Involvement of Rho and Rac small G proteins and Rho GDI in Ca2+-dependent exocytosis from PC12 cells

Background : The Rho small G protein family, which includes the Rho, Rac and Cdc42 subfamilies, is implicated in various cell functions such as cell shape change, cell motility and cytokinesis, through the reorganization of actin filaments. Rho GDI is an inhibitory regulator of the Rho small G protein family and inhibits the Rho family dependent cell functions. Reorganization of actin filaments is also known to regulate Ca²⁺-dependent exocytosis.
Results: We have examined here whether the Rho family members are also involved in Ca²⁺-dependent exocytosis. We have found, by the use of the human growth hormone (GH) co-expression assay system on PC12 cells, that overexpression of Rho GDI inhibits high K⁺-induced, Ca²⁺-dependent GH release. This inhibitory action of Rho GDI is restored by co-expression of a dominant active mutant of RhoA or Rac1, but not of a dominant active mutant of Cdc42. C3 transferase, known to ADP-ribosylate Rho and to inhibit its function, also inhibits this GH release. Overexpression of a dominant active mutant of RhoA or Rac1 alone shows only a small effect on GH release. Moreover, immunocytochemical studies show that the overexpression of Rho GDI prevents a partial disruption of the cortical actin network which accompanies exocytosis.
Conclusions: These results suggest that RhoA, Rac1 and Rho GDI are involved in Ca²⁺-dependent exocytosis at least partly through the reorganization of actin filaments, and that the activation of RhoA or Rac1 alone is not sufficient for this reaction.     

Agents That Inhibit Rho, Rac, and Cdc42 Do Not Block Formation of Actin Pedestals in HeLa Cells Infected with Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.     

Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes

Motile lymphocytes adopt a polarized morphology with different adhesion molecules (ICAM, CD43 and CD44) and ERM actin-binding proteins concentrated on the uropod, a slender posterior appendage with important functions in cell-cell interactions and lymphocyte recruitment. We have studied the role of Rho family of GTPases (Rho, Rac and Cdc42) in the control of lymphocyte polarity and migration by analyzing the effects of exogenously introduced Rho GTPase mutants. Transfection of T cell lines that constitutively display a polarized motile morphology with activated mutants of RhoA, Rac1 and Cdc42 impaired cell polarization. A guanosine nucleotide exchange factor for Rac, Tiam-1, induced the same effect as activated Rac1. Conversely, dominant negative forms of the three GTP-binding proteins induced a polarized phenotype in constitutively round-shaped T cells with redistribution of ICAM-3 and moesin to the uropod in an integrin-dependent manner. On the other hand, overexpression of dominant negative Cdc42 and activated mutants of all three Rho GTPases significantly inhibited SDF-1alpha-induced T cell chemotaxis. Together, these data demonstrate that Rho GTPases regulate lymphocyte polarization and chemokine-induced migration, and underscore the key role of Cdc42 in lymphocyte directional migration.     

Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells

BACKGROUND: Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumour cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumour cells in an in vitro fibronectin degradation/invasion assay. The Rho family small G proteins, consisting of the Rho, Rac and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We studied the roles of the Rho family small G proteins in invadopodia formation. RESULTS: We first demonstrated that invadopodia of RPMI7951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (PI3K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation. CONCLUSION: These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signalling pathway.     

Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins

The Rho GTPases, which belong to the Ras superfamily of low-molecular-mass GTP-binding proteins, are the preferred intracellular targets of bacterial protein toxins. The Rho GTPases RhoA/B/C, Rac1/2 and Cdc42 are the master regulators of the actin cytoskeleton. Clostridium difficile toxins A and B, the causative agents of the antibiotic-associated pseudomembranous colitis, are intracellularly acting cytotoxins which mono-glucosylate the Rho GTPases. Clostridium botulinum C3 toxin, which is not related to the clostridial neurotoxins, catalyses ADP-ribosylation of RhoA/B/C but not of other Rho GTPases. Glucosylation as well as ADP-ribosylation result in functional inactivation of Rho causing disassembly of the actin cytoskeleton.     

Pseudomonas aeruginosa ExoT is a Rho GTPase-activating protein

Transient intracellular expression of ExoT in CHO cells stimulated cell rounding and actin reorganization. Biochemical studies showed that ExoT was a GTPase-activating protein for RhoA, Rac1, and Cdc42. Together, these data show that ExoT interferes with Rho signal transduction pathways, which regulate actin organization, exocytosis, cell cycle progression, and phagocytosis.     

The first CH domain of affixin activates Cdc42 and Rac1 through alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor

Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell-substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin-linked kinase (ILK)-binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co-immunoprecipitated with alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of alphaPIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of alphaPIX, alphaPIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation.     

RhoA activation promotes transendothelial migration of monocytes via ROCK

Monocyte infiltration into inflamed tissue requires the initial arrest of the cells on the endothelium followed by firm adhesion and their subsequent migration. Migration of monocytes and other leukocytes is believed to involve a coordinated remodeling of the actin cytoskeleton. The small GTPases RhoA, Rac1, and Cdc42 are critical regulators of actin reorganization. In this study, we have investigated the role of Rho-like GTPases RhoA, Rac1, and Cdc42 in the adhesion and migration of monocytes across brain endothelial cells by expressing their constitutively active or dominant-negative constructs in NR8383 rat monocytic cells. Monocytes expressing the active form of Cdc42 show a reduced migration, whereas Rac1 expression did not affect adhesion or migration. In contrast, expression of the active form of RhoA in monocytes leads to a dramatic increase in their adhesion and migration across endothelial cells. The effect of RhoA was found to be mediated by its down-stream effector Rho kinase (ROCK), as pretreatment with the selective ROCK inhibitor Y-27632 prevented this enhanced adhesion and migration. These results demonstrate that RhoA activation in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells.     

In Vivo Rho GTPase-Activating Protein Activity of Pseudomonas aeruginosa Cytotoxin ExoS

ExoS is a bifunctional type III cytotoxin secreted by Pseudomonas aeruginosa, which comprises a C-terminal ADP ribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain. In vitro, ExoS is a Rho GAP for Rho, Rac, and Cdc42; however, the in vivo modulation of Rho GTPases has not been addressed. Using a transient transfection system and delivery by P. aeruginosa, interactions were examined between the Rho GAP domain of ExoS and Rho GTPases in CHO cells. Rho GTPases were expressed as green fluorescent protein (GFP) fusion proteins to facilitate quantitation. GFP fusions of wild-type and dominant active Rho, Rac, and Cdc42 localized to discrete regions of CHO cells and appeared functional based upon their modulation of the actin cytoskeleton. Coexpression of the Rho GAP domain of ExoS changed the intracellular distribution of GFP-Rac and GFP-Cdc42 from a predominately membrane location to a cytosolic location. Coexpression of the Rho GAP domain of ExoS did not change the distribution of GFP-Rho, which was primarily in the cytosol. Coexpression of dominant active Rac (DARac) and DACdc42 inhibited actin reorganization by the Rho GAP domain but did not maintain the formation of actin stress fibers, which indicated that Rho had been inactivated. Similar results were observed when ExoS was delivered into CHO cells by P. aeruginosa. These data indicate that in vivo the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibition of Rho.     

Rho regulates T cell receptor ITAM-induced lymphocyte spreading in an integrin-independent manner

T cell receptor (TCR) engagement increases integrin-mediated adhesion to APC, resulting in the stabilization of the T cell : APC interaction and the close apposition of the two cell membranes. Here we show that engagement of either the TCR or CD3 chimeras with immobilized antibodies causes the rapid spreading of T cells in an integrin-independent fashion. This effect concurs with the polymerization of the actin cytoskeleton and is dependent on the integrity of the immunoreceptor tyrosine-based activation motifs of the CD3 subunits. Expression of a dominant negative mutant of RhoA, as well as the Rho-specific inhibitor C3 toxin, abolished TCR-induced spreading. In contrast, constitutively active or dominant negative forms of Rac and Cdc42 did not affect cell spreading. We conclude that signals emanating from the TCR can directly induce T cell spreading, independently of integrins, and via a Rho-dependent reorganization of the actin cytoskeleton.     

Rho家族小GTP酶在黑素细胞树突形成和黏附中的作用

黑素细胞通过其树突将合成的黑素转运至角质形成细胞,进而发挥生理功能。黑素细胞树突形成是黑素转运过程中的重要环节,黑素转运必须在黑素细胞和角质形成细胞紧密接触后才能实现。黑素细胞形态学改变包括胞体大小和树突变化等细胞骨架的改变,细胞骨架变化主要与肌动蛋白和微管结构的重排有关。Rho家族小GTP酶包括20种成员,其中RhoA,Rac1和Cdc42对黑素细胞细胞骨架的变化和细胞黏附的调节起重要作用。     

Role of the Rho GTPase-activating protein RICS in neurite outgrowth

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase-activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS(-/-) neurons was higher than that in wild-type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS(-/-) mice bore longer neurites than those from wild-type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.     

Importance of spatial activation of Cdc42 and rac small G proteins by frabin for microspike formation in MDCK cells

BACKGROUND: Frabin is an actin filament (F-actin)-binding protein that shows GDP/GTP exchange activity for Cdc42 small G protein (Cdc42). Frabin furthermore induces indirect activation of Rac small G protein (Rac) in intact cells. We have recently shown that in nonepithelial cells, frabin induces the formation of both filopodia- and lamellipodia-like processes through the activation of Cdc42 and Rac, respectively. In epithelial cells such as MDCK cells, Cdc42 and Rac regulate cell-cell adherens junctions (AJs) via the accumulation of F-actin and E-cadherin, although neither Cdc42 nor Rac induces the formation of filopodia or lamellipodia. In this study, we have examined the effects of frabin on the reorganization of the actin cytoskeleton in MDCK cells. RESULTS: Frabin induces the formation of microspikes at the basal area of the lateral membranes through the activation of Cdc42 and Rac in MDCK cells, although a dominant active mutant of Cdc42 or Rac alone, or both, did not induce the formation of microspikes. Furthermore, frabin weakly increased the accumulation of F-actin and E-cadherin at cell-cell AJs and the formation of stress fibres through the activation of Cdc42 and Rac, under conditions where the dominant active mutant of Cdc42 or Rac markedly showed these effects. The Cdc42- and Rac-induced formation of stress fibres was dependent on the activation of Rho small G protein. CONCLUSION: These results indicate that the frabin-dependent spatial activation of Cdc42 and Rac is important for the formation of microspikes.     

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Background : We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin‐mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. Results : During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenin δ1 mRNAs with a splice‐site morpholino caused lateral widening and anterior‐posterior shortening of the presomitic mesoderm and somites and a shortened anterior‐posterior axis. These phenotypes indicate a cell‐migration effect. Co‐injection of low amounts of wild‐type Cdc42 or wild‐type Rac1 or dominant‐negative RhoA mRNAs, but not constitutively‐active Cdc42 mRNA, rescued these p120 catenin δ1‐depleted embryos. Conclusions : These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development. Developmental Dynamics 241:1545–1561, 2012. © 2012 Wiley Periodicals Inc.     

Novel membrane traffic steps regulate the exocytosis of the Menkes disease ATPase

The Menkes disease protein (ATP7A or MNK) is a P-type transmembrane ATPase that regulates translocation of cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. In this study, we show that endogenous MNK in cultured cell lines is localized to the distal Golgi apparatus and translocates to the plasma membrane in response to exogenous copper ions. This transport event is not blocked by expression of a dominant-negative mutant protein kinase D, an enzyme implicated in regulating constitutive trafficking from the trans-Golgi network (TGN) to the plasma membrane, whereas constitutive transport of CD4 is inhibited. In contrast, protein kinase A inhibitors block copper-stimulated MNK delivery to the plasma membrane. Expression of constitutively active Rho GTPases such as Cdc42, Rac1 and RhoA reveals a requirement for Cdc42 in the trafficking of MNK, to the cell surface. Furthermore, overexpression of WASp inhibits anterograde transport of MNK, further supporting regulation by the Cdc42 GTPase. These findings define a novel step in TGN-to-plasma membrane traffic required to export MNK to the cell surface.     

Association of frabin with specific actin and membrane structures

BACKGROUND: Frabin is an actin filament (F-actin)-binding protein with GDP/GTP exchange activity specific for Cdc42 small G protein. Expression of frabin forms filopodia-like microspikes through the direct activation of Cdc42, and lamellipodia through indirect activation of Rac small G protein. Frabin consists of the F-actin-binding domain (FAB), the Dbl homology domain (DH), the first pleckstrin homology domain (PH1), the FYVE-finger domain (FYVE), the second PH domain (PH2) from the N-terminus in this order. Although DH and PH1 show exchange activity, FAB, in addition to DH and PH1, is required for the formation of microspikes, whereas FYVE and PH2, in addition to DH and PH1, are required for the formation of lamellipodia. RESULTS: Various truncated mutants of frabin were co-expressed with a dominant active mutant (DA) of Cdc42, Rac1DA, or full-length frabin in L fibroblasts. FAB was recruited to the Cdc42DA-formed filopodia-like microspikes. FAB and a fragment containing DH, PH1, FYVE and PH2 were recruited to the Rac1DA-formed membrane ruffles. Furthermore, each of these fragments served as a dominant negative mutant of frabin when co-expressed with full-length frabin, and inhibited the full-length frabin-formed morphological changes. CONCLUSION: These results suggest that frabin recognizes a specific actin structure(s) through FAB and a specific membrane structure(s) through FAB and the region containing DH, PH1, FYVE and PH2. It is likely that frabin associates with the specific actin and membrane structures and activates Cdc42 and Rac in the vicinity of these structures, eventually leading to morphological changes.     

Facilitation of Ca2+-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1-V12, Rac1-L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin-permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1-N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21-activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1-V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1-V12-mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin-A inhibited secretion and eliminated the secretory effects of Rac1-V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca²⁺ influx, partly through regulation of cytoskeletal organization.     

Cytosolic delivery and characterization of the TcdB glucosylating domain by using a heterologous protein fusion

TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB(1-556) coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB(1-556) was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB(1-556) plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.