Multiple factors confer specific Cdc42 and Rac protein activation by dedicator of cytokinesis (DOCK) nucleotide exchange factors

DOCK (dedicator of cytokinesis) guanine nucleotide exchange factors (GEFs) activate the Rho-family GTPases Rac and Cdc42 to control cell migration, morphogenesis, and phagocytosis. The DOCK A and B subfamilies activate Rac, whereas the DOCK D subfamily activates Cdc42. Nucleotide exchange is catalyzed by a conserved DHR2 domain (DOCK(DHR2)). Although the molecular basis for DOCK(DHR2)-mediated GTPase activation has been elucidated through structures of a DOCK9(DHR2)-Cdc42 complex, the factors determining recognition of specific GTPases are unknown. To understand the molecular basis for DOCK-GTPase specificity, we have determined the crystal structure of DOCK2(DHR2) in complex with Rac1. DOCK2(DHR2) and DOCK9(DHR2) exhibit similar tertiary structures and homodimer interfaces and share a conserved GTPase-activating mechanism. Multiple structural differences between DOCK2(DHR2) and DOCK9(DHR2) account for their selectivity toward Rac1 and Cdc42. Key determinants of selectivity of Cdc42 and Rac for their cognate DOCK(DHR2) are a Phe or Trp residue within β3 (residue 56) and the ability of DOCK proteins to exploit differences in the GEF-induced conformational changes of switch 1 dependent on a divergent residue at position 27. DOCK proteins, therefore, differ from DH-PH GEFs that select their cognate GTPases through recognition of structural differences within the β2/β3 strands.     

The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway

The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.     

DOCK9 induces membrane ruffles and Rac1 activity in cancer HeLa epithelial cells

Dedicator-of-cytokinesis (DOCK) proteins are a family of guanine-nucleotide exchange factors (GEF) for Rho GTPases. The DOCK-D homology subfamily comprises DOCK9, DOCK10, and DOCK11. DOCK9 and DOCK11 are GEFs for Cdc42 and induce filopodia, while DOCK10 is a dual GEF for Cdc42 and Rac1 and induces filopodia and ruffles. We provide data showing that DOCK9, the only one of the DOCK-D members that is not considered hematopoietic, is nevertheless expressed at high levels in T lymphocytes, as do DOCK10 and DOCK11, although unlike these, it is not expressed in B lymphocytes. To investigate DOCK9 function, we have created a stable HeLa clone with inducible expression of HA-DOCK9. Induction of expression of HA-DOCK9 produced loss of elongation and polygonal shape of HeLa cells. Regarding membrane protrusions, HA-DOCK9 prominently induced filopodia, but also an increase of membrane ruffles. The latter was consistent with an increase in the levels of activation of Rac1, suggesting that DOCK9 carries a secondary ability to induce ruffles through activation of Rac1.     

DOCK10-mediated Cdc42 activation is necessary for amoeboid invasion of melanoma cells

BACKGROUND: Tumor cells can move in a three-dimensional (3D) environment in either mesenchymal-type or amoeboid modes. In mesenchymal-type movement, cells have an elongated morphology with Rac-induced protrusions at the leading edge. Amoeboid cells have high levels of actomyosin contractility, and movement is associated with deformation of the cell body through the matrix without proteolysis. Because signaling pathways that control the activation of GTPases for amoeboid movement are poorly understood, we sought to identify regulators of amoeboid movement by screening an siRNA library targeting guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. RESULTS: We identified DOCK10, a Cdc42 GEF, as a key player in amoeboid migration; accordingly, we find that expression of activated Cdc42 induces a mesenchymal-amoeboid transition and increases cell invasion. Silencing DOCK10 expression promotes conversion to mesenchymal migration and is associated with decreased MLC2 phosphorylation and increased Rac1 activation. Consequently, abrogating DOCK10 and Rac1 expression suppresses both amoeboid and mesenchymal migration and results in decreased invasion. We show that the Cdc42 effectors N-WASP and Pak2 are required for the maintenance of the rounded-amoeboid phenotype. Blocking Cdc42 results in loss of mesenchymal morphology, arguing that Cdc42 is also involved in mesenchymal morphology through different activation and effector pathways. CONCLUSIONS: Previous work has identified roles of Rho and Rac signaling in tumor cell movement, and we now elucidate novel roles of Cdc42 signaling in amoeboid and mesenchymal movement and tumor cell invasion.     

The roles of Cdc42 and Rac1 in the formation of plasma membrane protrusions in cancer epithelial HeLa cells

The inducible model of clones generated from the cervical cancer epithelial HeLa cell line has shown the role of DOCK10 as a guanine-nucleotide exchange factor for Rho GTPases Cdc42 and Rac1 and as an inducer of filopodia and plasma membrane (PM) ruffles. In this model, constitutively active (CA) mutants of Cdc42 and Rac1 promote filopodia and ruffles, respectively, as in other models. DOCK9 also induces filopodia and ruffles, although ruffling activity is less prominent. By exploiting this model further, the aim of this work is to provide a more complete understanding of the role of Cdc42 and Rac1, and their interactions with DOCK10 and DOCK9, in regulation of PM protrusions. New clones have been generated from HeLa, including single clones expressing one form of wild-type (WT) or dominant negative (DN) Cdc42 or Rac1, and double clones co-expressing one of them together with either DOCK10 or DOCK9. Expression of WT- and DN-Cdc42 induced filopodia. WT-Cdc42 and, especially, DN-Cdc42 also gave rise to veil protrusions, which were neutralized by DOCK10. Moreover, DN-Cdc42 stimulated the emergence of ruffles, further increased by DOCK10, and WT-Cdc42 also augmented ruffles in presence of DOCK9 and DOCK10. WT-Rac1 greatly increased PM blebbing, as did DN-Rac1 more moderately. In both cases, blebs were enhanced by DOCK10. DN-Rac1 and CA-Rac1 moderately raised filopodia, and DOCK10 and DOCK9 had opposed effects on filopodia (up and down, respectively) in presence of WT-Rac1. As conclusions, we highlight that Cdc42 promotes filopodia regardless of its conformational state, and Rac1 induces blebs in conformations other than CA, especially WT-Rac1, in the inducible HeLa clones. The model could be useful to learn more about the mechanisms underlying PM protrusions.

     

GC-GAP,a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.     

RhoGEF specificity mutants implicate RhoA as a target for Dbs transforming activity

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. Like many RhoGEFs, the in vitro catalytic activity of Dbs is not limited to a single substrate. It can catalyze the exchange of GDP for GTP on RhoA and Cdc42, both of which are expressed in most cell types. This lack of substrate specificity, which is relatively common among members of the RhoGEF family, complicates efforts to determine the molecular basis of their transforming activity. We have recently determined crystal structures of several RhoGEFs bound to their cognate GTPases and have used these complexes to predict structural determinants dictating the specificities of coupling between RhoGEFs and GTPases. Guided by this information, we mutated Dbs to alter significantly its relative exchange activity for RhoA versus Cdc42 and show that the transformation potential of Dbs correlates with exchange on RhoA but not Cdc42. Supporting this conclusion, oncogenic Dbs activates endogenous RhoA but not endogenous Cdc42 in NIH 3T3 cells. Similarly, a competitive inhibitor that blocks RhoA activation also blocks Dbs-mediated transformation. In conclusion, this study highlights the usefulness of specificity mutants of RhoGEFs as tools to genetically dissect the multiple signaling pathways potentially activated by overexpressed or oncogenic RhoGEFs. These ideas are exemplified for Dbs, which is strongly implicated in the transformation of NIH 3T3 cells via RhoA and not Cdc42.     

Regulation of Spine Density and Morphology by IQGAP1 Protein Domains

IQGAP1 is a scaffolding protein that regulates spine number. We now show a differential role for IQGAP1 domains in spine morphogenesis, in which a region of the N-terminus that promotes Arp2/3-mediated actin polymerization and branching stimulates spine head formation while a region that binds to Cdc42 and Rac is required for stalk extension. Conversely, IQGAP1 rescues spine deficiency induced by expression of dominant negative Cdc42 by stimulating formation of stubby spines. Together, our observations place IQGAP1 as a crucial regulator of spine number and shape acting through the N-Wasp Arp2/3 complex, as well as upstream and downstream of Cdc42.     

Structural insights into the small GTPase specificity of the DOCK guanine nucleotide exchange factors

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) regulates cytoskeletal dynamics by activating the GTPases Rac and/or Cdc42. Eleven human DOCK proteins play various important roles in developmental processes and the immune system. Of these, DOCK1–5 proteins bind to engulfment and cell motility (ELMO) proteins to perform their physiological functions. Recent structural studies have greatly enhanced our understanding of the complex and diverse mechanisms of DOCK GEF activity and GTPase recognition and its regulation by ELMO. This review is focused on gaining structural insights into the substrate specificity of the DOCK GEFs, and discuss how Rac and Cdc42 are specifically recognized by the catalytic DHR-2 and surrounding domains of DOCK or binding partners.     

Identification of a DOCK180-related guanine nucleotide exchange factor that is capable of mediating a positive feedback activation of Cdc42

Cdc42, a member of the Rho subfamily of small GTPases, influences a wide range of activities including the establishment of cell polarity, migration, and the regulation of cell growth and differentiation. Here we describe the identification of an approximately 220-kDa protein that formed a stable complex with activated forms of Cdc42 and thereby showed characteristics of a downstream target/effector for this GTPase. However, molecular cloning of the cDNA encoding this protein (p220) revealed that it was highly related to Zizimin-1 and identical in sequence to a gene product in the data base designated DOCK11, which are members of the DOCK180 family of guanine nucleotide exchange factors (GEFs) for Cdc42 and Rac. Biochemical characterization shows that p220 is a specific GEF for Cdc42, with the GEF activity originating from its DHR2 (for DOCK homology region 2) domain. Nucleotide-depleted Cdc42 forms a stable complex with the DHR2 domain, whereas the binding of activated Cdc42 requires both the DHR2 domain and residues 66-126 within the amino-terminal portion of p220. Moreover, the full-length protein shows markedly higher GEF activity than the isolated DHR2 domain, whereas removal of the amino-terminal 126 amino acids necessary for binding-activated Cdc42 dramatically diminishes the activity. These and other results point to activated Cdc42 providing a positive feedback regulation of the GEF activity of p220. Thus, we refer to p220/DOCK11 as activated Cdc42-associated GEF, befitting its functional activity.     

Dendritic spine geometry can localize GTPase signaling in neurons

Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation after synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Because Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased is unclear. Here we model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine, and, of interest, retention is enhanced by high diffusivity of Cdc42. Our findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia.     

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2

Numb has been implicated in cortical neurogenesis during nervous system development, as a result of its asymmetric partitioning and antagonizing Notch signaling. Recent studies have revealed that Numb functions in clathrin-dependent endocytosis by binding to the AP-2 complex. Numb is also expressed in postmitotic neurons and plays a role in axonal growth. However, the functions of Numb in later stages of neuronal development remain unknown. Here, we report that Numb specifically localizes to dendritic spines in cultured hippocampal neurons and is implicated in dendritic spine morphogenesis, partially through the direct interaction with intersectin, a Cdc42 guanine nucleotide exchange factor (GEF). Intersectin functions as a multidomain adaptor for proteins involved in endocytosis and cytoskeletal regulation. Numb enhanced the GEF activity of intersectin toward Cdc42 in vivo. Expression of Numb or intersectin caused the elongation of spine neck, whereas knockdown of Numb and Numb-like decreased the protrusion density and its length. Furthermore, Numb formed a complex with EphB2 receptor-type tyrosine kinase and NMDA-type glutamate receptors. Knockdown of Numb suppressed the ephrin-B1-induced spine development and maturation. These results highlight a role of Numb for dendritic spine development and synaptic functions with intersectin and EphB2.     

Specific recognition of Rac2 and Cdc42 by DOCK2 and DOCK9 guanine nucleotide exchange factors

Recognition of cognate Rho GTPases by guanine-nucleotide exchange factors (GEF) is fundamental to Rho GTPase signaling specificity. Two main GEF families use either the Dbl homology (DH) or the DOCK homology region 2 (DHR-2) catalytic domain. How DHR-2-containing GEFs distinguish between the GTPases Rac and Cdc42 is not known. To determine how these GEFs specifically recognize the two Rho GTPases, we studied the amino acid sequences in Rac2 and Cdc42 that are crucial for activation by DOCK2, a Rac-specific GEF, and DOCK9, a distantly related Cdc42-specific GEF. Two elements in the N-terminal regions of Rac2 and Cdc42 were found to be essential for specific interactions with DOCK2 and DOCK9. One element consists of divergent amino acid residues in the switch 1 regions of the GTPases. Significantly, these residues were also found to be important for GTPase recognition by Rac-specific DOCK180, DOCK3, and DOCK4 GEFs. These findings were unexpected because the same residues were shown previously to interact with GTPase effectors rather than GEFs. The other element comprises divergent residues in the beta3 strand that are known to mediate specific recognition by DH domain containing GEFs. Remarkably, Rac2-to-Cdc42 substitutions of four of these residues were sufficient for Rac2 to be specifically activated by DOCK9. Thus, DOCK2 and DOCK9 specifically recognize Rac2 and Cdc42 through their switch 1 as well as beta2-beta3 regions and the mode of recognition via switch 1 appears to be conserved among diverse Rac-specific DHR-2 GEFs.     

Distinct Roles for Rho Versus Rac/Cdc42 GTPases Downstream of Vav2 in Regulating Mammary Epithelial Acinar Architecture

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.     

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation.     

Dissociation mechanism of GDP from Cdc42 via DOCK9 revealed by molecular dynamics simulations

Cell division control protein 42 homolog (Cdc42) influences a variety of cellular responses such as cell migration and polarity. Deregulation of Cdc42 has been associated with several human diseases and developmental disorders. Over-activation of Cdc42 through guanine nucleotide exchange factor (GEF) is a critical event for Cdc42 involved cancer metastasis. Members of DOCK family of GEF are important activators of Cdc42. However, this activation mechanism is still unknown. Molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) calculations were employed to investigate the central step of the activation of Cdc42: the dissociation mechanism of GDP from Cdc42 via DOCK9. Simulation results show that Mg²⁺ ion has a remarkable influence on the conformational change of switch I of Cdc42 through residue Pro34 which functions as a “clasp” to control the flexibility of switch I. In the GDP dissociation process, the Mg²⁺ ion leave first to result in a suitable conformation of Cdc42 for following DOCK9 binding to. When DOCK9 binds to Cdc42, it changes the orientations of residues Lys16, Thr17, Cys18 and Phe28 of Cdc42 to weaken the interactions between Cdc42 and GDP to release GDP. This study first elucidates the dissociation mechanism of GDP from Cdc42 via DOCK9 and identifies the essential residues of Cdc42 in this process. These simulation results are consistent with the recent findings of biochemical and amino acid mutational studies, and the observations are beneficial to understand the activation mechanism of Cdc42 and to provide insights for designing compounds targeting on Cdc42 related cancer metastasis.     

SNX26, a GTPase-activating Protein for Cdc42, Interacts with PSD-95 Protein and Is Involved in Activity-dependent Dendritic Spine Formation in Mature Neurons

SNX26, a brain-enriched RhoGAP, plays a key role in dendritic arborization during early neuronal development in the neocortex. In mature neurons, it is localized to dendritic spines, but little is known about its role in later stages of development. Our results show that SNX26 interacts with PSD-95 in dendritic spines of cultured hippocampal neurons, and as a GTPase-activating protein for Cdc42, it decreased the F-actin content in COS-7 cells and in dendritic spines of neurons. Overexpression of SNX26 resulted in a GTPase-activating protein activity-dependent decrease in total protrusions and spine density together with dramatic inhibition of filopodia-to-spine transformations. Such effects of SNX26 were largely rescued by a constitutively active mutant of Cdc42. Consistently, an shRNA-mediated knockdown of SNX26 significantly increased total protrusions and spine density, resulting in an increase in thin or stubby type spines at the expense of the mushroom spine type. Moreover, endogenous expression of SNX26 was shown to be bi-directionally modulated by neuronal activity. Therefore, we propose that in addition to its key role in neuronal development, SNX26 also has a role in the activity-dependent structural change of dendritic spines in mature neurons.     

Cdc42/Rho GTPases in fungi: variations on a common theme

Cdc42/Rho GTPases are universally important regulators of cellular morphogenesis. Whereas their functions are well characterized in budding yeast, they are only beginning to be understood in filamentous fungi. The recent systematic analysis of Cdc42, Rac1 and Rho function in Aspergillus niger provides the first global perspective on their respective roles in hyphal morphogenesis. Surprisingly, the partitioning of these roles between Cdc42 and Rac1 seems to vary even among related fungi. These observations highlight the variable use of a common signalling module in filamentous fungi.     

The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity

The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF-like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC-induced stimulation of dendritic branching. In contrast, CALEB/NGC-induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.     

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.