Rho Family GTPases and Neuronal Growth Cone Remodelling: Relationship between Increased Complexity Induced by Cdc42Hs, Rac1, and Acetylcholine and Collapse Induced by RhoA and Lysophosphatidic Acid

Abstract

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...

Critical Activities of Rac1 and Cdc42Hs in Skeletal Myogenesis: Antagonistic Effects of JNK and p38 Pathways

“…Several lines of evidence have suggested a functional antagonism between the Rac1/Cdc42 and RhoA-C subfamilies in controlling cytoskeletal structures (Kozma et al, 1997;Leeuwen et al, 1997;Moorman et al, 1999). As far as myogenesis is concerned, a similar balance between RhoG/Rac1/ Cdc42Hs and RhoA activities emerges in the light of the present study.…”

Mechanism of activation of Pak1 kinase by membrane localization

“…One could imagine a scenario in which weak inhibition of nucleotide exchange on endogenous Cdc42 by the N17 mutant might lead, through a feedback mechanism, to superstimulation of a GEF for the novel Rho-family GTPase. Others have noted a complex interplay in neuronal cells between the activation or inhibition of Cdc42 and Rac1 on the one hand and RhoA on the other, consistent with the idea that inhibition of one Rho-family GTPase can lead to the activation of other family members (Kozma et al, 1997).…”

Dual Regulation of Actin Rearrangement through Lysophosphatidic Acid Receptor in Neuroblast Cell Lines: Actin Depolymerization by Ca2+-α-Actinin and Polymerization by Rho

“…LPA-induced actin polymerization results in process retraction and cell rounding in neuronal cells such as N1E-115, or stress fiber formation in fibroblast cells (Ridley and Hall, 1992;Jalink et al, 1993). These cellular responses are mediated by actomyosin interactions through activation of the Rho pathway, produc- ing contractile forces that induce cell shape changes (Jalink et al, 1994;Tigyi et al, 1996;Kozma et al, 1997;Hirose et al, 1998;Kranenburg et al, 1999;Fukushima et al, 2000;Weiner et al, 2001). Herein, we have also shown that Rho is activated by LPA in TR cells and LPA-induced process retraction is blocked by a ROCK inhibitor or CD, which indicates the possible involvement of Rho and actomyosin for process retraction in TR cells (Figure 10).…”