced by rho. For example, the activity of myosin is regulated by the inyosin light chain kinase, and the interaction of myosin with actin is controlled by the actin-binding protein caldcsmon. Both caldesmon and the myosin light chain kinase are in turn regulated by the concentration of frec calcium as well as by phosphorylation. That rho may, in fact, regulate inyosin activity in nonmuscle cells comes from a recent study of smooth muscle contractiotd7). In permeabilized smooth muscle, activated rho (bound to GTPyS) was found to enhance the calcium sensitivity of contraction. Since earlier work had demonstrated that GTPyS increases the level of phosphorylation of the myosin light chain kinase, it seems likely that increased calcium sensitivity is due to enhanced phosphorylation of this kinase. In view of the extensive similarities between smooth muscle and nonmuscle with respect to the regulation of myosin activity, it will be important to determine whether rho regulates the phosphorylation of the myosin light chain kinase in nonmuscle cells and thereby enhances actomyosin contractility. A second possible mechanism for the action of rho, suggested by Ridley and Hall, is that rho may promote the assembly of focal adhesions. Support for this possibility comes from a recent study by Symons and Mitchison@). These investigators found that microinjcctjon of GTPyS into fibroblasts increased their adhesion to the underlying substrate. Of course, the GTPyS would be expected to activate many G proteins in these cells, and any one of them could be contributing to increased adhesion. Rather little is known about the factors that regulate the formation of focal adhesions and stress fibers, but evidence has been presented that activation of protein lunase C is required in some cells(9).In addition, recent work from this lab has indicated a role for tyrosine phosphorylation in the assembly of these structures(l0'. It will be interesting to determine whether rho modulates kinases or phosphatases that regulate formation of focal adhesions. In a second paper in Cell, Ridley and coworkers examined the role of a member of the rho subfamily, racl , in cytoskeletal function@). Expression of a constitutively activated mutant form of racl was found to stimulate pronounced membrane ruffling in rat fibroblasts. Direct microinjection of
this activated form of racl into these cells caused an increase in actin accumulation in membrane ruffles within 5 minutes. The extensive membrane ruffling activity was associated with macropinocytosis and the accumulation of many large vesicles in the cytoplasm. Immunofluorescence revealed rac localized on the surface of the vesicles and at the plasma membrane. Cells microinjected with rac 1 also developed stress fibers but at a much slower rate than in cells injected with rho. This development of stress fibers was inhibited by the C3 transferase, leading the authors to conclude that rho was acting downstreaiii of rac 1 in this process. The C3 transferase did not affect membrane ruffling induced by racl, indicating that there are at least two pathways involving rac and that rho is not involved in the pathway leading to membrane ruffling. Many growth factors induce membrane ruffling. In this second paper, it was demonstrated that the ruffling induced by various growth factors was inhibited by introduction of a dominant inhibitory mutant form of rac 1, indicating that rac 1 is involved in the signal transduction pathway initiated by growth factor receptors and leading to the reorganization of the actin cytoskeleton. Microinjcction of H-ras, the constitutively activated, oncogenic form of ras also induces membrane ruffling(''). Again. the authors show that the ruffling induced by H-ras is blocked by the presence of the dominant inhibitory mutant form of racl. These results indicate that racl is acting downstream of ras in this signalling cascade that leads to ruffling. Membrane ruffling is a striking phenomenon usually associated with increased migration of cells. Little is known, however, about the mechanics of extending these thin veils of plasma membrane. The identification of racl as a critical component in the signalling pathways that lead to ruffle formation is a major advance. Determining the downstream
components with which rac interacts should shed lighl on how the actin cytoskeleton can generate thcse impressive cell extensions and so rapidly reorganize from a relatively stable to a highly dynamic state.
References 1 Ridley, A. J. and Hall, A. (1992). The small GTP-binding protcin rho regulates the assenibly of 1)cal adhz\ions and actin stress fibers in response to grouth factors. C e / / 70,?W3YY 2 Kidley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. and Hall, A. ( I 992). The small GTP-binding protein rac i-cgulatcs growth factor-induced rrirrnhrane ruffling. Cel/70,401-410. 3 Rubin, E. J., Gill, D. M., Boquet, P., and Popoff, M. R. (1YX8). Functional modification nf a 21-kilodalloii Ci protein when AI)P-rihi Clo.rtridiamhotu/irwnz. M d C d / . Hioi. 8. 31 8-426. 4 Chardin. P., Boquet, P., Maduale, P., Popoff, M.R., Kubin, E. J., and Gill, D. M. (1989). The mammalian G protein rhoC is ADP-ribosylated by Clostridiurn bozulirrrrm exoenzyine C3 and affects actin microfilaments i n Vero cclls. EMKO.1. 8, 10x7- 1092. 5 Paterson, H. F., Self, A. J., Garrett. M. D., Just. I., Aktories, K., and Hall, A. (1990). Micioin.lectinn of recomhioant p21""' inducer rapid changrh in cell morphology. J. C'eliLIiol 111, 1001-1007. 6Burridge,K. (1981). Are stress fibers contractile'?Nature284.691-6Y2. 7 Hirata, K., Kikuchi, A., Sasaki. T., Kuroda, S.. Kaihuchi, K., Matsuura, Y., Seki, H.. Saida, K., and Takai, Y. (1992). Involvement of rho p21 in the GTP-enhanced c d. ]L. .i u m ion senbitivily of smooth muscle contration J. Biol Chef??. 267,8719-8722. 8 Symons, M. and Mitchison, T. M. (1992). A GTPase controls cell-substrate adhesion inXenopu.7 XTC fibroblasts. J. CellBiol. 118, 1235.1244. (1992). Protein kinase C in focal adhesion
omer, L. H. (IYY?). Tyrosine phosphorylation of paxillin and pplXfak accompanies cell ndhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell B i d 119. in press 11 Bar-Sagi, D., and Feramisco, J. R. (1986). Induction of me fluid-phase pinoeytosi$ in quierccnt fibioblarts by ras moteins. i m .
ence of stress fibers, this finding in itself is not too surprising. However, the authors go on to show that the effect of microinjecting activated rho into serum-starved, quiescent cells is mimicked by the addition of serum and, furthermore, that this response to serum is mediated by rho. Surprisingly, the factor in serum responsible for this effect is shown not to be a polypeptide growth factor but lysophosphatidic acid. How might rho affect the cytoskeleton? Several lines of evidence suggest that it promotes contractility. Paterson and coworkers observed that fibroblasts at low density injected with rho appcared to ont tract'^). The reappearance of prominent stress fibers in quiescent cells may also be consistent with increased contractility since it has been argued previously that stress fibers are under a state of isometric contraction and that this contraction contributes to their formation(@). Contractility in nonmuscle cells depends on the interaction of actin and myosin. This interaction is under several levels of control, any of which could be influenced by rho. For example, the activity of myosin is regulated by the inyosin light chain kinase, and the interaction of myosin with actin is controlled by the actin-binding protein caldcsmon. Both caldesmon and the myosin light chain kinase are in turn regulated by the concentration of frec calcium as well as by phosphorylation. That rho may, in fact, regulate inyosin activity in nonmuscle cells comes from a recent study of smooth muscle contractiotd7). In permeabilized smooth muscle, activated rho (bound to GTPyS) was found to enhance the calcium sensitivity of contraction. Since earlier work had demonstrated that GTPyS increases the level of phosphorylation of the myosin light chain kinase, it seems likely that increased calcium sensitivity is due to enhanced phosphorylation of this kinase. In view of the extensive similarities between smooth muscle and nonmuscle with respect to the regulation of myosin activity, it will be important to determine whether rho regulates the phosphorylation of the myosin light chain kinase in nonmuscle cells and thereby enhances actomyosin contractility. A second possible mechanism for the action of rho, suggested by Ridley and Hall, is that rho may promote the assembly of focal adhesions. Support for this possibility comes from a recent study by Symons and Mitchison@). These investigators found that microinjcctjon of GTPyS into fibroblasts increased their adhesion to the underlying substrate. Of course, the GTPyS would be expected to activate many G proteins in these cells, and any one of them could be contributing to increased adhesion. Rather little is known about the factors that regulate the formation of focal adhesions and stress fibers, but evidence has been presented that activation of protein lunase C is required in some cells(9).In addition, recent work from this lab has indicated a role for tyrosine phosphorylation in the assembly of these structures(l0'. It will be interesting to determine whether rho modulates kinases or phosphatases that regulate formation of focal adhesions. In a second paper in Cell, Ridley and coworkers examined the role of a member of the rho subfamily, racl , in cytoskeletal function@). Expression of a constitutively activated mutant form of racl was found to stimulate pronounced membrane ruffling in rat fibroblasts. Direct microinjection of
this activated form of racl into these cells caused an increase in actin accumulation in membrane ruffles within 5 minutes. The extensive membrane ruffling activity was associated with macropinocytosis and the accumulation of many large vesicles in the cytoplasm. Immunofluorescence revealed rac localized on the surface of the vesicles and at the plasma membrane. Cells microinjected with rac 1 also developed stress fibers but at a much slower rate than in cells injected with rho. This development of stress fibers was inhibited by the C3 transferase, leading the authors to conclude that rho was acting downstreaiii of rac 1 in this process. The C3 transferase did not affect membrane ruffling induced by racl, indicating that there are at least two pathways involving rac and that rho is not involved in the pathway leading to membrane ruffling. Many growth factors induce membrane ruffling. In this second paper, it was demonstrated that the ruffling induced by various growth factors was inhibited by introduction of a dominant inhibitory mutant form of rac 1, indicating that rac 1 is involved in the signal transduction pathway initiated by growth factor receptors and leading to the reorganization of the actin cytoskeleton. Microinjcction of H-ras, the constitutively activated, oncogenic form of ras also induces membrane ruffling(''). Again. the authors show that the ruffling induced by H-ras is blocked by the presence of the dominant inhibitory mutant form of racl. These results indicate that racl is acting downstream of ras in this signalling cascade that leads to ruffling. Membrane ruffling is a striking phenomenon usually associated with increased migration of cells. Little is known, however, about the mechanics of extending these thin veils of plasma membrane. The identification of racl as a critical component in the signalling pathways that lead to ruffle formation is a major advance. Determining the downstream
components with which rac interacts should shed lighl on how the actin cytoskeleton can generate thcse impressive cell extensions and so rapidly reorganize from a relatively stable to a highly dynamic state.
References 1 Ridley, A. J. and Hall, A. (1992). The small GTP-binding protcin rho regulates the assenibly of 1)cal adhz\ions and actin stress fibers in response to grouth factors. C e / / 70,?W3YY 2 Kidley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. and Hall, A. ( I 992). The small GTP-binding protein rac i-cgulatcs growth factor-induced rrirrnhrane ruffling. Cel/70,401-410. 3 Rubin, E. J., Gill, D. M., Boquet, P., and Popoff, M. R. (1YX8). Functional modification nf a 21-kilodalloii Ci protein when AI)P-rihi Clo.rtridiamhotu/irwnz. M d C d / . Hioi. 8. 31 8-426. 4 Chardin. P., Boquet, P., Maduale, P., Popoff, M.R., Kubin, E. J., and Gill, D. M. (1989). The mammalian G protein rhoC is ADP-ribosylated by Clostridiurn bozulirrrrm exoenzyine C3 and affects actin microfilaments i n Vero cclls. EMKO.1. 8, 10x7- 1092. 5 Paterson, H. F., Self, A. J., Garrett. M. D., Just. I., Aktories, K., and Hall, A. (1990). Micioin.lectinn of recomhioant p21""' inducer rapid changrh in cell morphology. J. C'eliLIiol 111, 1001-1007. 6Burridge,K. (1981). Are stress fibers contractile'?Nature284.691-6Y2. 7 Hirata, K., Kikuchi, A., Sasaki. T., Kuroda, S.. Kaihuchi, K., Matsuura, Y., Seki, H.. Saida, K., and Takai, Y. (1992). Involvement of rho p21 in the GTP-enhanced c d. ]L. .i u m ion senbitivily of smooth muscle contration J. Biol Chef??. 267,8719-8722. 8 Symons, M. and Mitchison, T. M. (1992). A GTPase controls cell-substrate adhesion inXenopu.7 XTC fibroblasts. J. CellBiol. 118, 1235.1244. (1992). Protein kinase C in focal adhesion
omer, L. H. (IYY?). Tyrosine phosphorylation of paxillin and pplXfak accompanies cell ndhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell B i d 119. in press 11 Bar-Sagi, D., and Feramisco, J. R. (1986). Induction of me fluid-phase pinoeytosi$ in quierccnt fibioblarts by ras moteins. i m .
