The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phasecontrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42Hs T17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs T17N, while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs T17N and Rac1 T17N, respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs T17N or Rac1 T17N. Together these results suggest that there is competition between the ACh-and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.The Ras-related Rho family p21 GTPases Cdc42, Rac, and Rho are involved in many aspects of cell growth and morphology (10,20,45). Rac activates the neutrophil oxidative burst (1), and Cdc42 is involved in bud site selection and cell polarity in the yeast Saccharomyces cerevisiae (13,23). In particular, these GTPases have been shown to induce morphological changes associated with actin polymerization. In Swiss 3T3 fibroblasts, Rac1 induces membrane ruffling and lamellipodium formation (42), RhoA induces stress fiber formation (40), and Cdc42Hs induces the formation of microspikes and filopodia (26,36), all of which are dependent on filamentousactin organization.The neuronal growth cone, a specialized structure at the distal end of a developing neurite, is responsible for pathfinding in the developing nervous system (4,8,12,21). The growth cone guides the extending neurite towards its target by constantly protruding and retracting filopodia and lamellipodia. Filopodia appear to have a sensory role in growth cone guidance: developing grasshopper neurons deprived of their filopodia by cytochalasin treatment exhibit aberrant behavior in reaching their target organs or do not reach them at all (9,16,24). Lamellipodia are implicated in neurite extension and cellular movement via membrane extension (4, 46). The growth cone receives a variety of signals from molecules on the surfaces of other cells, the extracellular matrix, diffusible chemoattractants, and chemorepellents (25). These environmental cues direct the developing neurite to extend in the correct direction by reorganization of the internal actin cytoskeleton (8,14,29,44...

Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein–protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-581267N mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.