Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration

BACKGROUND: Mammalian Scribble (Scrib) plays a conserved role in polarization of epithelial and neuronal cells. Polarization is essential for migration of a variety of cell types; however, the function of Scrib in this context remains unclear. Scrib has been shown to interact with betaPIX, a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42. Cdc42 controls cell polarity from yeast to mammals during asymmetric cell division and epithelial cell polarization, as well as during cell migration. Cdc42 is, in particular, required for polarization and orientation of astrocytes in a scratch-induced polarized migration assay. Using this assay, we characterized Scrib function during polarized cell migration. RESULTS: Depletion of Scrib by siRNA or expression of dominant-negative constructs inhibits astrocyte polarization. Like Cdc42, Scrib controls protrusion formation, cytoskeleton polarization, and centrosome and Golgi reorientation. Scrib interacts and colocalizes with betaPIX at the front edge of polarizing astrocytes. Perturbation of Scrib localization or of Scrib-betaPIX interaction inhibits betaPIX polarized recruitment. We further show that betaPIX is required for astrocyte polarization and that both the Scrib-binding motif and the GEF activity of betaPIX are essential for its function. Scrib and betaPIX control Cdc42 activation and localization during astrocyte polarization. Thereby, Scrib regulates Cdc42-dependent APC and Dlg1 recruitment to the leading edge to promote cell orientation. CONCLUSION: We conclude that Scrib plays a key role in the establishment of cell polarity during migration. By interacting with betaPIX, Scrib controls localization and activation of the small GTPase Cdc42 and regulates Cdc42-dependent polarization pathways.     

Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells

Cdc42 is a small GTPase involved in the regulation of the cytoskeleton and cell polarity. To test whether Cdc42 has an essential role in the formation of filopodia or directed cell migration, we generated Cdc42-deficient fibroblastoid cells by conditional gene inactivation. We report here that loss of Cdc42 did not affect filopodium or lamellipodium formation and had no significant influence on the speed of directed migration nor on mitosis. Cdc42-deficient cells displayed a more elongated cell shape and had a reduced area. Furthermore, directionality during migration and reorientation of the Golgi apparatus into the direction of migration was decreased. However, expression of dominant negative Cdc42 in Cdc42-null cells resulted in strongly reduced directed migration, severely reduced single cell directionality, and complete loss of Golgi polarization and of directionality of protrusion formation toward the wound, as well as membrane blebbing. Thus, our data show that besides Cdc42 additional GTPases of the Rho-family, which share GEFs with Cdc42, are involved in the establishment and maintenance of cell polarity during directed migration.     

Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro

Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.     

Localized cdc42 activation,detected using a novel assay,mediates microtubule organizing center positioning in endothelial cells in response to fluid shear stress

Fluid flow regulates morphology, physiology, and pathophysiology of vascular endothelial cells (reviewed in Ref. 1). The small GTPase Cdc42 mediates polarity in several systems including migrating cells and early embryos, which involve reorientation of the microtubule organizing center (MTOC) and Golgi apparatus toward the direction of movement. Here, we show that Cdc42 is activated by fluid shear stress and that activation is a consequence of integrins binding to extracellular matrix. A novel fluorescence energy transfer assay to visualize Cdc42 activation in single cells shows that Cdc42 activity is polarized in the direction of flow. Localized activation of Cdc42 as well as the activity of Par6 and protein kinase Czeta direct the reorientation of the MTOC to a position on the downstream side of the nucleus relative to the direction of flow. Thus, shear-stimulated integrin dynamics induce polarized Cdc42 activity, which induces MTOC localization through the Par6-protein kinase Czeta complex.     

T1alpha/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.     

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.     

Peptide YY and neuropeptide Y induce villin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity

Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.     

Small rho GTPases regulate antigen presentation in dendritic cells

Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity. However, the molecular mechanisms maintaining DC function remain to be elucidated. In this study, we report on the role of small Rho GTPases: Cdc42, Rac1, and RhoA in the regulation of DC adherence, Ag presentation, migration, chemotaxis, and endocytosis. Murine DC were transfected with vaccinia virus-based constructs, encoding dominant-negative or constitutively active (ca) mutant forms of Rho GTPases. We demonstrate that Cdc42 plays a major role in the regulation of DC adhesion, because caCdc42-transfected DC had significant up-regulation of adhesion to extracellular matrix, which was blocked by the Rho GTPase inhibitor toxin B (ToxB). In contrast, caRho-transfected DC only modestly elevated DC adhesion, and caRac had no effect. Additionally, caCdc42 and caRho increased the ability of DC to present OVA peptide to specific T cells. This effect was abrogated by ToxB. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced DC migration. Furthermore, uptake of dextran 40 by DC was significantly enhanced by Rho GTPase activators cytotoxic necrotizing factor 1 and PMA, and reduced by ToxB. caCdc42 also increased endocytotic activity of DC, whereas dominant-negative Cdc42 blocked it. Thus, Rho GTPases Cdc42, RhoA, and Rac1 regulate DC functions that are critical for DC-mediated immune responses in vivo.     

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.     

Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells

The small GTPase Cdc42 has been implicated as an important regulator of cell migration. However, whether Cdc42 plays similar role in all cancer cells irrespective of metastatic potential remains poorly defined. Here, we show by using three different breast cancer cell lines with different metastatic potential, the role of Cdc42 in cell migration/invasion and its relationship with a number of downstream signaling pathways controlling cell migration. Small interfering RNA (siRNA)-mediated knockdown of Cdc42 in two highly metastatic breast cancer cell lines (MDA-MB-231 and C3L5) resulted in enhancement, whereas the same in moderately metastatic (Hs578T) cell line resulted in inhibition of intrinsic cellular migration/invasion. Furthermore, Cdc42 silencing in MDA-MB-231 and C3L5 but not Hs578T cells was shown to be accompanied by increased RhoA activity and phosphorylation of protein kinase C (PKC)-δ, extracellular signal regulated kinase1/2 (Erk1/2), and protein kinase A (PKA). Pharmacological inhibition of PKCδ, MEK-Erk1/2, or PKA was shown to inhibit migration of both control and Cdc42-silenced MDA-MB-231 cells. Furthermore, introduction of constitutively active Cdc42 was shown to decrease migration/invasion of MDA-MB-231 and C3L5 but increase migration/invasion of Hs578T cells. This decreased migration/invasion of MDA-MB-231 and C3L5 cells was also shown to be accompanied by the decrease in the phosphorylations of PKCδ, Erk1/2, and PKA. These results suggested that endogenous Cdc42 could exert a negative regulatory influence on intrinsic migration/invasion and some potentially relevant changes in phosphorylation of PKCδ, Erk1/2, and PKA of some aggressive breast cancer cells.     

Rudhira/BCAS3 is a cytoskeletal protein that controls Cdc42 activation and directional cell migration during angiogenesis

Cell migration is a common cellular process in angiogenesis and tumor metastasis. Rudhira/BCAS3 (Breast Cancer Amplified Sequence 3) is a conserved protein expressed in the embryonic vasculature and malignant tumors. Here, we show for the first time that Rudhira plays an active role in directional cell migration. Rudhira depletion in endothelial cells inhibits Matrigel-induced tube formation and retards healing of wounded cell monolayers. We demonstrate that during wound healing, Rudhira rapidly re-localizes and promotes Cdc42 activation and recruitment to the leading edge of migrating cells. Rudhira deficient cells show impaired downstream signaling of Cdc42 leading to dramatic changes in actin organization and classic cell polarity defects such as loss of microtubule organizing center (MTOC) and Golgi re-orientation. Biochemical assays and co-localization studies show that Rudhira interacts with microtubules as well as intermediate filaments. Thus, Rudhira could control directional cell migration and angiogenesis by facilitating crosstalk between cytoskeletal elements.     

Newly formed E‐cadherin contacts do not activate Cdc42 or induce filopodia protrusion in human keratinocytes1

Background information. The appropriate regulation of cell–cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E‐cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin‐mediated cell–cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E‐cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell–cell contacts has been inferred from the presence of filopodia, the typical F‐actin structure induced by Cdc42 activation, as cells approach each other to establish cell–cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. Results. In the present study, we set out to address (a) whether Cdc42 is activated by new E‐cadherin cell–cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E‐cadherin clustering decreased Cdc42‐GTP levels. In the absence of serum‐ and migration‐induced Cdc42 activation, we demonstrated that cell–cell contacts do not induce filopodia or require Cdc42 function to assemble. Conclusion. We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re‐positioning or basolateral trafficking.     

Cdc42 Regulates Microtubule‐Dependent Golgi Positioning

The molecular mechanisms underlying cytoskeleton‐dependent Golgi positioning are poorly understood. In mammalian cells, the Golgi apparatus is localized near the juxtanuclear centrosome via dynein‐mediated motility along microtubules. Previous studies implicate Cdc42 in regulating dynein‐dependent motility. Here we show that reduced expression of the Cdc42‐specific GTPase‐activating protein, ARHGAP21, inhibits the ability of dispersed Golgi membranes to reposition at the centrosome following nocodazole treatment and washout. Cdc42 regulation of Golgi positioning appears to involve ARF1 and a binding interaction with the vesicle‐coat protein coatomer. We tested whether Cdc42 directly affects motility, as opposed to the formation of a trafficking intermediate, using a Golgi capture and motility assay in permeabilized cells. Disrupting Cdc42 activation or the coatomer/Cdc42 binding interaction stimulated Golgi motility. The coatomer/Cdc42‐sensitive motility was blocked by the addition of an inhibitory dynein antibody. Together, our results reveal that dynein and microtubule‐dependent Golgi positioning is regulated by ARF1‐, coatomer‐, and ARHGAP21‐dependent Cdc42 signaling.     

Faciogenital Dysplasia Protein (FGD1) Regulates Export of Cargo Proteins from the Golgi Complex via Cdc42 Activation

Mutations in the FGD1 gene are responsible for the X-linked disorder known as faciogenital dysplasia (FGDY). FGD1 encodes a guanine nucleotide exchange factor that specifically activates the GTPase Cdc42. In turn, Cdc42 is an important regulator of membrane trafficking, although little is known about FGD1 involvement in this process. During development, FGD1 is highly expressed during bone growth and mineralization, and therefore a lack of the functional protein leads to a severe phenotype. Whether the secretion of proteins, which is a process essential for bone formation, is altered by mutations in FGD1 is of great interest. We initially show here that FGD1 is preferentially associated with the trans-Golgi network (TGN), suggesting its involvement in export of proteins from the Golgi. Indeed, expression of a dominant-negative FGD1 mutant and RNA interference of FGD1 both resulted in a reduction in post-Golgi transport of various cargoes (including bone-specific proteins in osteoblasts). Live-cell imaging reveals that formation of post-Golgi transport intermediates directed to the cell surface is inhibited in FGD1-deficient cells, apparently due to an impairment of TGN membrane extension along microtubules. These effects depend on FGD1 regulation of Cdc42 activation and its association with the Golgi membranes, and they may contribute to FGDY pathogenesis.     

Disruption of the Diaphanous-related formin Drf1 gene encoding mDia1 reveals a role for Drf3 as an effector for Cdc42

BACKGROUND: Mammalian Diaphanous-related formins (Drfs) act as Rho small GTPase effectors during growth factor-induced cytoskeletal remodeling and cell division. While both p140 mDia1 (herein called Drf1) and p134 mDia2 (Drf3) have been shown to bind in vitro to activated RhoA-C, and Drf3 has also been shown to bind to Cdc42, little is known about the cellular function of these GTPase effector pairs. Thus, we have begun targeting the murine Drf genes to address their various contributions to small GTPase signaling in cytoskeletal remodeling and development. RESULTS: Drf1 +/+, +/-, and -/- cell lines were derived from embryonic stem cells. While some Drf1 +/- lines had fewer actin stress fibers, several Drf1 +/- and -/- cells were more motile and had more abundant lamella and filopodia. Because the apparent "gain-of-function" corresponded with elevated levels of Drf3 protein expression, we hypothesized that the effects on the actin cytoskeleton were due to Cdc42 utilization of Drf3 as an effector. In this study, we found that inactive Drf3 variants and microinjected Drf3 antibodies interfered with Cdc42-induced filopodia. In addition, we observed that Drf3 contains a previously unidentified CRIB-like motif within its GTPase binding domain (GBD). By fluorescent resonance energy transfer (FRET) analysis, we demonstrate that this motif is required for Cdc42 binding and Drf3 recruitment to the leading edge and, surprisingly, to the microtubule organizing center (MTOC) of migrating fibroblasts. CONCLUSIONS: Our observations extend the role of the mammalian Drfs in cell signaling and demonstrate that Cdc42 not only activates Drf3, but guides the effector to sites at the cell cortex where it remodels the actin cytoskeleton.     

Nudel binds Cdc42GAP to modulate Cdc42 activity at the leading edge of migrating cells

Cdc42GAP promotes inactivation of Cdc42, a small GTPase whose activation at the leading edge by guanine nucleotide exchange factors is critical for cell migration. How Cdc42GAP is regulated to ensure proper levels of active Cdc42 is poorly understood. Here we show that Nudel, a cytoplasmic dynein regulator, competes with Cdc42 for binding Cdc42GAP. Consequently, Nudel can inhibit Cdc42GAP-mediated inactivation of Cdc42 in a dose-dependent manner. Both Nudel and Cdc42GAP exhibit leading-edge localization in migrating cells. The localization of Nudel requires its phosphorylation by Erk1/2. Depleting Nudel by RNAi or overexpression of a nonphosphorylatable mutant abolishes Cdc42 activation and cell migration. Our data thus uncover Nudel as a regulator of Cdc42 during cell migration. Nudel facilitates cell migration by sequestering Cdc42GAP at the leading edge to stabilize active Cdc42 in response to extracellular stimuli. Excess active Cdc42 may in turn control its own activity by recruiting Cdc42GAP from Nudel.     

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.     

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.