Cdc42-specific Guanine Nucleotide Exchange Factor Research Articles

A conserved PI(4,5)P2-binding domain is critical for immune regulatory function of DOCK8.

DOCK8 is a Cdc42-specific guanine-nucleotide exchange factor that is essential for development and functions of various subsets of leukocytes in innate and acquired immune responses. Although DOCK8 plays a critical role in spatial control of Cdc42 activity during interstitial leukocyte migration, the mechanism remains unclear. We show that the DOCK homology region (DHR)-1 domain of DOCK8 binds specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and is required for its recruitment to the plasma membrane. Structural and biochemical analyses reveal that DOCK8 DHR-1 domain consists of a C2 domain-like core with loops creating the upper surface pocket, where three basic residues are located for stereospecific recognition of phosphoinositides. Substitution of the two basic residues, K576 and R581, with alanine abolished PI(4,5)P2 binding in vitro, ablated the ability of DOCK8 to activate Cdc42 and support leukocyte migration in three-dimensional collagen gels. Dendritic cells carrying the mutation exhibited defective interstitial migration in vivo. Thus, our study uncovers a critical role of DOCK8 in coupling PI(4,5)P2 signaling with Cdc42 activation for immune regulation.

Tuba Activates Cdc42 during Neuronal Polarization Downstream of the Small GTPase Rab8a.

The acquisition of neuronal polarity is a complex molecular process that depends on changes in cytoskeletal dynamics and directed membrane traffic, regulated by the Rho and Rab families of small GTPases, respectively. However, during axon specification, a molecular link that couples these protein families has yet to be identified. In this paper, we describe a new positive feedback loop between Rab8a and Cdc42, coupled by Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), that ensures a single axon generation in rodent hippocampal neurons from embryos of either sex. Accordingly, Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Although Rab8 and Tuba do not interact physically, the activity of Rab8 is essential to generate a proximal to distal axonal gradient of Tuba in cultured neurons. Tuba-associated and Rab8a-associated polarity defects are also evidenced in vivo, since dominant negative (DN) Rab8a or Tuba knock-down impairs cortical neuronal migration in mice. Our results suggest that Tuba coordinates directed vesicular traffic and cytoskeleton dynamics during neuronal polarization.SIGNIFICANCE STATEMENT The morphologic, biochemical, and functional differences observed between axon and dendrites, require dramatic structural changes. The extension of an axon that is 1 µm in diameter and grows at rates of up to 500 µm/d, demands the confluence of two cellular processes: directed membrane traffic and fine-tuned cytoskeletal dynamics. In this study, we show that both processes are integrated in a positive feedback loop, mediated by the guanine nucleotide-exchange factor (GEF) Tuba. Tuba connects the activities of the Rab GTPase Rab8a and the Rho GTPase Cdc42, ensuring the generation of a single axon in cultured hippocampal neurons and controlling the migration of cortical neurons in the developing brain. Finally, we provide compelling evidence that Tuba is the GEF that mediates Cdc42 activation during the development of neuronal polarity.

Identification of CDC42 Effectors Operating in FGD1-Dependent Trafficking at the Golgi.

Loss of function mutations in the FGD1 gene cause a rare X-linked disease, faciogenital dysplasia (FGDY, also known as Aarskog-Skott syndrome), which is associated with bone and urogenital abnormalities. The FGD1 gene encodes à CDC42-specific guanine nucleotide exchange factor. The mutations are frequently located in the DH module of FGD1 preventing its transformation to the active form. We previously reported that Golgi-associated FGD1 regulates post-Golgi transport of some conventional and bone-specific proteins in a CDC42-dependent manner. However, the downstream targets of FGD1/CDC42 signaling that operate to support transport from the Golgi remain elusive. Here, we demonstrate that Golgi-localized CDC42 effectors might be involved in FGD1-mediated post-Golgi transport, probably through coordination of Golgi membrane and cytoskeleton dynamics. Overexpression of effector-specific CDC42 mutants (exhibiting preferential affinities for PAK1, IQGAP1, N-WASP, or PAR6) only partially rescue membrane trafficking in FGD1-deficient cells, indicating that the orchestrated activities of several downstream targets of CDC42 are required to support FGD1-mediated export from the Golgi. Our findings provide new insights into understanding the molecular mechanisms behind FGD1/CDC42-dependent transport events and uncover new targets whose potential might be explored for correction of membrane trafficking in FGDY.

Abstract 4369: Functional role of RhoGEF FGD4 in promotion of aggressive prostate cancer

Abstract Hyperactivation of Rho family of GTPases including Rac, Cdc42 and Rho, as a result of deregulation of Rho guanine nucleotide exchange factors (GEF) is commonly noted in various cancers including prostate cancer. A number of GEFs are overexpressed in prostate and breast cancer cells and tissues, and show an association with poor prognosis. Earlier, we showed that a prolonged treatment with androgen receptor antagonist Casodex resulted in up regulation of a GEF FGD4, traditionally associated with hereditary motor and sensory neuropathies including Charcot-Marie-Tooth disease. FGD4 is a Cdc42 specific GEF and contains an actin binding domain FAB, in addition to the conserved FYVE domain, a DH domain for catalytic activity and two PH domains for targeting proteins to the membranes through phosphoinositide binding. Our study revealed that Casodex treatment of prostate cancer cells lead to down regulation of a microRNA cluster miR-17-92a that targets FGD4. Here we show that FGD4 is upregulated in cancerous prostate tissues compared to benign prostatic hyperplasia and PIN. FGD4 protein expression and altered localization gradually increase with increasing tumor grade and aggressiveness. Inhibition of FGD4 expression using siRNAs, reduced cell proliferation, resulted in a G2/M arrest, inhibited cell migration and reduced Cdc42 activity. Inhibition of FGD4 also improved sensitivity of androgen sensitive LNCaP cells to Casodex and PC-3 cells to the microtubule stabilizing drug docetaxel. Our data demonstrate a tumor promoting function of FGD4 in prostate cancer cells and that inhibition of FGD4 expression has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells. Citation Format: Alexia Bossan, Richard Ottman, Domenico Coppola, Ratna Chakrabarti. Functional role of RhoGEF FGD4 in promotion of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4369.

Mechanisms of Impaired Neutrophil Migration by MicroRNAs in Myelodysplastic Syndromes.

In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8-induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.

Emerging role of Cdc42-specific guanine nucleotide exchange factors as regulators of membrane trafficking in health and disease

It is widely accepted that the Golgi complex operates as a main sorting station in the biosynthetic pathway. On the other hand, the Golgi complex harbors numerous signaling molecules that generate the platform for the coordination of the transduction of specific signals and of membrane transport events. A part of these processes, which require the complex integration of transport-, cytoskeleton- and polarity-associated mechanisms, is tightly regulated by molecular machineries comprising guanine nucleotide exchange factors (GEF) and their down-stream effectors, such as the small GTPase Cdc42. Dysfunction of several Cdc42-specific GEFs has been shown to cause a number of human diseases, which are associated with impaired intracellular trafficking at the level of the Golgi complex as well as in other compartments. Here we briefly overview how mutations in Cdc42-specific GEFs have an impact on the organization of intracellular trafficking fluxes and how such trafficking aberrations could be associated with a number of human disorders.

Dynamin Binding Protein (Tuba) Deficiency Inhibits Ciliogenesis and Nephrogenesis in Vitro and in Vivo

Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for ciliogenesis and regulated by multiple Rho and Rab family GTPases, such as Cdc42. Cdc42 deficiency resulted in a disruption of renal ciliogenesis and a polycystic kidney disease phenotype in zebrafish and mice. Here we investigate the role of Dynamin binding protein (also known as Tuba), a Cdc42-specific guanine nucleotide exchange factor, in ciliogenesis and nephrogenesis using Tuba knockdown Madin-Darby canine kidney cells and tuba knockdown in zebrafish. Tuba depletion resulted in an absence of cilia, with impaired apical polarization and inhibition of hepatocyte growth factor-induced tubulogenesis in Tuba knockdown Madin-Darby canine kidney cell cysts cultured in a collagen gel. In zebrafish, tuba was expressed in multiple ciliated organs, and, accordingly, tuba start and splice site morphants showed various ciliary mutant phenotypes in these organs. Co-injection of tuba and cdc42 morpholinos at low doses, which alone had no effect, resulted in genetic synergy and led to abnormal kidney development with highly disorganized pronephric duct cilia. Morpholinos targeting two other guanine nucleotide exchange factors not known to be in the Cdc42/ciliogenesis pathway and a scrambled control morpholino showed no phenotypic effect. Given the molecular nature of Cdc42 and Tuba, our data strongly suggest that tuba and cdc42 act in the same ciliogenesis pathway. Our study demonstrates that Tuba deficiency causes an abnormal renal ciliary and morphogenetic phenotype. Tuba most likely plays a critical role in ciliogenesis and nephrogenesis by regulating Cdc42 activity.

Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway.

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9.

Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge.

Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses

AbstractTo migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans. In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially.

In vivo fluorescence resonance energy transfer imaging reveals differential activation of Rho-family GTPases in glioblastoma cell invasion

Two-photon excitation microscopy was used to visualized two different modes of invasion at perivascular and intraparenchymal regions of rat C6 glioblastoma cells that were orthotopically implanted into rat brains. Probes based on the principle of Förster resonance energy transfer (FRET) further revealed that glioblastoma cells penetrating the brain parenchyma showed higher Rac1 and Cdc42 activities and lower RhoA activity than those advancing in the perivascular regions. This spatial regulation of Rho-family GTPase activities was recapitulated in three-dimensional spheroid invasion assays with rat and human glioblastoma cells, in which multipod glioblastoma cells that invaded the gels and led the other glioblastoma cells exhibited higher Rac1 and Cdc42 activities than the trailing glioblastoma cells. We also studied the Cdc42-specific guanine nucleotide exchange factor Zizimin1 (also known as DOCK9) as a possible contributor to this spatially controlled activation of Rho-family GTPases, because it is known to play an essential role in the extension of neurites. We found that shRNA-mediated knockdown of Zizimin1 inhibited formation of pseudopodia and concomitant invasion of glioblastoma cells both under a 3D culture condition and in vivo. Our results suggest that the difference in the activity balance of Rac1 and Cdc42 versus RhoA determines the mode of glioblastoma invasion and that Zizimin1 contributes to the invasiveness of glioblastoma cells with high Rac1 and Cdc42 activities.

Septation of Infectious Hyphae Is Critical for Appressoria Formation and Virulence in the Smut Fungus Ustilago Maydis

Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently.

The Cdc42 Guanine Nucleotide Exchange Factor FGD1 Regulates Osteogenesis in Human Mesenchymal Stem Cells

Loss of function mutations in FGD1 result in faciogenital dysplasia, an X-linked human developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor that specifically activates Cdc42, a Rho family small GTPase that regulates a variety of cellular behaviors. We have found that FGD1 is expressed in human mesenchymal stem cells (hMSCs) isolated from adult bone marrow. hMSCs are multipotent cells that can differentiate into many cell types, including fibroblasts, osteoblasts, adipocytes, and chondrocytes, and are thought to play a role in maintaining musculoskeletal tissues throughout life. We demonstrate an active role of FGD1 in osteogenic differentiation of hMSCs. During osteogenic differentiation of hMSCs in culture, we observed up-regulation of both FGD1 expression and Cdc42 activity. Activating FGD1/Cdc42 signaling by overexpression of either FGD1 or constitutively active Cdc42 promoted hMSC osteogenesis, while inhibiting Cdc42 signaling by either dominant negative mutants of FGD1 or Cdc42 suppressed osteogenesis. These results demonstrate an important role for FGD1/Cdc42 signaling in hMSC osteogenesis and suggest that the defects in bone remodeling in faciogenital dysplasia may persist throughout adult life and serve as a potential pathway that may be targeted for enhancing bone regeneration.

Tuba, a Cdc42 GEF, is required for polarized spindle orientation during epithelial cyst formation

The Cdc42 guanosine triphosphatase is essential for cell polarization in several organisms and in vitro for the organization of polarized epithelial cysts. A long-standing question concerns the identity of the guanine nucleotide exchange factor (GEF) that controls this process. Using Madin-Darby canine kidney cells grown in Matrigel, we screened 70 GEFs by RNA interference. Of these, six positives were identified that caused a multilumen phenotype, including Tuba, a Cdc42-specific GEF localized below the apical cortex. Loss of Tuba abolishes Cdc42 enrichment at the apical cortex. Normal lumen formation is rescued by human Tuba or active Cdc42 but not by a GEF-negative Tuba mutant. Silencing Cdc42 causes a similar phenotype, including multilumen formation and reduced atypical protein kinase C (aPKC) activity. Lumen disorganization after depletion of Tuba or Cdc42 or inhibition of aPKC is caused by defective spindle orientation. Together, our findings implicate Tuba as a key activator of the Cdc42 GTPase during epithelial ductal morphogenesis, which in turn activates apical aPKC to ensure that spindles orient parallel to the lateral plane.

The Cdc42 GEF Intersectin 2 controls mitotic spindle orientation to form the lumen during epithelial morphogenesis

Epithelial organs are made of tubes and cavities lined by a monolayer of polarized cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. The Rho guanosine triphosphatase (GTPase) Cdc42, which is a master regulator of cell polarity, regulates the formation of the central lumen in epithelial morphogenesis. However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the three-dimensional Madin-Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localizes to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects.

Adenomatous Polyposis Coli and Asef Function Downstream of Hepatocyte Growth Factor and Phosphatidylinositol 3-Kinase

Mutations of the tumor suppressor adenomatous polyposis coli (APC) are responsible for sporadic and familial colorectal tumors. APC negatively regulates Wnt signaling by inducing beta-catenin degradation. It has also been shown that APC plays a role in the organization of cytoskeletal networks. APC interacts with Asef and Asef2, Rac1- and Cdc42-specific guanine nucleotide exchange factors (GEFs), and stimulates their GEF activity; thereby regulating cell morphology, adhesion, and migration. Truncated mutant APCs present in colorectal tumor cells activate Asef and Asef2 constitutively and contribute to their aberrant migratory properties. We show here that hepatocyte growth factor (HGF), as well as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), induce the accumulation and colocalization of APC and Asef in membrane ruffles and lamellipodia of epithelial cells. Both APC and Asef were found to be required for HGF-induced cell migration. Furthermore, we show that the effects of HGF, bFGF, and EGF on APC and Asef are mediated by the activation of phosphatidylinositol 3-kinase (PI3-kinase) and require the PH domain of Asef. These results suggest that Asef and APC function downstream of HGF and PI3-kinase, and play critical roles in growth factor-mediated regulation of cell morphology and migration.

Faciogenital Dysplasia Protein Fgd1 Regulates Invadopodia Biogenesis and Extracellular Matrix Degradation and Is Up-regulated in Prostate and Breast Cancer

Invadopodia are proteolytically active membrane protrusions that extend from the ventral surface of invasive tumoral cells grown on an extracellular matrix (ECM). The core machinery controlling invadopodia biogenesis is regulated by the Rho GTPase Cdc42. To understand the upstream events regulating invadopodia biogenesis, we investigated the role of Fgd1, a Cdc42-specific guanine nucleotide exchange factor. Loss of Fgd1 causes the rare inherited human developmental disease faciogenital dysplasia. Here, we show that Fgd1 is required for invadopodia biogenesis and ECM degradation in an invasive cell model and functions by modulation of Cdc42 activation. We also find that Fgd1 is expressed in human prostate and breast cancer as opposed to normal tissue and that expression levels matched tumor aggressiveness. Our findings suggest a central role for Fgd1 in the focal degradation of the ECM in vitro and, for the first time, show a connection between Fgd1 and cancer progression, proposing that it might function during tumorigenesis.

Asef2 and Neurabin2 cooperatively regulate actin cytoskeletal organization and are involved in HGF-induced cell migration

The tumor suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumors. APC interacts with the Rac1- and Cdc42-specific guanine-nucleotide exchange factors (GEF), Asef and Asef2, which contain an APC-binding region (ABR) in addition to Dbl homology, Pleckstrin homology (PH) and Src homology 3 (SH3) domains. APC stimulates the GEF activity of Asef and Asef2, and thereby regulates cell adhesion and migration. Here we show that Asef2, but not Asef, interacts with Neurabin2/Spinophilin, a scaffold protein that binds to Filamentous actin (F-actin). In response to hepatocyte growth factor (HGF) treatment of HeLa cells, Asef2, Neurabin2 and APC were induced to accumulate and colocalize in lamellipodia and membrane ruffles. Neurabin2 did not affect the GEF activity of Asef2. RNA interference experiments showed that Asef2, Neurabin2 and APC are involved in HGF-induced cell migration. Furthermore, knockdown of Neurabin2 resulted in the suppression of Asef2-induced filopodia formation. These results suggest that Asef2, Neurabin2 and APC cooperatively regulate actin cytoskeletal organization and are required for HGF-induced cell migration.

Coordination of Cytokinesis and Cell Separation by Endosomal Targeting of a Cdc42-specific Guanine Nucleotide Exchange Factor inUstilago maydis

The small GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in most eukaryotic cells. In Ustilago maydis, Cdc42 and the guanine nucleotide exchange factor (GEF) Don1 regulate cytokinesis and cell separation. Don1 belongs to the FGD1 family of Cdc42-specific GEFs that are characterized by a C-terminal lipid-binding FYVE domain. Although the FGD1/frabin family of Rho-GEFs is evolutionary conserved from fungi to mammals the role of the FYVE domain for its biological function is unknown. Here, we show that the FYVE domain is specific for phosphatidylinositol-3-phosphate (PtdIns(3)P) and targets Don1 to endosomal vesicles. During cytokinesis asymmetric accumulation of Don1-containing vesicles occurs at the site of septation. We could show that FYVE-dependent localization is critical for the function of Don1 at normal expression levels but can be compensated for by overexpression of Don1 lacking a functional FYVE domain. Our results demonstrate that endosomal compartmentalization of a Cdc42-specific exchange factor is involved in the coordination of cytokinesis and cell separation.

Frabin and other related Cdc42‐specific guanine nucleotide exchange factors couple the actin cytoskeleton with the plasma membrane

Frabin, together with, at least, FGD1, FGD2, FGD3 and FGD1-related Cdc42-GEF (FRG), is a member of a family of Cdc42-specific gua-nine nucleotide exchange factors (GEFs). These proteins have multiple phosphoinositide-binding domains, including two pleckstrin homology (PH) domains and an FYVE or FERM domain. It is likely that they couple the actin cytoskeleton with the plasma membrane. Frabin associates with a specific actin structure(s) and induces the direct activation of Cdc42 in the vicinity of this structure(s), resulting in actin reorganization. Furthermore, frabin associates with a specific membrane structure(s) and induces the indirect activation of Rac in the vicinity of this structure(s), resulting in the reorganization of the actin cytoskeleton. This reorganization of the actin cytoskeleton induces cell shape changes such as the formation of filopodia and lamellipodia.