Table of contents

Rac and cytoskeletal rearrangement

The chemotactic ability of Dictyostelium cells has been used to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B61L or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increases levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B61L and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B17N do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, it is suggested that DdRac1 regulates (1) the basal levels of F-actin assembly, (2) its dynamic reorganization in response to chemoattractants, and (3) cellular polarity during chemotaxis (Chung, 2000).

The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulates actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response is prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors is the formation of actin stress fibers, a process requiring endogenous rho proteins. Growth factors act through rac to stimulate this rho-dependent response. It is proposed that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin (Ridely, 1992b).

Besides activating JNK and SAPK MAP kinase cascades, Rac and Cdc-42 regulate formation of lamellipodia and filopodia, both of which involve formation of polymerized actin structures and the assembly of associated integrin complexes. The modification of cytoskeleton mediated by Rac and Cdc-42 is activated by extracellular factors such as lysophosphatidic acid, bombesin, PDGF, EGF (Drosophila homolog: Spitz) and insulin. Rac and Cdc-42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets. Rac and Cdc42 contain a Serine 40C effector site. Mutation of this site results in proteins that can no longer interact with the Serine/Threonine kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, Rac and Cdc42 still induce cytoskeletal changes and G1 cell cycle progression. In contrast, Rac containing an F37A effector site substitution no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. Thus Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade (Lamarche, 1996).

Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of hematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulates the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulates lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibits CSF-1-induced lamellipodium formation, and also induces cell rounding. V12Cdc42 induces the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevents CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulates actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induces the loss of actin cables. Bac1 cells have cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These "focal complexes" are present in growing and CSF-1-starved cells, but are disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin does not disperse until long after focal phosphotyrosine and vinculin staining has disappeared. It is concluded that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix (Allen, 1997).

In mast cells, activation of GTP-binding proteins induces centripetal reorganization of actin filaments. This effect is due to disassembly, relocalization, and polymerization of F-actin and is dependent on two small GTPases, Rac and Rho. Activities of Rac and Rho are also essential for the secretory function of mast cells. In response to GTP-gamma-S and/or calcium, only a proportion of permeabilized mast cells is capable of secretory response. Actin organization of secreting and nonsecreting cell populations are compared. The cytoskeletal and secretory responses are strongly correlated, indicating a common upstream regulator of the two functions. The secreting cell population preferentially displays both relocalization and polymerization of actin. However, when actin relocalization or polymerization is inhibited by either phalloidin or cytochalasin (respectively), secretion is unaffected. Moreover, the ability of the constitutively active mutants of Rac and Rho to enhance secretion is also unaffected in the presence of cytochalasin. Therefore, Rac and Rho control these two functions by divergent, parallel signaling pathways. Cortical actin disassembly occurs in both secreting and nonsecreting populations and does not, by itself, induce exocytosis. A model for the control of exocytosis is proposed that includes at least four GTP-binding proteins and suggests the presence of both shared and divergent signaling pathways from Rac and Rho (Norman, 1996).

Neurons contain distinct compartments including dendrites, dendritic spines, axons and synaptic terminals. The molecular mechanisms that generate and distinguish these compartments, although largely unknown, may involve the small GTPases Rac and Cdc42, which appear to regulate actin polymerization. Having shown that perturbations of Rac1 activity block the growth of axons but not dendrites in Drosophila neurons, an examination of transgenic mice expressing constitutively active human Rac1 in Purkinje cells was carried out to see whether this also applies to mammals. These mice were ataxic and had a reduction of Purkinje-cell axon terminals in the deep cerebellar nuclei, whereas the dendritic trees grew to normal height and branched extensively. Unexpectedly, the dendritic spines of Purkinje cells in developing and mature cerebella are much reduced in size but increased in number. These 'mini' spines often form supernumerary synapses. These differential effects of perturbing Rac1 activity indicate that there may be distinct mechanisms for the elaboration of axons, dendrites and dendritic spines (Luo, 1996).

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Microinjection of Cdc42Hs and Rac1 promote 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 is inhibited by coinjection with the dominant negative mutant Cdc42HsT17N, while the Cdc42Hs response can be competed by coinjection with RhoA. 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 is inhibited (respectively) by preinjection with the dominant negative mutants Cdc42HsT17N and Rac1T17N. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, is inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth is inhibited by injection with Cdc42HsT17N or Rac1T17N. 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 (Kozima, 1997).

Macropinocytosis has emerged as a key antigen uptake pathway by which dendritic cells can rapidly and non-specifically sample large amounts of surrounding fluid. Exogenous antigen taken up by this route can be processed and presented to T cells on both MHC class I and class II molecules. Macropinosomes are large (~0.2-5.0 ┬Ám diameter) endocytic structures that form when membrane ruffles fuse together, non-selectively engulfing fluid-phase material. Particularly striking in dendritic cells is the fact that membrane ruffling and macropinocytosis are constitutive, whereas in fibroblasts, epithelial cells and most other cell types studied, these events require stimulation by growth factors. Moreover, in cultured human monocyte-derived dendritic cells at least, macropinocytosis is downregulated by inflammatory stimuli. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental program leading to dendritic cell maturation and migration, and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. The possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells was tested. Dendritic cell populations have been estabilished that show constitutive macropinocytosis that is downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibit the formation of macropinosomes but, surprisingly, do not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminates actin plaques/podosomes and actin cables, respectively, but have little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS have equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells does not reactivate macropinocytosis. It is concluded that Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions (West, 2000).

Vulval development in the nematode C. elegans can be divided into a fate specification phase controlled in part by let-60 Ras, and a fate execution phase involving stereotypical patterns of cell division and migration controlled in part by lin-17 Frizzled. Since the small GTPase Rac has been implicated as a downstream target of both Ras and Frizzled and influences cytoskeletal dynamics, the role of Rac signaling during each phase of vulval development was investigated. The Rac gene ced-10 and the Rac-related gene mig-2 are redundantly required for the proper orientation of certain vulval cell divisions, suggesting a role in spindle positioning. ced-10 Rac and mig-2 are also redundantly required for vulval cell migrations and play a minor role in vulval fate specification. Constitutively active and dominant-negative mutant forms of mig-2 cause vulval defects that are very similar to those seen in ced-10;mig-2 double loss-of-function mutants, indicating that they interfere with the functions of both ced-10 Rac and mig-2. Mutations in unc-73 (a Trio-like guanine nucleotide exchange factor) cause similar vulval defects, suggesting that UNC-73 is an exchange factor for both CED-10 and MIG-2. The similarities and differences between the cellular defects seen in Rac mutants and let-60 Ras or lin-17 Frizzled mutants are discussed (Kishore, 2002).

Although the pathways through which Rac controls different cellular processes in vivo are still poorly defined, a large number of candidate Rac regulators and targets have been identified biochemically. These observations raise the question of whether Rac signals through multiple (perhaps redundant) pathways concomitantly, or whether Rac signals through different pathways in different cells to control different biological processes. A comparison of different Rac-dependent processes in C. elegans seems to support the latter model. For example, UNC-73 Trio is required for CED-10- and MIG-2-mediated vulva fate execution, but it is not required for CED-10-mediated cell corpse engulfment. Conversely, the adaptor proteins CED-2 CrkII and CED-5 Dock180 are required for cell corpse engulfment, but not for vulval fate execution since ced-2lf and ced-5lf mutants have wild-type vulval development. C. elegans vulval development will be a useful model system for elucidating specific Rac pathways involved in cell-fate specification, division axis orientation, and cell migration, and for testing the relationship between Rac and the Ras and Wnt signaling pathways (Kishore, 2002).

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. An ephrinB-EphB receptor trans-synaptic signaling pathway has been identified that regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin (Drosophila ortholog: Trio) to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin or dominant-negative Rac1, or inhibition of PAK each eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity (Penzes, 2003).

The role of the Rac1 effector p21-activated kinase PAK was examined. Several PAK proteins are expressed in the brain, and previous studies have shown that some of the effects of Rac1 on the cytoskeleton are mediated by PAK. In addition, genetic analysis in Drosophila has shown that PAK1 is genetically associated with Trio, the fly ortholog of kalirin, in the pathway through which Trio affects axon growth and guidance. Binding of activated Rac1 to PAK induces PAK autophosphorylation, which strongly correlates with its activation. To test whether ephrinB treatment induces activation of PAKs, an antibody detecting autophosphorylated PAK (P-PAK) was used. In addition, this experiment can be regarded as a way to visualize endogenous Rac1 activation. Treatment of hippocampal neurons with clustered ephrinB1 induce a dramatic increase in the number and size of clusters stained with the P-PAK antibody. This effect was confirmed by Western analysis with the P-PAK antibody of extracts of 4-week-old high-density cortical neurons treated with ephrinB1. Moreover, in hippocampal neurons, ephrinB1 treatment induces activation of PAK at synapses, as shown by P-PAK immunostaining coincident with synaptophysin (Penzes, 2003).

To test whether kalirin-7 was required for ephrinB1-induced PAK phosphorylation, the effect was examined of overexpressing the GEF inactive kal7-mut in hippocampal neurons on the ability of clustered ephrinB1 to induce phosphorylation of PAK. Therefore, DIV7 hippocampal neurons were transfected with myc-kal7-mut, and 2 days later the neurons were treated with clustered ephrinB1 for 2 hr, followed by fixation and immunostaining for myc and P-PAK. While ephrinB1 treatment induces an increased phosphorylation of PAK in nontransfected neurons, in neurons expressing kal7-mut, the level of P-PAK is visibly reduced compared to adjacent nontransfected neurons. Quantification of the ratios of P-PAK fluorescence intensities to total cell areas of nontransfected control neurons relative to the same ratios for neurons expressing myc-kal7-mut confirmed this observation (Penzes, 2003).

PAKs phosphorylate proteins involved in regulating the actin cytoskeleton and gene expression. To test whether PAK is an essential downstream component of ephrinB signaling in spine morphogenesis, GFP-transfected hippocampal neurons were treated with a fusion protein of the PAK1 inhibitory domain (PID) fused with the cell-penetrating peptide (TAT-PID) along with ephrinB1. These neurons exhibit a reduction in the number and size of spines, compared to the ephrinB1-treated neurons, while also showing a reduced phosphorylation level of PAK, confirming its inhibition by PID. Together, these data demonstrate that Rac1 and PAK are key downstream components of ephrinB regulation of spine morphogenesis (Penzes, 2003).

During development, it is necessary to coordinate accurately the formation and location of presynaptic active zones with those of the postsynaptic structures. This could be achieved by signaling from presynaptic ephrinB, clustered at active zones on axons, to activate postsynaptic EphB2, resulting in synaptogenesis on the apposing dendrites. Even in mature neurons, dendritic spines are very dynamic structures, and recent studies have demonstrated that LTP induces morphological changes in spines, which may contribute to plasticity in adult neurons. The rapid and dramatic effect of ephrinB on spine maturation suggests that ephrinB-EphB2 signaling may be a key component in the regulation of spine morphogenesis during plasticity. Other extracellular signals have been shown to regulate spine morphogenesis, such as K+ depolarization, glutamate action on NMDA receptors, and BDNF. It is possible that kalirin mediates the intracellular effects of these signals as well (Penzes, 2003).

Rac and Cell Polarity

MDCK cells expressing RhoA or Rac1 mutants under control of the tetracycline repressible transactivator were used to examine short-term effects of known amounts of each mutant before, during, or after development of cell polarity. At low cell density, Rac1V12 cells have a flattened morphology and intact cell-cell contacts, whereas Rac1N17 cells are tightly compacted. Abnormal intracellular aggregates form between Rac1N17, F-actin, and E-cadherin in these nonpolarized cells. At all subsequent stages of polarity development, Rac1N17 and Rac1V12 colocalize with E-cadherin and F-actin in an unusual beaded pattern at lateral membranes. In polarized cells, intracellular aggregates form with Rac1V12, F-actin, and an apical membrane protein (GP135). At low cell density, RhoAV14 and RhoAN19 are localized in the cytoplasm, and cells are generally flattened and more fibroblastic than epithelial in morphology. In polarized RhoAV14 cells, F-actin is diffuse at lateral membranes and prominent in stress fibers on the basal membrane. GP135 is abnormally localized to the lateral membrane and in intracellular aggregates, but E-cadherin distribution appears normal. In RhoAN19 cells, F-actin, E-cadherin, and GP135 distributions are similar to those in controls. Expression of either RhoAV14 or RhoAN19 in Rac1V12 cells disrupts Rac1V12 distribution and causes cells to adopt the more fibroblastic, RhoA mutant phenotype. It is suggested that Rac1 and RhoA are involved in the transition of epithelial cells from a fibroblastic to a polarized structure and function by direct and indirect regulation of actin and actin-associated membrane protein organizations (Jou, 1998a).

A novel effector of Rac and Cdc42, hPar-6 (see Drosophila par-6), has been identified that is the human homolog of a cell-polarity determinant in C. elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKC, and forms a stable ternary complex with either Rac1 or Cdc42 and PKC. This association results in stimulation of PKC kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKC-dependent pathway distinct from the pathway mediated by Raf (Qui, 2000).

Many direct targets of Rac1 and Cdc42 have been identified, but none has been shown to have a direct role in cell transformation by Rac1 and Cdc42. hPar-6 is a novel effector of Rac1 and Cdc42 that promotes PKCzeta-dependent transformation by both GTPases. Although it has been suggested that PAK1 may also contribute to transformation by Rac1 in Rat1 fibroblasts, PAK1 does not enhance transformation by activated Raf or activated Rac1 in NIH-3T3 cells, and studies using effector domain mutants indicate that interaction of PAK1 with Rac1 does not correlate with cell-cycle progression or transformation. Thus, hPar-6 appears to be the first effector shown to directly mediate transformation by Rac1 and Cdc42. The identification of PKCzeta as a downstream effector of hPar-6 represents the first elucidation of a signaling pathway linking Rac1/Cdc42 to cell transformation. A model is presented depicting two separate pathways downstream of Ras that lead to cell polarity and growth control: these pathways can contribute to cell transformation. One pathway is comprised of Rac/Cdc42, hPar-6 and PKCzeta, and the other is mediated by Raf, MEK and MAP kinase (Qui, 2000).

The mechanism by which hPar-6 regulates the kinase activity of PKCzeta is currently under investigation. Subcellular targeting by interaction with specific proteins provides an attractive mechanism for PKC isozyme-specific regulation. It is possible that hPar-6 and PKCzeta are translocated by Rac1 or Cdc42 to the membrane, where PKCzeta could interact with an activator. One candidate activator is the phosphatidylinositol 3-kinase (PI3-kinase) target PDK1, since PDK1 and PKCzeta associate in vivo via their catalytic domains, and both PI3-kinase and PDK1 stimulate PKCzeta activity. Consistent with this model, it has been demonstrated that PI3-kinase can act as a link between Ras and Rac in transformation and that membrane-targeted PKCzeta is constitutively active. The observation that hPar-6 alone exhibits little, if any, transforming activity is also consistent with the membrane-targeting model. It should also be noted that although overexpression of hPar-6 alone (i.e., in the absence of Rac1[G12V]) is sufficient to activate PKCzeta kinase activity, overexpression of hPar-6 and PKCzeta only marginally promotes focus formation, suggesting that activated Rac1 is necessary to target PKCzeta to substrates involved in transformation. However, the possibility that Rac1 activates some other pathway that is also necessary for transformation cannot be ruled out. In addition to being activated by hPar-6, PKCzeta might in turn phosphorylate hPar-6. In this regard, it should be noted that there is a putative PKCzeta-phosphorylation site in mammalian Par-6 (Qui, 2000).

The mechanism underlying transformation by hPar-6 and PKCzeta is not yet clear. Stimulation of cell proliferation and inhibition of apoptosis are, however, important characteristics of cell transformation. In this regard, it has been shown that Rac1 and Cdc42 induce cyclin D1 transcription and accumulation, phosphorylation and inactivation of the tumor suppressor protein Rb, and activation of the transcription factor E2F. Inactivation of Rb may be necessary for Rac1/Cdc42 stimulation of cell proliferation, and it is possible that hPar-6 and PKCzeta have a role in this pathway. In addition, Ras, Rac1, Cdc42 and PKCzeta are all able to activate the transcription factor NF-kappaB. NF-kappaB activation is associated with mitogenesis, anti-apoptotic activity and cell transformation. Thus, the hPar-6-PKCzeta pathway might mediate NF-kappaB activation, and thereby contribute to cell transformation by Rac1 and Ras. Another possibility is that the hPar-6-PKCzeta pathway may mediate growth control by Rac1/Cdc42 by inducing downregulation of the pro-apoptotic protein Par-4 (prostate apoptosis response-4; unrelated to the C. elegans Par gene product). Par-4 interacts with PKCzeta and overexpression of PKCzeta downregulates Par-4, an event that appears important for Ras transformation and tumor progression. Thus, cyclin D1, Rb, E2F, NF-kappaB and Par-4 all warrant further investigation as possible downstream targets of the hPar-6-PKCzeta pathway (Qui, 2000).

Polarity is a fundamental feature of all eukaryotic cells. Rac, Cdc42, Par-6 and atypical PKCs appear to be conserved in diverse metazoans, including Drosophila, C. elegans, Xenopus, mouse and humans. The CRIB motif of Par-6 is also conserved, suggesting that it interacts with Rac and/or Cdc42 in these different species. In C. elegans, inhibition of Cdc42 function by RNA-mediated gene interference (RNAi) produces defects in cell polarity similar to those observed in par and pkc-3 mutants, while in mammalian cells, Par-6 is localized to tight junctions, together with atypical PKC and ASIP, the mammalian homolog of Par-3. Moreover in C. elegans, Par-6 interacts with Par-3, and in Drosophila the Par-3 homolog has an important role in the asymmetric cleavage of epithelial cells and neuroblasts. Taken together, these observations suggest that Rac or Cdc42, Par-6, atypical PKC, and perhaps Par-3, constitute a conserved pathway that regulates cell polarity. As hPar-6 and PKCzeta mediate cell transformation by Rac1 and Cdc42, there may be a link between cell-polarity signaling and growth control: aberrant cell-polarity signaling could lead to oncogenic transformation. In the light of the important roles of Rac1/Cdc42 in Ras-induced transformation, hPar-6 and PKCzeta could represent potential targets for anti-cancer therapeutics (Qui, 2000).

The Wnt planar cell polarity (Wnt/PCP) pathway signals through small Rho-like GTPases to regulate the cytoskeleton. The core PCP proteins have been mapped to the Wnt/PCP pathway genetically, but the molecular mechanism of their action remains unknown. This study investigated the function of the mammalian PCP protein Vang-like protein 2 (Vangl2). RNAi knockdown of Vangl2 impaired cell-cell adhesion and cytoskeletal integrity in the epithelial cell lines HEK293T and MDCK. Similar effects were observed when Vangl2 was overexpressed in HEK293T, MDCK or C17.2 cells. The effects of Vangl2 overexpression could be blocked by knockdown of the small GTPase Rac1 or by dominant-negative Rac1. In itself, knockdown of Rac1 impaired cytoskeletal integrity and reduced cell-cell adhesion. Vangl2 bound and re-distributed Rac1 within the cells but did not alter Rac1 activity. Moreover, both transgenic mouse embryos overexpressing Vangl2 in neural stem cells and loop-tail Vangl2 loss-of-function embryos displayed impaired adherens junctions, a cytoskeletal unit essential for neural tube rigidity and neural tube closure. In vivo, Rac1 was re-distributed within the cells in a similar way to that observed in vitro. It is propose that Vangl2 affects cell adhesion and the cytoskeleton by recruiting Rac1 and targeting its activity in the cell to adherens junctions (Lindqvist, 2010).

Novel role of Rac-Mid1 signaling in medial cerebellar development

Rac (see Drosophila Rac1) signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. This study generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). Impaired tangential migration at E16.5 was observed, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 (see Drosophila Dacapo) and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development (Nakamura, 2017).

Rac and axon guidance

Rac GTPases act as molecular switch in various morphogenic events. However, the regulation of their activities during the development of multicellular organisms is not well understood. Caenorhabditis elegans rac genes ced-10 and mig-2 have been shown to act redundantly to control P cell migration and the axon outgrowth of D type motoneurons. ced-10 and mig-2 also control amphid axon outgrowth and amphid dendrite fasciculation in a redundant fashion. Biochemical and genetic data indicate that unc-73, which encodes a protein related to Trio-like guanine nucleotide exchange factor, acts as a direct activator of ced-10 and mig-2 during P cell migration and axon outgrowth of D type motoneurons and amphid sensory neurons. Furthermore, rac regulators ced-2/crkII and ced-5/dock180 function genetically upstream of ced-10 and mig-2 during axon outgrowth of D type motoneurons and act upstream of mig-2 but not ced-10 during P cell migration. However, neither ced-2/crkII nor ced-5/dock180 is involved in amphid axon outgrowth. Therefore, distinct rac regulators control ced-10 and mig-2 differentially in various cellular processes (Wu, 2002).

Biochemical data suggest that the GEF1 domain of UNC-73 has a guanine nucleotide exchange activity for both CED-10 and MIG-2 in vitro, consistent with the finding that the GEF1 domain of the Drosophila Trio acts on Drosophila Rac1 and Drosophila Mtl (MIG-2 like). RHO-1 has been shown to function in P cell migration, likely through activation by UNC-73 GEF2. Thus, UNC-73 likely functions as a common exchange factor for CED-10, MIG-2, and RHO-1 during P cell migration, with the GEF1 domain acting on CED-10 and MIG-2 and the GEF2 domain on RHO-1. UNC-73, CED-10, and MIG-2 also act in axon outgrowth of D-type motoneurons and amphid sensory neurons. Biochemical data and genetic analyses suggest that UNC-73 likely activates CED-10 and MIG-2 through the GEF1 domain to control the axon outgrowth of D-type motoneurons and amphid sensory neurons. Genetic analysis indicates that UNC-73 GEF2 likely functions in these axonal outgrowth processes as well. For example, axon outgrowth defects of unc-73(gm33) mutants are more severe than those of unc-73(rh40) mutants. The rh40 mutation abolishes UNC-73 GEF1 activities for RAC, and the gm33 mutation affects both GEF1 and GEF2 domains of UNC-73. UNC-73 GEF2 has a GEF activity for human RhoA but not RAC. Therefore, UNC-73 GEF2 probably act through rho-1 but not ced-10 or mig-2 to control axon outgrowth of DDs, VDs, and amphid neurons. An additional GEF besides UNC-73 may be important for the activation of CED-10 and MIG-2 during the axon outgrowth of DDs, VDs, and amphid neurons, since defects in these axon outgrowths in unc-73(rh40) mutants are not as severe as those in unc-73(rh40);mig-2(mu28), unc-73(rh40); ced-10 and ced-10;mig-2(mu28) double mutants. Therefore, multiple GEFs may regulate various GTPase activities during the development of DD, VD, and amphid axons, patterning axons by regulating their directional extension (Wu, 2002).

Rac GTPases control cell shape by regulating downstream effectors that influence the actin cytoskeleton. UNC-115, a putative actin-binding protein similar to human abLIM/limatin, has been implicated in axon pathfinding. The role of UNC-115 as a downstream cytoskeletal effector of Rac signaling in axon pathfinding has now been discovered. unc-115 double mutants with ced-10 Rac, mig-2 Rac or unc-73 GEF but not with rac-2/3 Rac display synthetic axon pathfinding defects, and loss of unc-115 function suppresses the formation of ectopic plasma membrane extensions induced by constitutively-active rac-2 in neurons. Furthermore, UNC-115 can bind to actin filaments. Thus, UNC-115 is an actin-binding protein that acts downstream of Rac signaling in axon pathfinding (Struckhoff, 2003).

UNC-115 is a molecule similar to the human actin-binding protein abLIM/limatin and consists of three N-terminal LIM domains, which are thought to mediate protein-protein interactions, and a C-terminal villin headpiece domain, an actin-binding domain found in a variety of proteins. UNC-115 is required for pathfinding of many but not all axons in C. elegans, and dominant-negative abLIM/limatin can perturb RGC axon pathfinding in the developing mouse visual system. UNC-115 might act as a cytoskeletal adapter protein that interacts with actin via the VHD and with other molecules via the LIM domains. UNC-115 acts downstream of Rac signaling during axon pathfinding, suggesting that UNC-115 might adapt Rac activity to the growth cone actin cytoskeleton (Struckhoff, 2003).

In C. elegans, unc-115 is expressed in most if not all neurons throughout development, yet many neurons display normal or near-normal axon pathfinding and development in unc-115 null mutants. This paradoxical observation is explained by the demonstration that unc-115 acts in the rac-2/3 branch of the tripartite rac cascade and has overlapping function with the ced-10 and mig-2 pathways in axon pathfinding. However, ced-10(n1993) is not a null allele and unc-115 might enhance ced-10(n1993). Therefore, unc-115 might act in both the rac-2/3 and ced-10 pathways. If this is the case, then there must be other genes that act in parallel to unc-115 in the ced-10 pathway, since the unc-115 null phenotype is viable and fertile and does not resemble the ced-10 null. Although unc-115 might act in the rac-2/3 pathway in axon pathfinding, unc-115 might act with all three racs in the suppression of ectopic axons, since unc-115 mutants alone display ectopic axon formation that is not enhanced by mutations in the three rac genes (Struckhoff, 2003).

There are genetic relationships between the rac genes, unc-73 and unc-115 in axon pathfinding. Although other results indicate that UNC-73 controls the three Racs in axon development, this does not exclude the possibility that UNC-73 has additional, Rac-independent effects on axon pathfinding. In addition to the GEF1 Rac GEF domain, UNC-73 has a second GEF domain (GEF2) that acts on the Rac-related small GTPase Rho. Possibly, rho-1, the single C. elegans gene that encodes Rho, acts downstream of unc-73 in parallel to the racs to control PDE axon development (Struckhoff, 2003).

Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. An examination was carried out to see whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse is accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases are capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolishes CNS myelin-induced collapse and allows remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1 induces growth cone collapse and promotes neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 are effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse (Kuhn, 1999).

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, a gain-of-function UNC-40 molecule, MYR::UNC-40 (an UNC-40 fusion protein in which the extracellular and transmembrane domains are deleted and replaced by sequences encoding a membrane-targeting myristoylation signal) is generated. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein: Drosophila homolog - unc-115). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. These results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton (Gitai, 2003).

Netrins have been shown to promote outgrowth and guidance: vertebrate Netrin-1 was originally identified based on its ability to enhance axon outgrowth into a collagen matrix, and Netrin-1 knockout mice have defects in axon outgrowth in addition to axon guidance. Netrin can also orient axon outgrowth. Both of these effects of Netrin are dependent on the DCC receptor. The results of this study suggest that MYR::UNC-40 activates cytoplasmic signaling of the UNC-40 pathway in a constitutive, ligand-independent manner. The in vivo activation of signaling by the deletion of the extracellular and transmembrane domains suggests that these domains normally function to prevent UNC-40 activation but are disinhibited when UNC-6 binds to UNC-40. A similar disinhibition model has been proposed for the role of Netrin in activating the DCC-UNC-5 complex for axon repulsion (Gitai, 2003).

Double and triple mutant analysis indicates that all of the myr::unc-40 suppressors, unc-34, ced-10, and unc-115 are likely to participate in the endogenous unc-40 signaling pathway. These results suggest that myr::unc-40 activates the endogenous unc-40 signaling pathway, consistent with its acting as a constitutively active form of unc-40. unc-34, ced-10, and unc-115 were found to signal downstream of unc-40 in two parallel, partially redundant pathways. unc-34/Enabled also plays a partially redundant role in the sax-3/Robo pathway. The activation of parallel signaling modules with some functional overlap or redundancy may be a general feature of axon guidance signaling. It is worth noting that this apparent genetic redundancy could result from disrupting cell biological processes that are actually distinct. The activation of multiple pathways for cytoskeletal remodeling by guidance receptors may contribute to accurate guidance through various physical environments (Gitai, 2003).

MYR::UNC-40 is capable of inducing axon outgrowth, misguidance, branching, and cell body deformation. All of these phenotypes can be suppressed by unc-34, ced-10, and unc-115 or by deletions in the P1 and P2 motifs. These results suggest that distinct effects on cell morphology can be induced by the same signaling pathways, consistent with the observation that Netrin can signal through DCC to regulate cell migration, axon outgrowth, axon attraction, and axon repulsion (Gitai, 2003).

MYR::UNC-40 activity generates new outgrowths even in the adult stage, well past the normal period of neuronal development. It thus seems likely that downstream effectors of UNC-40 persist and remain functional into adulthood. Indeed, reporter gene fusions to unc-115 and ced-10 are expressed throughout the life of C. elegans. One possibility is that these genes function later in development to increase the size of the neuron as the size of the animal increases (Gitai, 2003).

These results identified two distinct pathways that mediate UNC-40 signaling: UNC-34/Enabled acts in one and CED-10/Rac and UNC-115/abLIM act in the other. Rac proteins have previously been shown to play roles in axon guidance, and Rac function is essential for repulsive axon guidance signaling by the Semaphorin receptor, Plexin. The involvement of a Rac protein in Netrin attraction is consistent with the observation that Rac promotes lamellipodial extension, since growth cones have a flattened area with some similarities to lamellipodia. Indeed, recent reports demonstrate that Netrin stimulation can activate Rac in vitro. It is interesting that ced-10 is important in the unc-40 pathway, but both mig-2, which encodes another C. elegans Rac-like protein, and unc-73, which encodes a Guanine Nucleotide Exchange Factor (GEF), are not. In preliminary studies, a mutation in rac-2(ok326), the third Rac-like gene in C. elegans, appears to partially suppress the excess outgrowth of MYR::UNC-40, suggesting that UNC-40 may signal to several, but not all, Rac proteins (Gitai, 2003).

The mechanisms by which Rac proteins cause changes in the actin cytoskeleton during axon guidance are largely unknown. The results suggest that UNC-115 acts as an element in the Rac signaling pathway. The UNC-115 protein contains three LIM domains and a villin headpiece domain. UNC-115 has been proposed to bind actin through its villin headpiece domain; thus, UNC-115 may provide a link between Rac and actin. A different LIM domain-containing protein, LIM-kinase, acts downstream of Rac through a PAK intermediate. The role of UNC-115 in axon guidance is not specific to C. elegans; a dominant-negative form of a vertebrate UNC-115 homolog, abLIM, can cause axon defects in retinal ganglion cells (Gitai, 2003).

Directed-turning toward an axonal attractant requires propagation of spatial information about the source of the attractant to downstream signaling events. Localized signaling might be achieved by localized nucleation of a signaling complex around the activated receptor. The activation of the UNC-34- and CED-10/UNC-115-dependent pathways by UNC-40 correspond to the specific conserved P1 and P2 motifs within the UNC-40 cytoplasmic domain. It is suggested that these actin-regulatory activities may remain closely associated with the activated receptor. UNC-40 may thus function as a scaffold for assembling several independent activities that regulate the cytoskeleton (Gitai, 2003).

The ephrin/Eph system plays a central role in neuronal circuit formation; however, its downstream effectors are poorly understood. α-chimerin Rac GTPase-activating protein mediates ephrinB3/EphA4 forward signaling. A spontaneous mouse mutation, miffy (mfy), was discovered that results in a rabbit-like hopping gait, impaired corticospinal axon guidance, and abnormal spinal central pattern generators. Using positional cloning, transgene rescue, and gene targeting, loss of α-chimerin was found to lead to mfy phenotypes similar to those of EphA4−/− and ephrinB3−/− mice. α-chimerin interacts with EphA4 and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, downregulation of α-chimerin suppresses ephrinB3-induced growth cone collapse in cultured neurons. These findings indicate that ephrinB3/EphA4 signaling prevents growth cone extension in motor circuit formation via α-chimerin-induced inactivation of Rac. They also highlight the role of a Rho family GTPase-activating protein as a key mediator of ephrin/Eph signaling (Iwasato, 2007).

Axon migrations are guided by extracellular cues that induce asymmetric outgrowth activity in the growth cone. Several intracellular signaling proteins have been implicated in the guidance response. However, how these proteins interact to generate asymmetric outgrowth activity is unknown. Evidence is presented that in C. elegans, the CED-10/Rac1 GTPase binds to and causes asymmetric localization of MIG-10/lamellipodin, a protein that regulates actin polymerization and has outgrowth-promoting activity in neurons. Genetic analysis indicates that mig-10 and ced-10 function together to orient axon outgrowth. The RAPH domain of MIG-10 binds to activated CED-10/Rac1, and ced-10 function is required for the asymmetric MIG-10 localization that occurs in response to the UNC-6/netrin guidance cue. Asymmetric localization of MIG-10 in growth cones is associated with asymmetric concentrations of f-actin and microtubules. These results suggest that CED-10/Rac1 is asymmetrically activated in response to the UNC-6/netrin signal and thereby causes asymmetric recruitment of MIG-10/lamellipodin. It is proposed that the interaction between activated CED-10/Rac1 and MIG-10/lamellipodin triggers local cytoskeletal assembly and polarizes outgrowth activity in response to UNC-6/netrin (Quinn, 2008).

Rac and Synapses

Dynamic control of excitatory synapse development by a Rac1 GEF/GAP regulatory complex

The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. This study, carried out in cultured mammalian cells, identified a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases (Um, 2014).

The small GTPase Rac1 (see Drosophila Rac1) orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. This study, carried out in cultured mammalian cells, identified a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors (see Drosophila Eph) utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases (Um, 2014).

Thrombospondin receptor alpha2delta-1 promotes synaptogenesis and spinogenesis via postsynaptic Rac1

Astrocytes control excitatory synaptogenesis by secreting thrombospondins (TSPs), which function via their neuronal receptor, the calcium channel subunit alpha2delta-1 (see Drosophila straightjacket). alpha2delta-1 is a drug target for epilepsy and neuropathic pain; thus the TSP-alpha2delta-1 interaction is implicated in both synaptic development and disease pathogenesis. However, the mechanism by which this interaction promotes synaptogenesis and the requirement for alpha2delta-1 for connectivity of the developing mammalian brain are unknown. This study shows that global or cell-specific loss of alpha2delta-1 yields profound deficits in excitatory synapse numbers, ultrastructure, and activity and severely stunts spinogenesis in the mouse cortex. Postsynaptic but not presynaptic alpha2delta-1 is required and sufficient for TSP-induced synaptogenesis in vitro and spine formation in vivo, but an alpha2delta-1 mutant linked to autism cannot rescue these synaptogenesis defects. Finally, this study reveals that TSP-alpha2delta-1 interactions control synaptogenesis postsynaptically via Rac1, suggesting potential molecular mechanisms that underlie both synaptic development and pathology (Risher, 2018).

Rac and Junctions

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. Roles of the RhoA and Rac1 GTPases were examined in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturb TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy reveals abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appear normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupt the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane are unaffected. Expression levels and protein complexes of occludin and ZO-1 appear normal in all mutant cells, although ZO-1 is more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane (Jou, 1998b).

During neuromuscular junction formation, agrin secreted from motor neurons causes muscle cell surface acetylcholine receptors (AChRs) to cluster at synaptic sites by mechanisms that are insufficiently understood. The Rho family of small guanosine triphosphatases (GTPases), including Rac and Cdc42, can mediate focal reorganization of the cell periphery in response to extracellular signals. The role of Rac and Cdc42 in coupling agrin signaling to AChR clustering was investigated. Agrin causes marked muscle-specific activation of Rac and Cdc42 in differentiated myotubes, as detected by biochemical measurements. Moreover, this activation is crucial for AChR clustering, since the expression of dominant interfering mutants of either Rac or Cdc42 in myotubes blocks agrin-induced AChR clustering. In contrast, constitutively active Rac and Cdc42 mutants cause AChR to aggregate in the absence of agrin. Activation of AChR clustering was detected using three separate measurements and was demonstrated both with endogenous Rac in nontransfected muscle cultures and with ectopically expressed Rac and Cdc42 in transfected myotubes. The most direct demonstration involves agrin-induced increases in the selective binding of activated (GTP-bound) Rac and Cdc42 to a Rac/Cdc42-binding domain derived from PAK, a downstream effector molecule that is thought to link Rac activation to actin polymerization. Additional evidence for agrin stimulation of Rac/Cdc42 was obtained by recording the activation of a second downstream component, JNK, and the conclusion that JNK activation by agrin is indeed mediated by Rac and Cdc42 was confirmed by showing that this activation is blocked by overexpression of dominant negative mutants of either G protein. By indicating that agrin-dependent activation of Rac and Cdc42 constitutes a critical step in the signaling pathway leading to AChR clustering, these findings suggest a novel role for these Rho-GTPases: the coupling of neuronal signaling to a key step in neuromuscular synaptogenesis. How might Rac and Cdc42 mediate the agrin-initiated clustering of AChR and other constituents of the neuromuscular junction? The ability of activated Rac/Cdc42 to induce reorganization of cortical actin by modulating the dynamics of actin polymerization is well documented. Moreover, surface AChR is thought to be attached to the actin cytoskeleton via a complex in which rapsyn links AChR to the actin-binding protein utrophin, and recent findings indicate that agrin-induced AChR aggregation involves the clustering of these diffusely distributed complexes at sites of MuSK activation. Thus it is thought that agrin-induced activation of Rac/Cdc42 produces highly localized reorganization of cortical actin cytoskeleton, resulting in redistribution of actin-anchored AChR-containing complexes into clusters (Weston, 2000).

Rac and Differentiation

Rho family proteins play a critical role in muscle differentiation. The Rho family of GTP-binding proteins consists of the Rho, Rac, and Cdc42 subfamilies and has been demonstrated to regulate numerous aspects of cytoskeleton function. Rho family proteins also play a critical role in transcriptional regulation of the c-fos gene by modulating the transcription factor SRF. Since SRF also binds to the CArG box, which is a critical cis element in the promoters of many muscle-specific genes, an examination was made to determine whether the Rho family plays an important role in the expression of muscle-specific genes. To test whether Rho family G proteins are involved in transcription of muscle-specific genes, the role of Rho family proteins was examined in the transcription of the skeletal alpha-actin gene, which depends on the CArG box. A luciferase reporter plasmid containing the skeletal alpha-actin promoter (bp -394 to +24) was transiently transfected into C2C12 myoblasts together with dominant interfering mutants of Rac1 (Rac1N17), RhoA (RhoAN17), and Cdc42 (Cdc42N17) or the GDP dissociation inhibitor RhoGDI. Luciferase activities were measured at 36 h after induction of C2C12 cell differentiation (by changing the culture medium from GM to DM). The transcriptional activity of the skeletal alpha-actin gene is much higher in differentiated myotubes than in undifferentiated myoblasts and is reduced by cotransfection of either dominant interfering plasmid and RhoGDI. RhoGDI, which inhibits the functions of all Rho family members, most strongly inhibits the transcriptional activity of the skeletal alpha-actin gene during muscle differentiation; among the three dominant interfering mutants the inhibitory activity is strongest in Rac1N17. Transfection of these interfering mutants of the Rho family proteins and RhoGDI do not show such a strong inhibitory effect on transcription of nonmuscle gene promoters such as the SV40-derived promoter. The effects of wild-type and mutationally activated forms of Rho family G proteins (wild-type Rac1, Rac1V12, RhoAV12, or Cdc42V12) were tested on the activity of the skeletal alpha-actin promoter. Although overexpression of wild-type Rac1 has no significant effects on the activity of the skeletal alpha-actin promoter, all constitutively active mutants of Rho family proteins activate the promoter to various degrees. Rac1V12 most strongly activates the transcription, by more than 10-fold. It is concluded that dominant negative forms of Rho family proteins and RhoGDI, a GDP dissociation inhibitor, suppress transcription of muscle-specific genes, while mutationally activated forms of Rho family proteins strongly activate their transcription (Takano, 1998).

C2C12 cells overexpressing RhoGDI (C2C12RhoGDI cells) do not differentiate into myotubes, and expression levels of myogenin, MRF4, and contractile protein genes (but not MyoD and myf5 genes) are markedly reduced in C2C12RhoGDI cells. The promoter activity of the myogenin gene is suppressed by dominant negative mutants of Rho family proteins and is reduced in C2C12RhoGDI cells. Expression of myocyte enhancer binding factor 2 (MEF2), which has been reported to be required for the expression of the myogenin gene, is reduced at the mRNA and protein levels in C2C12RhoGDI cells. These results suggest that the Rho family proteins play a critical role in muscle differentiation, possibly by regulating the expression of the myogenin and MEF2 genes (Takano, 1998).

The mouse small intestinal epithelium undergoes continuous renewal throughout life. Previous studies suggest that differentiation of this epithelium is regulated by instructions that are received as cells migrate along crypt-villus units. The nature of the instructions and their intracellular processing remain largely undefined. In this report, genetic mosaic analysis was used to examine the role of Rac1 GTPase-mediated signaling in controlling differentiation. A constitutively active mutation (Rac1Leu61) or a dominant negative mutation (Rac1Asn17) was expressed in the 129/Sv embryonic stem cell-derived component of the small intestine of mice. Rac1Leu61 induces precocious differentiation of members of the Paneth cell and enterocytic lineages in the proliferative compartment of the fetal gut, without suppressing cell division. Forced expression of the dominant negative mutation inhibits epithelial differentiation, without affecting cell division, and slows enterocytic migration along crypt-villus units. The effects produced by Rac1Leu61 or Rac1Asn17 in the epithelium do not spread to adjacent normal epithelial cells. These results provide in vivo evidence that Rac1 is involved in the import and intracellular processing of signals that control differentiation of a mammalian epithelium (Stappenbeck, 2000).

These findings support the notion that expression of Rac1 mutations perturbs the proper entry and/or processing of (unspecified) extracellular cues that normally help define the state of epithelial differentiation in vivo. This idea is consistent with the formulation that differentiation of intestinal epithelial cells is regulated in large part by cell non-autonomous mechanisms, and that position-dependent cues are received during the course of cellular migration along crypt-villus units. It is not known whether the constitutively active or dominant negative Rac1 perturbs receipt or processing of signals imported from the mesenchyme underlying the small intestinal epithelium, or from adjacent epithelial cells. The effect of the constitutive active mutation on differentiation is not 'exported' to juxtaposed normal cells. This suggest that the protein acts directly within expressing cells, and not by re-sculpting the local extracellular environment so as to generate instructions sufficient to enforce differentiation of non-expressing cells (Stappenbeck, 2000).

Rac and photoreceptor response

While small GTPases have been investigated in a wide variety of cells, few studies have addressed their role in photoreceptors. In vertebrate retinal rods, the light stimulus is transmitted from rhodopsin via the pathway mediated by the heterotrimeric G protein transducin. To increase their sensitivity to light, photoreceptors accumulate remarkably high concentrations of rhodopsin and transducin in specialized cellular compartments, the outer segments (OS). Transport of these proteins from the inner segments is regulated by the small GTPases Rab6 and Rab8, which do not enter OS. This study asks whether small G proteins have other functions in photoreceptors. OS are shown to contain the small GTPase Rac-1, a member of the Rho family. In contrast to other cells, Rac-1 in OS is exclusively associates with the membranes and resides in lipid rafts. Most importantly, Rac-1 is activated by light. This activation is specifically blocked by a synthetic peptide corresponding to the Asn-Pro-X-X-Tyr motif found in rhodopsin, and Rac-1 coprecipitates with rhodopsin on Concanavalin A Sepharose. These data provide the first direct evidence for the existence of a novel pathway activated by rhodopsin (Balasubramanian, 2003).

The key biological function of photoreceptor OS is generating a photoresponse, which occurs via the classic transducin-mediated pathway. What could be the potential role of Rac-1 in this system? Although a regulatory role in phototransduction cannot be ruled out at this point, Rac-1 could be involved in a different process(es). Light sets in motion slow molecular events important for light adaptation, for example, active transport of proteins: transducin moves from the outer to inner segments, and arrestin travels in the opposite direction. Although, in general, protein traffic in photoreceptors is not well understood, it has been shown that rhodopsin movement to OS occurs via microtubules. Since Rac-1 is known to regulate microtubules, actin cytoskeleton, and vesicular trafficking, it is a good candidate for being involved in various transport processes, such as protein trafficking and moving and/or shedding of the disks, which is a light-sensitive process. In Drosophila, expression of constitutively active Rac-1 restores morphological changes resulting from a rhodopsin null mutation via an unidentified mechanism. This work brings out light-mediated activation of Rac-1 as a potential mechanism regulating cell morphology and perhaps other functions in normal vertebrate photoreceptors (Balasubramanian, 2003 and references therein).

Dihydroceramide desaturase regulates the compartmentalization of Rac1 for neuronal oxidative stress

Disruption of sphingolipid homeostasis is known to cause neurological disorders, but the mechanisms by which specific sphingolipid species modulate pathogenesis remain unclear. The last step of de novo sphingolipid synthesis is the conversion of dihydroceramide to ceramide by dihydroceramide desaturase (human DEGS1; Drosophila Ifc). Loss of ifc leads to dihydroceramide accumulation, oxidative stress, and photoreceptor degeneration, whereas human DEGS1 variants are associated with leukodystrophy and neuropathy. This work demonstrates that DEGS1/ifc regulates Rac1 compartmentalization in neuronal cells and that dihydroceramide alters the association of active Rac1 with organelle-mimicking membranes. The Rac1-NADPH oxidase (NOX) complex was identified as the major cause of reactive oxygen species (ROS) accumulation in ifc-knockout (ifc-KO) photoreceptors and in SH-SY5Y cells with the leukodystrophy-associated DEGS1(H132R) variant. Suppression of Rac1-NOX activity rescues degeneration of ifc-KO photoreceptors and ameliorates oxidative stress in DEGS1(H132R)-carrying cells. Therefore, it is concluded that DEGS1/ifc deficiency causes dihydroceramide accumulation, resulting in Rac1 mislocalization and NOX-dependent neurodegeneration (Tzou, 2021).

Rac. learning, memory and forgetting

Hippocampal activation of Rac1 regulates the forgetting of object recognition memory

Forgetting is a universal feature for most types of memories. The best-defined and extensively characterized behaviors that depict forgetting are natural memory decay and interference-based forgetting. In Drosophila, training-induced activation of the small G protein Rac1 mediates natural memory decay and interference-based forgetting of aversive conditioning memory. In mice, the activation of photoactivable-Rac1 in recently potentiated spines in a motor learning task erases the motor memory. These lines of evidence prompted an investigation of the role for Rac1 in time-based natural memory decay and interference-based forgetting in mice. The inhibition of Rac1 activity in hippocampal neurons through targeted expression of a dominant-negative Rac1 form extended object recognition memory from less than 72 hr to over 72 hr, whereas Rac1 activation accelerated memory decay within 24 hr. Interference-induced forgetting of this memory was correlated with Rac1 activation and was completely blocked by inhibition of Rac1 activity. Electrophysiological recordings of long-term potentiation provided independent evidence that further supported a role for Rac1 activation in forgetting. Thus, Rac1-dependent forgetting is evolutionarily conserved from invertebrates to vertebrates (Liu, 2016).

Rac and apoptosis

Fas receptor ligation activates the small G-protein Rac1, Jun N-terminal kinase (see Drosophila basket/JNK)/p38 kinases (p38-K), and the transcription factor GADD153. Cellular treatment with synthetic C6-ceramide results in the phosphorylation of these same proteins. A signaling cascade from the Fas receptor via ceramide, Ras, Rac1, and JNK/p38-K to GADD153 has been demonstrated, employing either transfection of transdominant inhibitory N17Ras, N17Rac1, c-Jun, or treatment with a specific p38-K inhibitor. The critical function of this signaling cascade is indicated by prevention of Fas- or C6-ceramide-induced apoptosis after inhibition of Ras, Rac1, or JNK/p38-K (Brenner, 1997b).

Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta; this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen with 32PO4 labeling as well as in the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses (Maundrell, 1997).

Rac and cellular oncogenic transformation

Although substantial evidence supports a critical role for the activation of Raf-1 (a target of Ras) and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation (see Drosophila Ras1), recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, three complementary approaches were used to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant causes a greater than 35-fold enhancement of transforming activity. Second, coexpression of dominant negative mutants of Rac1 and RhoA reduces oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhance oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, kinase-deficient MAPKs inhibit Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation (Khosravi-Far, 1995).

The heterotrimeric G-protein, G alpha12, together with the closely-related G alpha13, are both members of the G12 class of alpha-subunits important in mediating the signaling from seven transmembrane domain-spanning receptors. Recent evidence implicating both G alpha12 and G alpha13 in the activation of signaling pathways involving members of the RHO gene family has led to an examination of the role of Rac1, RhoA and Cdc42Hs in the transforming properties of G alpha12. Asparagine 17 (Asn 17) dominant inhibitory mutants of Rac1, and to a lesser extent RhoA, block focus forming ability of the GTPase-deficient mutant of G alpha12 (G alpha12 Leu 229) in NIH3T3 cells. In turn, wild-type G alpha12 cooperates well with Rac1 Val 12 but not with the RhoA Leu 63 mutant in transforming NIH3T3 cells. Interestingly, the morphology of foci induced by G alpha12 and RhoA mutants is strikingly similar and is distinct from those displayed by Rac1 Val 12 mutant. The fact that G alpha12's ability to induce mitogenesis in NIH3T3 cells is not significantly perturbed by C3 ribosyltransferase suggests that RhoA does not play a major role in G alpha12-induced mitogenic events. Activated mutant of Rac1 stimulates the activity of the stress-induced c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs). Transient co-transfection of Rac1 Val 12 mutant with the wild-type G alpha12 in COS7 cells leads to the further activation of an exogenously expressed hemagglutinin(HA)-tagged JNK. Furthermore, the cooperation between G alpha12 and Rac1 in cellular transformation is correlated with their ability to stimulate transcription from c-fos serum response element (SRE) (Tolkacheva, 1997).

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of either fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change, remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, it is proposed that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton (He, 1998).

Table of contents

Rac1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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