Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. βPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-βPix-actin pathway for this mechanism.Alignment of the mitotic spindles with a predetermined axis, which confines the plane of cell division, occurs in many types of cells and is crucial for morphogenesis and embryogenesis. Cell geometry (30, 32, 47), cell polarity (6, 24, 35), and cell-cell adhesions (20, 22, 48) are proposed to be the determinants for the axis of the spindles. In most cases, spindle alignment along the predetermined axis requires both astral microtubules and the actin cytoskeleton and is believed to involve dynein-dependent microtubule pulling forces functioning at the cell cortex (4, 12, 31).We have previously shown that in nonpolarized adherent cells, such as HeLa cells, integrin-mediated cell-substrate adhesion orients the spindles parallel to the substratum, which ensures that both daughter cells remain attached to the substrate after cell division (42). This mechanism requires the actin cytoskeleton, astral microtubules, the microtubule plus-end-tracking protein EB1, and myosin X. Furthermore, our recent study has shown that the lipid second messenger phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] is also essential to this mechanism. PtdIns(3,4,5)P3 is accumulated in the midcortex of metaphase cells, which is important for the localized accumulation of dynactin, a dynein-binding partner, at the midcortex. We have proposed that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on the spindle to the midcortex and orients the spindle parallel to the substratum (43). However, the molecular mechanisms that regulate the actin cytoskeleton and PtdIns(3,4,5)P3 in the spindle orientation control remain unknown.The Rho family of GTPases, including Rho, Rac, and Cdc42, plays central roles in the regulation of not only the actin cytoskeleton but also microtubules in the control of various activities of cell motility, including cell adhesion, cell migration, and cell cycle progression (9, 33, 41). Rho family GTPases are also reported to regulate several mitotic events. RhoA plays a crucial role in contractile ring function and localizes to the cleavage furrow along with its effectors, ROCK, citron kinase, and mDia, during cytokinesis (18, 11). Cdc42 and its effector, mDia3, are reported to regulate the alignment of chromosomes during prometaphase and metaphase (49). Interestingly, Cdc42 is also required for proper spindle positioning in polarized cells such as budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans one-cell stage embryos, and mouse oocytes, which undergo asymmetric cell division (1, 23, 13, 28). However, how Cdc42 regulates spindle orientation and whether it has a role in spindle orientation in nonpolarized cells remain unknown.Here, we show that Cdc42 is required for the mechanism that orients the spindle parallel to the substratum in nonpolarized adherent cells. Moreover, our results show that Cdc42 regulates both PtdIns(3,4,5)P3 and the actin cytoskeleton through PI(3)K- and p21-activated kinase 2 (PAK2)/βPix-signaling pathways, respectively. Both pathways are required for the localized accumulation of dynein/dynactin complexes in the midcortex in metaphase cells and, thus, for the proper spindle orientation parallel to the substratum.     

The Function of Two Rho Family GTPases Is Determined by Distinct Patterns of Cell Surface Localization

Rho family GTPases are critical regulators in determining and maintaining cell polarity. In Saccharomyces cerevisiae, Rho3 and Cdc42 play important but distinct roles in regulating polarized exocytosis and overall polarity. Cdc42 is highly polarized during bud emergence and is specifically required for exocytosis at this stage. In contrast, Rho3 appears to play an important role during the isotropic growth of larger buds. Using a novel monoclonal antibody against Rho3, we find that Rho3 localizes to the cell surface in a dispersed pattern which is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we demonstrate that a small region at the N terminus is necessary and sufficient to confer Rho3 localization and function onto Cdc42. Analysis of this domain reveals two essential elements responsible for distinguishing function. First, palmitoylation of a cysteine residue by the Akr1 palmitoyltransferase is required both for the switch of function and the switch of localization properties of this domain. Second, two basic residues distal to the palmitoylation site are required for regulating binding affinity with the Exo70 and Sec3 effectors. This demonstrates the importance of localization and effector binding in determining how these GTPases evolved specific functions at distinct stages of polarized growth.Cell polarity is a highly conserved feature of eukaryotic cells and is important for a number of events in animal cell biology, including embryonic development, cell migration, and epithelial function (21). The budding yeast Saccharomyces cerevisiae provides a simple model system in which to understand cell polarity, as much of the machinery that is responsible for polarity between yeast and animal cells is highly conserved. Rho/Cdc42 family GTPases are examples of this conservation and have been shown to be critical determinants of polarity in both yeast and animal cells. Rho GTPases are thought to exert their effects on cell polarization through regulation of a number of cellular processes, including the cytoskeleton and polarized delivery of new membrane to sites of active growth.Previous studies have demonstrated that Rho3 and Cdc42 have direct roles in regulating exocytosis which are independent of their role in regulating the polarity of the actin cytoskeleton (1, 2). Studies from a number of laboratories have shown that a multisubunit vesicle tethering complex known as the exocyst is likely to be a critical effector for Rho/Cdc42 signaling during polarized exocytosis (1, 2, 11, 22). A number of models have been suggested to describe the action of Rho GTPases in regulating exocytic function (28, 30). Analysis of specific loss-of-function alleles of RHO3 and CDC42 demonstrated that defects in secretion could be distinguished not only from actin polarity but from the polarization of the exocytic machinery as well. This led to the suggestion that Rho GTPases act by local activation rather than recruitment of the exocytic machinery (25).Genetic analysis suggests that the pathway by which Cdc42 regulates secretion is closely linked to that of Rho3. Secretion-defective alleles in each of these GTPases are suppressed by a common set of genes, and the mutants exhibit synthetic lethality when combined in the same cell (1). Recent work has provided direct evidence that the Exo70 subunit of the exocyst both genetically and physically interacts with both Rho3 and Cdc42 (29).Although Rho3 and Cdc42 share a effector and have overlapping functions, there are different characteristics in how these two proteins regulate exocytosis in yeast. Analysis of the Rho3 and Cdc42 secretory mutants by electron microscopy and secretory assays revealed that cdc42-6 mutants showed defects only in cells with small or emerging buds; in contrast, rho3-V51 mutants exhibited secretory defects throughout bud growth (1, 2). These phenotypes suggested that the exocytic function of Rho3 and Cdc42 is required at overlapping but distinct stages of bud growth.Most small GTPases require multiple elements to promote their association with the membrane on which they engage their downstream targets (26). Modification of the C-terminal CAAX motif by prenylation is common to both Rho3 and Cdc42, with Rho3 predicted to be farnesylated and Cdc42 shown to be geranylgeranylated (14, 17, 19). However, as with other small GTPases, C-terminal prenylation by itself is not sufficient for stable membrane association (10, 18). As with many other small GTPases, a second site of interaction is thought to be required for both Rho3 and Cdc42 GTPases. Sequence alignment of Rho3 and Cdc42 revealed that Rho3 has a long N-terminal extension, which contains a site (a cysteine at position 5) for palmitoylation (24). In contrast, Cdc42 is not palmitoylated but instead contains a polybasic domain adjacent to the CAAX motif at its C terminus, which is thought to act as a membrane targeting signal via the electrostatic interactions with phospholipids at the plasma membrane (6, 12).In this study we examine how the function and localization of two Rho GTPases are specified at distinct stages of polarized growth in yeast. Using a novel monoclonal antibody, we find that the pattern of cell surface localization observed for the Rho3 GTPase is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we find that the N terminus plays a particularly important role in this specification. The functional effect imparted by the N terminus appears to have two key elements. One element involves palmitoylation of a cysteine in the N terminus of Rho3 that is critical in generating the dispersed pattern of localization observed for Rho3. A second element regulates the affinity of the GTPases for a common effector, the exocyst complex. Taken together, this work provides a model for how these GTPases have evolved distinct functions by adopting sequence elements that affect both the pattern of localization and the ability to engage the downstream effector in a way that allows each GTPase to function at different stages of polarized growth.     

Protein Kinase Cδ-Mediated Phosphorylation of Phospholipase D Controls Integrin-Mediated Cell Spreading

Integrin signaling plays critical roles in cell adhesion, spreading, and migration, and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to the proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin are dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here we demonstrate that protein kinase Cδ (PKCδ) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCδ is activated and translocated to the edges of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCδ activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCδ and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation, and cell spreading after integrin activation. Together, these results suggest that PKCδ is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.Integrin-mediated cell adhesion, spreading, and migration, which are essential for cellular differentiation, proliferation, survival, chemotaxis, and wound healing, require cell polarization with an environmental stimulus (32). To regulate these integrin-mediated functions accurately, coordinated and spatial control of localized cytoskeletal rearrangement is required. The key downstream signaling molecules of integrin-mediated actin cytoskeletal rearrangements include small GTPases of the Rho family, such as Rho, Cdc42, and Rac (57, 58). Recently it was suggested that integrin indirectly regulates the recruitment of small G proteins and their localized activation at a specific plasma membrane region called the cholesterol-enriched membrane microdomain. Furthermore, the membrane targeting of these molecules appears to be required for the activation of downstream effectors that induce actin reorganization (8, 9, 48). However, in the absence of integrin signaling, despite the GTP loading status, activated Rac and Cdc42 remain in the cytosol and cannot activate downstream effectors, such as p21-activated kinase (PAK) (8). This regulation of the localization of small GTPases to a specific site is supported by the observation that Rac1 is localized and activated at the leading edges of migrating cells, while Cdc42 is also activated in cellular protrusions and in the peripheral region (33, 51). The differentially localized activation of small GTPases results in coordinated spatially confined signaling leading to cytoskeletal rearrangement, which is critical for the regulation of integrin-mediated cell spreading and directional cell migration.The hydrolysis of phosphatidylcholine by phospholipase D1 (PLD1) and PLD2 generates the messenger lipid phosphatidic acid (PA) in response to a variety of signals, which include hormones, neurotransmitters, and growth factors (17). It has been shown that PA affects actin cytoskeletal rearrangement and hence lamellipodium extension and integrin-mediated cell spreading as well as migration. PLD activity has been found in detergent-insoluble membrane fractions in which a wide variety of cytoskeletal proteins, such as F-actin, α-actinin, vinculin, paxillin, and talin, were enriched (34). Furthermore, the stimulation of PLD with physiologic and pharmacologic agonists results in its association with actin filaments (34). In addition, actin polymerization and stress fiber formation are tightly coupled to the activation of PLD (14). The formation of lamellipodium structures and membrane ruffles is blocked by PLD inhibition (53, 60), and PLD activity is critical for epithelial cell, leukocyte, and neutrophil adhesion and migration (41, 43, 52). Furthermore, we have previously shown that the activity of PLD is upregulated, and that the activated PLD is translocated to lamellipodia, after integrin activation (3). The PLD product PA acts as a lipid anchor for the membrane translocation of Rac, and this PA-mediated localized activation of Rac is critical for integrin-mediated cell spreading and migration through Rac downstream signaling activation and actin cytoskeleton rearrangement (3). However, the mechanisms that regulate the activation and localization of PLD, which induce the localized downstream activation of integrin signaling, have not been elucidated.Members of the protein kinase C (PKC) family of serine-threonine kinases are known to play important roles in the transduction of signals from the activation of integrin to cell adhesion and spreading, as well as in cell migration via actin reorganization (11, 25, 61, 66). Several studies have shown that the activities of several PKC isozymes are modulated and are crucially required for integrin-mediated cell spreading and migration. The PKCα, -δ, and -ɛ isotypes were activated and then translocated from the cytosol to the membrane after integrin activation, and inhibition of these PKC isozymes prevented cell spreading (10, 47, 66). In addition, the activation of PKCα, -δ, and -ɛ rescued the spreading of α5 integrin-deficient cells on fibronectin (10), and PKCβΙ mediated platelet cell spreading and migration on fibrinogen (2). It has also been demonstrated that PKCθ activity is involved in endothelial cell migration (65). These results suggest that the kinase activities of diverse members of PKC are involved in the integrin-mediated signaling pathway leading to the actin cytoskeletal rearrangement required for cell spreading and migration. Several PKC substrates are known to influence the actin cytoskeleton directly (42). However, the natures of the isoform-specific functions of PKC members and of their specific downstream effectors for actin cytoskeletal rearrangement induction by integrin signaling remain to be elucidated.In this study, we found that PKCδ is an upstream modulator of localized PLD activation in the integrin signaling pathway. We demonstrate for the first time that PKCδ activity (not PKCα or PKCɛ activity) is critical for integrin-mediated PLD activation, and we found that PLD2 is phosphorylated at Thr566 by PKCδ in the integrin signaling pathway. Furthermore, we show that this phosphorylation is critical for integrin-mediated targeting of PLD to membrane ruffles, Rac translocation to the membrane, and lamellipodium formation during cell spreading. These findings strongly suggest a bridge between PKCδ and the signaling of actin cytoskeletal rearrangement by the integrin signaling pathway via PLD activation, and they provide a novel molecular mechanism for localized PLD activation via PKCδ phosphorylation, which is critical for the actin cytoskeletal rearrangements required for integrin-mediated cell spreading.     

Regulation of Immature Dendritic Cell Migration by RhoA Guanine Nucleotide Exchange Factor Arhgef5

There are a large number of Rho guanine nucleotide exchange factors, most of which have no known functions. Here, we carried out a short hairpin RNA-based functional screen of Rho-GEFs for their roles in leukocyte chemotaxis and identified Arhgef5 as an important factor in chemotaxis of a macrophage phage-like RAW264.7 cell line. Arhgef5 can strongly activate RhoA and RhoB and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, in transfected human embryonic kidney 293 cells. In addition, Gβγ interacts with Arhgef5 and can stimulate Arhgef5-mediated activation of RhoA in an in vitro assay. In vivo roles of Arhgef5 were investigated using an Arhgef-5-null mouse line. Arhgef5 deficiency did not affect chemotaxis of mouse macrophages, T and B lymphocytes, and bone marrow-derived mature dendritic cells (DC), but it abrogated MIP1α-induced chemotaxis of immature DCs and impaired migration of DCs from the skin to lymph node. In addition, Arhgef5 deficiency attenuated allergic airway inflammation. Therefore, this study provides new insights into signaling mechanisms for DC migration regulation.Leukocyte chemotaxis underlies leukocyte migration, infiltration, trafficking, and homing that are not only important for normal leukocyte functions, but also have a important role in inflammation-related diseases. Leukocyte chemotaxis is regulated by leukocyte chemoattractants that include bacterial by-products such as formylmethionylleucylphenylalanine, complement proteolytic fragments such as C5a, and the superfamily of chemotactic cytokines, chemokines. These chemoattractants bind to their specific cell G protein-coupled receptors and are primarily coupled to the G_i family of G proteins to regulate leukocyte chemotaxis. Previous studies have established that the Rho family of small GTPases regulates leukocyte migration (1, 2). Rac, Cdc42, and RhoA are the three best studied Rho small GTPases. In myeloid cells, Cdc42 regulates directionality by directing where F-actin and lamellipodia are formed, and Rac regulates F-actin formation in the lamellipodia, which provides a driving force for cell motility (3–6). On the other hand, RhoA regulates the formation and contractility of the actomyosin structure at the back that provides a pushing force (5, 7). Rho guanine nucleotide exchange factors (GEF)³ are key regulators for the activity of these small GTPases. GEFs activate small GTPases by promoting the loading of GTP to the small GTPases, a rate-limiting step in GTPase regulation (8–11). Previous biochemical and genetic studies have revealed how Cdc42 and Rac may be regulated by chemokine receptors in leukocytes. Chemokine receptors can regulate Cdc42 via a Rho-GEF PIXα, which is regulated by Gβγ from the G_i proteins via the interactions between Gβγ and Pak1 and between Pak1 and PIXα in myeloid cells 12. On the other hand, in neutrophils chemokine receptors regulate Rac2 via another Rho-GEF P-Rex1, which is directly regulated by Gβγ (13–15). Two Rho-GEFs have been implicated in regulation of RhoA in neutrophils. GEF115 was found in the leading edges of polarized mouse neutrophils, whereas PDZ Rho-GEF was found in the uropods of differentiated HL-60 cells. Both Rho-GEFs were believed to mediate pertussis toxin-resistant activation of RhoA in these cells. However, a significant portion of RhoA activity in leukocytes are pertussis toxin-sensitive, which is presumably regulated by the α and/or βγ subunits from the G_i proteins. The signaling mechanism for this pertussis toxin-sensitive RhoA regulation by chemokine receptors remains largely elusive.Molecular cloning and genomic sequencing have identified more than 70 Rho-GEFs in mammals (16–20). Many of these Rho-GEFs have been shown to activate RhoA in in vitro and overexpression assays (16–20). However, it is not known if any of them regulate RhoA in vivo, we have found that PIXα is a specific GEF for Cdcd42 in neutrophils (12) despite its potent activity on Rac in in vitro and overexpression assays (21, 22). Therefore, we used a siRNA-based loss of function screen in an attempt to identify the GEFs that regulate myeloid cell migration and RhoA activity. One of the candidates, Arhgef5, was found to be directly activated by Gβγ to regulate RhoA and has an important role in immature DC migration. In addition, Arhgef5 deficiency attenuated allergic airway inflammation in a mouse model.     

Cell Migration Is Regulated by Platelet-Derived Growth Factor Receptor Endocytosis

Cell migration requires spatial and temporal processes that detect and transfer extracellular stimuli into intracellular signals. The platelet-derived growth factor (PDGF) receptor is a cell surface receptor on fibroblasts that regulates proliferation and chemotaxis in response to PDGF. How the PDGF signal is transmitted accurately through the receptor into cells is an unresolved question. Here, we report a new intracellular signaling pathway by which DOCK4, a Rac1 guanine exchange factor, and Dynamin regulate cell migration by PDGF receptor endocytosis. We showed by a series of biochemical and microscopy techniques that Grb2 serves as an adaptor protein in the formation of a ternary complex between the PDGF receptor, DOCK4, and Dynamin, which is formed at the leading edge of cells. We found that this ternary complex regulates PDGF-dependent cell migration by promoting PDGF receptor endocytosis and Rac1 activation at the cell membrane. This study revealed a new mechanism by which cell migration is regulated by PDGF receptor endocytosis.Chemoattractants bind to cell surface receptors, resulting in the cytoskeletal reorganization that permits the migration of cells toward a stimulus. In fibroblasts, the platelet-derived growth factor receptor β (PDGFRβ) is a cell surface receptor tyrosine kinase (RTK) that regulates cell proliferation and chemotaxis in response to PDGF. PDGF binding activates PDGF receptor autophosphorylation, which in turn mediates a series of intracellular signaling cascades initiated by the association of SH2 domain-containing adaptor proteins (25). The adaptor protein Grb2 at the plasma membrane binds to Ras exchange factor Sos1, activating mitogen-activated protein kinase (MAPK) and cell proliferation signals (19). Grb2 also plays a critical role in receptor internalization via its interaction with dynamin, an exchange factor that facilitates receptor entry into endocytic vesicles (32). Grb2 regulates ubiquitination and the degradation of the receptor via its interaction with Cbl, an E3 ubiquitin ligase (33). While the role of Grb2 in modulating receptor levels and facilitating growth factor-dependent mitogenic signals is defined, its role in coordinating receptor-dependent chemotaxis has not been elucidated.The small GTPase Rac1 plays a crucial role in PDGF-mediated chemotaxis by regulating cortical actin at the leading edge of cells. PDGF receptor activation promotes GTP loading and the translocation of Rac1 to the cell membrane via guanine exchange factors (GEFs). The DOCK family of Rac1 GEFs, also called CDM proteins (for Caenorhabditis elegans ced-5, vertebrate DOCK180, and Drosophila myoblast city), are regulators of cell migration and have been implicated in various biological processes, such as lymphocyte migration, phagocytosis, and cancer progression (6, 10, 30, 35). In migrating fibroblasts, DOCK proteins localize to the cell''s leading edge via their interaction with the phospholipid PIP3, but a direct molecular link to PDGF has not been established (5). Biochemical studies show that Rac activation requires the DHR2/docker domain of DOCK proteins and the expression of the PH domain-containing protein Ced-12/ELMO. Previously we identified DOCK4 in a screen for novel tumor suppressor genes using representational difference analysis on mouse tumor cell lines (35). DOCK4, like other CDM proteins, binds ELMO and exerts its biochemical effects on the small GTPases Rac and Rap1 (30, 35). An interesting observation is that the amino acid sequence toward the C terminus is not conserved among individual DOCK family members. The alternate splicing of the DOCK4 gene has been reported, but how amino acid sequence variation alters the signaling properties of DOCK4 for the regulation of cell migration is unknown.Members of the Nck family of adaptor proteins, CrkII and Nck, have been reported to bind to the C terminus of DOCK180 (12, 29). Here, we show that the third member of the family of Nck adaptors, namely Grb2, binds to wild-type DOCK4. We found that a ternary complex formed by Grb2-DOCK4-Dynamin2 interacts with PDGF-activated PDGFβ receptor and promotes growth factor-dependent migration without altering cell proliferation. PDGF-dependent migration requires receptor endocytosis and is regulated by the formation of a DOCK4-Grb2-Dynamin2-PDGFRβ complex at the cell''s leading edge. These studies provide novel mechanistic insights into PDGFRβ regulation and cell migration.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP

Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood. Here we show that ERK activity is required for fibronectin-stimulated Rho-GTP loading, Rho-kinase function, and the maturation of focal adhesions in spreading cells. We identify p190A RhoGAP as a major target for ERK signaling in adhesion assembly and identify roles for ERK phosphorylation of the C terminus in p190A localization and activity. These observations reveal a novel role for ERK signaling in adhesion assembly in addition to its established role in adhesion disassembly.Cell migration is a highly coordinated process essential for physiological and pathological processes (69). Signaling through Rho family GTPases (e.g., Rac, Cdc42, and Rho) is crucial for cell migration. Activated Rac and Cdc42 are involved in the production of a dominant lamellipodium and filopodia, respectively, whereas Rho-stimulated contractile forces are required for tail retraction and to maintain adhesion to the matrix (57, 58, 68). Rac- and Cdc42-dependent membrane protrusions are driven by the actin cytoskeleton and the formation of peripheral focal complexes; Rho activation stabilizes protrusions by stimulating the formation of mature focal adhesions and stress fibers. Active Rho influences cytoskeletal dynamics through effectors including the Rho kinases (ROCKs) (2, 3).Rho activity is stimulated by GEFs that promote GTP binding and attenuated by GTPase-activating proteins (GAPs) that enhance Rho''s intrinsic GTPase activity. However, due to the large number of RhoGEFs and RhoGAPs expressed in mammalian cells, the molecular mechanisms responsible for regulation of Rho activity in time and space are incompletely understood. p190A RhoGAP (hereafter p190A) is implicated in adhesion and migration signaling. p190A contains an N-terminal GTPase domain, a large middle domain juxtaposed to the C-terminal GAP domain, and a short C-terminal tail (74). The C-terminal tail of ∼50 amino acids is divergent between p190A and the closely related family member p190B (14) and thus may specify the unique functional roles for p190A and p190B revealed in gene knockout studies (10, 11, 41, 77, 78). p190A activity is dynamically regulated in response to external cues during cell adhesion and migration (5, 6, 59). Arthur et al. (5) reported that p190A activity is required for the transient decrease in RhoGTP levels seen in fibroblasts adhering to fibronectin. p190A activity is positively regulated by tyrosine phosphorylation (4, 5, 8, 17, 31, 39, 40, 42): phosphorylation at Y1105 promotes its association with p120RasGAP and subsequent recruitment to membranes or cytoskeleton (8, 17, 27, 31, 71, 75, 84). However, Y1105 phosphorylation is alone insufficient to activate p190A GAP activity (39). While the functions of p190A can be irreversibly terminated by ubiquitinylation in a cell-cycle-dependent manner (80), less is known about reversible mechanisms that negatively regulate p190A GAP activity during adhesion and motility.The integration of Rho family GTPase and extracellular signal-regulated kinase (ERK) signaling is important for cell motility (48, 50, 63, 76, 79). Several studies have demonstrated a requirement for ERK signaling in the disassembly of focal adhesions in migrating cells, in part through the activation of calpain proteases (36, 37) that can downregulate focal adhesion kinase (FAK) signaling (15), locally suppress Rho activity (52), and sever cytoskeletal linkers to focal adhesions (7, 33). Inhibition of ERK signaling increases focal adhesion size and retards disassembly of focal adhesions in adherent cells (57, 64, 85, 86). It is also recognized that ERK modulates Rho-dependent cellular processes, including membrane protrusion and migration (18, 25, 64, 86). Interestingly, ERK activated in response to acute fibronectin stimulation localizes not only to mature focal adhesions, but also to peripheral focal complexes (32, 76). Since these complexes can either mature or be turned over (12), ERK may play a distinct role in focal adhesion assembly. ERK is proposed to promote focal adhesion formation by activating myosin light chain kinase (MLCK) (21, 32, 50).Here we find that ERK activity is required for Rho activation and focal adhesion formation during adhesion to fibronectin and that p190A is an essential target of ERK signaling in this context. Inspection of the p190A C terminus reveals a number of consensus ERK sites and indeed p190A is phosphorylated by recombinant ERK only on its C terminus in vitro, and on the same C-terminal peptide in vivo. Mutation of the C-terminal ERK phosphorylation sites to alanine increases the biochemical and biological activity of p190A. Finally, inhibition of MEK or mutation of the C-terminal phosphorylation sites enhances retention of p190A in peripheral membranes during spreading on fibronectin. Our data support the conclusion that ERK phosphorylation inhibits p190A allowing increases in RhoGTP and cytoskeletal changes necessary for focal adhesion formation.     

The Molecular Basis of Phospholipase D2-Induced Chemotaxis: Elucidation of Differential Pathways in Macrophages and Fibroblasts

We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body''s innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by ∼60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid [PA]) and another that involves protein-protein interactions. The first is evidenced by partial abrogation of chemotaxis with lipase activity-defective constructs (PLD2-K758R) and by n-butanol treatment of cells. The second is evidenced by PLD association with the growth factor receptor-bound protein 2 (Grb2) through residue Y¹⁶⁹, located within a Src homology 2 (SH2) consensus site. The association Grb2-PLD2 could be visualized by fluorescence microscopy in RAW/LR5 macrophages concentrated in actin-rich membrane ruffles, making possible that Grb2 serves as a docking or intermediary protein. The Grb2/PLD2-mediated chemotaxis process also depends on Grb2''s ability to recognize other motility proteins, like the Wiskott-Aldrich syndrome protein (WASP). Cell transfection with WASP, PLD2, and Grb2 constructs yields the highest levels of cell migration response, particularly in a macrophage cell line (RAW/LR5) and only modestly in the fibroblast cell line COS-7. Further, RAW/LR5 macrophages utilize for cell migration an additional pathway that involves S6 kinase (S6K) through PLD2-Y²⁹⁶, known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.Normalcy of migration is found in the ability of leukocytes to move toward foreign invaders of the body in phagocytic and immunogenic responses. Fibroblasts and endothelial cells migrate to aid wound repair and deposit collagen around the wounded area (32, 41). Migration is also important in embryogenesis and angiogenesis (37). Cellular migration may be involved not only in the normal physiological state of some cells but also in the pathological state, as in metastasis, whereby tumor cells migrate from the initial tumor site into the circulatory system and establish new colonies (32). Before migration can occur, cells must orient themselves to establish a distinct cell front and rear. The orientation of the cell depends on the inflammation site and location of the chemical stimuli or chemoattractants being released (32, 35). A localized enrichment of filamentous actin (F-actin) under the cell membrane occurs shortly after cell stimulation and is responsible for the formation of the lamellipodia and other morphological changes, such as membrane protrusions or ruffles that occur right before chemotaxis (28).Monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), and colony-stimulating factor (CSF-1, also known as macrophage colony-stimulating factor [M-CSF]) are chemoattractants found to increase cell motility of monocytes and macrophages (52). MIP-1α acts through G-protein-coupled cell surface receptors that are highly abundant on those cells (42) and activate the phosphatidylinositol 3-kinase (PI3K) pathway and phospholipase C (PLC) (49). CSF-1 plays essential roles in the ability of monocytes to survive, proliferate, differentiate, and mature (49) and is also a monocyte/macrophage chemoattractant. CSF-1 acts by binding to cell surface receptors (CSF-1R) encoded by the c-fms proto-oncogene (48). Upon the binding of M-CSF, the CSF-1R dimerizes, and the tyrosine kinase domain is activated, resulting in transphosphorylation of the receptor. Grb2 and PI3K bind to two of the four phosphotyrosyl residues created, and the signal is transmitted to the cell interior (8).Epidermal growth factor (EGF) triggers proliferation and differentiation in fibroblasts (6, 37). EGF also serves other biological functions, such as cell rounding, ruffling, actin cytoskeletal reorganization, filopodium extension, and cell motility (37). Tyrosines that are autophosphorylated at the EGF receptor (EGFR) C terminus upon ligand binding enable binding to Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains (37). Receptor kinase activity, along with at least one of the C-terminal tyrosine autophosphorylation sites, is required for cell movement (9). In addition, PLCγ and protein kinase C (PKC) have been linked to EGF and its ability to enhance cell motility (9, 10).As for the downstream signaling mechanism of these cell surface receptors, the recognized key players are the small GTPases, Rho, Rac, and cdc42. They are activated during actin cytoskeleton rearrangement and in cellular migration (7). New evidence has implicated other signaling proteins. Phospholipase D (PLD) has been found to play a role in leukocyte chemotaxis and adhesion (35). PLD is also involved in the regulation of essential cellular functions, largely due to the production of second messengers such as phosphatidic acid (PA) and ultimately diacylglycerol (DAG) (2, 11, 21, 22, 25, 38, 47, 53). Once produced, PA is involved in many cellular functions, including cytoskeletal rearrangement, phagocytosis, vesicle trafficking, exocytosis, and neuronal and cardiac stimulation (1, 11, 21, 30, 38). PA mediates chemotaxis, as increasing concentrations of PA enhanced the rate of cell migration of phagocytes (35). In the murine lymphoma cell line EL4, Knoepp et al. found that activated PLD2 promotes phosphorylation of FAK and Akt, leading to cell-substrate adhesion (7, 29). However, while inactivated PLD2 inhibits adhesion, migration, proliferation, and tumor invasion, it does not alter the basal level of FAK and Akt phosphorylation (29). Although PA does play a role in cell migration, the specific mechanisms involved are not completely understood, and more precise structure-function studies are needed.We have reported earlier an association of PLD2 and Grb2 important for DNA synthesis/cell proliferation at the level of Y¹⁷⁹ (13) and Y⁵¹¹ (23). This study uncovers that migrating cells also use PLD2 and Grb2 but through a different mechanism, involving residue Y¹⁶⁹ and the presence of the Wiskott-Aldrich syndrome protein (WASP). We also present new evidence implicating PLD2-Y²⁹⁶, known to be phosphorylated by EGFR kinase (24), in a pathway utilizing S6 kinase (S6K). This additional pathway serves to boost the ability of RAW/LR5 cells to migrate more readily than the other type of cell utilized in this study, COS-7 fibroblasts.     

Ustilago maydis Rho1 and 14-3-3 Homologues Participate in Pathways Controlling Cell Separation and Cell Polarity

Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3ɛ and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.Morphological switching is a unique attribute of all dimorphic fungi, which alternate between budding and filamentous growth. In some cases, as with mating, this is a prerequisite for genetic diversity for this subfamily of fungi. In addition, many dimorphic fungal pathogens rely on this ability in order to effectively invade their host. In general, the transition between these alternate life forms means a complete turnover of cellular and proteomic components, which often involves cell cycle arrest and/or cytoskeletal rearrangement. Although the cellular proteomes associated with these two processes share many components, there are both temporal and spatial regulations that are manifested during the transitional phase (4).Temporal-spatial regulation of the proteome during the dimorphic transition requires cooperation and synchronized communication among different regulatory pathways. Two highly intricate, yet well-established, signaling cascades that regulate fungal morphogenesis are the mitogen-activated protein kinase (MAPK) (34, 46) and protein kinase A pathways (11). These signaling cascades detect and perpetuate extracellular stimuli, e.g., pheromones and nutrients, which lead to phase transitions in dimorphic fungi. Although the mechanisms are not as fully understood, members of two highly conserved families of proteins, Rho/Rac GTPases and 14-3-3 proteins, have also been shown to control filamentation. Constituents of the Rho/Rac protein family have been shown to regulate actin organization (26, 35, 36), cytokinesis (3, 49, 52), cell integrity (42, 56), pathogenicity (29), signal transduction (22, 44, 56), and cell migration (8). Their activity is dependent upon the reversible binding of guanine nucleotides catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (15, 22, 23, 25, 44). Upon activation, Rho-GTPases stimulate downstream effector proteins such as p21-activated kinases (PAKs) (29, 51) or Rho kinase (ROCK) (36). Based on in silico analysis of the genome sequence, the fungal pathogen of maize, Ustilago maydis, contains six different Rho/Rac encoding genes: cdc42, rac1, and four additional genes predicted to encode Rho-like proteins (20). Of these, only the roles of Cdc42 and Rac1 have been examined in depth. Cdc42 was shown to regulate cytokinesis, while Rac1 regulates hyphal development in U. maydis (26). We examine here the place of a Rho1 homologue, Rho1, in U. maydis cell morphology, polarity, and development.Similarly, the highly conserved, ubiquitously expressed 14-3-3 proteins that are found in most eukaryotes have also been shown to contribute to cellular differentiation and cytoskeletal organization. Like Rho-GTPases, 14-3-3 proteins play multiple roles, in cytoskeletal function, cell cycle regulation, apoptosis, and the regulation of a variety of signaling pathways (17, 19, 33, 50). These acidic proteins have been found in each cellular compartment and most organisms examined possess multiple isoforms: seven isoforms are found in mammals, and as many as fifteen isoforms have been identified in plants (31). Interestingly, the yeast Saccharomyces cerevisiae, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans, each possess only two 14-3-3 isoforms (50), while Candida albicans contains a single isoform (37). They function typically by binding their particular ligands at phosphoserine or phosphothreonine residues (50). It is not clear what 14-3-3 proteins do in the processes mentioned above, whether they act as scaffolds or effectors. Inspection of the U. maydis genome sequence revealed that this organism could be ideal for the study of 14-3-3 proteins because, unlike most other organisms, the U. maydis genome contains only a single 14-3-3 homologue. Due to its predicted binding of phosphorylated proteins, we named this homologue Pdc1 (for phosphorylation domain coupling protein [10]). Recently, the protein (also designated Bmh1 [32]) was also shown to be involved in cell cycle regulation.Despite the functional differences between Rho-GTPases and 14-3-3 proteins, we provide evidence that members of these two families participate in the same regulatory cascade(s) that control morphogenesis in the dimorphic fungus U. maydis. We are able to demonstrate that both Pdc1 and Rho1 are essential for cell viability. In addition, overexpression of Rho1 led to the reduction of filamentation. Overexpression of Rac1 triggers filamentation in U. maydis (13, 29). We show here that deleting Rac1 eliminates the lethal effect imposed by either Rho1 or Pdc1 depletion. Our results have led us to predict that both Rho1 and Pdc1 are negative regulators of Rac1 in U. maydis and that they play important roles in polarized growth and cytokinesis.     

New Insights into How the Rho Guanine Nucleotide Dissociation Inhibitor Regulates the Interaction of Cdc42 with Membranes

The subcellular localization of the Rho family GTPases is of fundamental importance to their proper functioning in cells. The Rho guanine nucleotide dissociation inhibitor (RhoGDI) plays a key regulatory role by influencing the cellular localization of Rho GTPases and is essential for the transforming activity of oncogenic forms of Cdc42. However, the mechanism by which RhoGDI helps Cdc42 to undergo the transition between a membrane-associated protein and a soluble (cytosolic) species has been poorly understood. Here, we examine how RhoGDI influences the binding of Cdc42 to lipid bilayers. Despite having similar affinities for the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42 in solution, we show that when RhoGDI interacts with Cdc42 along the membrane surface, it has a much higher affinity for GDP-bound Cdc42 compared with its GTP-bound counterpart. Interestingly, the rate for the dissociation of Cdc42·RhoGDI complexes from membranes is unaffected by the nucleotide-bound state of Cdc42. Moreover, the membrane release of Cdc42·RhoGDI complexes occurs at a similar rate as the release of Cdc42 alone, with the major effect of RhoGDI being to impede the re-association of Cdc42 with membranes. These findings lead us to propose a new model for how RhoGDI influences the ability of Cdc42 to move between membranes and the cytosol, which highlights the role of the membrane in helping RhoGDI to distinguish between the GDP- and GTP-bound forms of Cdc42 and holds important implications for how it functions as a key regulator of the cellular localization and signaling activities of this GTPase.The Rho family GTPases are a tightly regulated class of signaling proteins that controls a number of important cellular processes. Known most prominently for their ability to remodel the actin cytoskeleton in mammalian cells (1–3), members of this GTPase family have been shown to play essential roles in cell migration, epithelial cell polarization, phagocytosis, and cell cycle progression (4–11). The Rho family member Cdc42 was discovered for its essential role in bud formation in Saccharomyces cerevisiae (12). However, after its identification in higher organisms (13), Cdc42 has been implicated in a diverse array of signaling pathways including those involved in the regulation of cell growth and in the induction of malignant transformation (14). Indeed, point mutations which enable Cdc42 to undergo the spontaneous exchange of GDP for GTP cause NIH3T3 cells to form colonies in soft agar and grow in low serum, two hallmarks of cellular transformation (15). The introduction of activated Cdc42 mutants into nude mice gives rise to tumor formation (16). Moreover, cellular transformation by oncogenic Ras, one of the most commonly mutated proteins in human cancers, requires the activation of Cdc42 (17).At the molecular level, there are a number of mechanisms that possibly contribute to the roles played by Cdc42 in cell growth control and cellular transformation. These include the ability of Cdc42 to activate the c-Jun NH₂-terminal kinase and p38/Mpk2 signaling pathways (18–20) as well as spatially regulate proteins implicated in the establishment of microtubule-dependent cell polarity including glycogen synthase kinase-3β and adenomatous polyposis coli (21), extend the lifetime of epidermal growth factor receptor-signaling activities by sequestering Cbl, a ubiquitin E3 ligase (22), and influence intracellular trafficking events (23, 24). To mediate such a wide range of cellular responses, two parameters must be properly regulated; that is, the activation state of Cdc42 and its subcellular localization. As is the case with other GTPases, the activation of Cdc42 occurs as an outcome of GDP-GTP exchange, which then enables it to undergo high affinity interactions with effector proteins (25–27). Upon the hydrolysis of GTP to GDP, Cdc42 is converted back to a signaling-inactive state. Two families of proteins work in opposing fashion to regulate the GTP-binding/GTPase cycle of Cdc42. GTPase-activating proteins recognize the GTP-bound form of Cdc42 and accelerate the hydrolysis of GTP to GDP, rendering Cdc42 inactive (28, 29). Guanine nucleotide exchange factors (GEFs)² stimulate the dissociation of GDP from Cdc42, thereby promoting the formation of its signaling-active, GTP-bound state (29, 30).Of equal importance to its activation status is the spatial regulation of Cdc42. This is highly contingent on the particular cellular membranes that serve as sites of binding and/or recruitment of Cdc42 (31–33). The vast majority of in vitro studies performed on Cdc42 have been carried out in the absence of lipids, which is an important omission considering that virtually all of the physiological functions of Cdc42 occur on a membrane surface (34). Cdc42, along with most other Rho family GTPases, undergoes a series of carboxyl-terminal modifications which result in the covalent attachment of a 20-carbon geranylgeranyl lipid anchor (35–37). Directly preceding this lipid tail is a sequence of basic residues that further stabilizes the association of Cdc42 with the membrane surface (31, 33, 38). A ubiquitously expressed 22-kDa protein called Rho guanine nucleotide dissociation inhibitor (RhoGDI) was found to form a soluble (cytosolic) complex with Cdc42 and other Rho GTPases and to apparently promote their release from membranes (39, 40). RhoGDI was originally discovered and named for its ability to block the GEF- and EDTA-stimulated nucleotide exchange activity of Rho family GTPases (39, 41, 42) and then subsequently shown to inhibit the GTP-hydrolytic activity of Cdc42 (43) and to be capable of interacting with the GDP- and GTP-bound forms of Cdc42 in solution with equal affinity (44). The x-ray crystal structure of a complex between RhoGDI and Cdc42-GDP revealed two types of binding interactions (45). An amino-terminal regulatory arm of RhoGDI was shown to form a helix-loop-helix motif that binds to both of the switch domains of Cdc42, leading to the inhibition of GTP hydrolysis and GDP dissociation (45, 46). The carboxyl-terminal two-thirds of RhoGDI assumes an immunoglobulin-like domain, forming a hydrophobic pocket that in effect provides a membrane substitute for the geranylgeranyl moiety of Cdc42. After release from membranes, the lipid anchor of Cdc42 binds in the hydrophobic pocket of RhoGDI, thereby helping to maintain Cdc42 in solution (45–47).Prior work from our laboratory has demonstrated an essential role for RhoGDI in Cdc42-mediated cellular transformation. Based on the x-ray crystal structure for the Cdc42·RhoGDI complex, Arg-66 of Cdc42 makes multiple contacts with RhoGDI. When this residue was changed to alanine, Cdc42 was unable to bind to RhoGDI but was still capable of interacting with its other regulatory and effector proteins. Interestingly, when the R66A mutant of Cdc42 was examined in the constitutively active Cdc42(F28L) background, the resulting Cdc42 double mutant was no longer able to transform cells (48). Knocking down RhoGDI by small interfering RNA also blocked transformation by Cdc42. These findings highlighted a key role for RhoGDI in the ability of Cdc42 to stimulate signaling pathways of importance to cellular transformation, presumably by influencing the membrane association of Cdc42 and ensuring its proper cellular localization.In the present study we have set out to better understand how RhoGDI regulates the signaling functions of Cdc42 and, in particular, how RhoGDI affects the association of Cdc42 with membranes. We show how the membrane plays a previously unappreciated role in allowing RhoGDI to distinguish between the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42. By assaying the binding of Cdc42 to insect cell membranes and compositionally defined liposomes through different approaches including a sensitive, real-time fluorescence resonance energy transfer (FRET) readout, we have been able to establish how RhoGDI influences the ability of Cdc42 to transition between a membrane-bound and soluble species. This has led us to propose a new mechanism describing how RhoGDI performs its important regulatory function.     

HIV-1 Nef Inhibits Ruffles,Induces Filopodia,and Modulates Migration of Infected Lymphocytes

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.Human immunodeficiency virus type 1 (HIV-1) mostly replicates in T-cell areas of secondary lymphoid organs (SLOs) and induces pathological changes in their architecture. Such changes are likely due to a combination of events, including destruction of T cells, chronic immune activation, and alteration of T-cell motility toward and inside the SLOs (27, 37, 50, 53). Indeed, to fulfill their immune surveillance role, T cells continuously circulate in and out of blood, lymph nodes (LNs), and tissues (60).Lymphocyte recruitment from the bloodstream into LNs depends on three distinct processes, i.e., attachment to high endothelial venules (HEVs), extravasation, and cell migration (10, 60). Adhesion to the endothelium and extracellular matrix (ECM) is a crucial step, regulated in part by β1 integrins, α4β1 (VLA-4) and α5β1, that bind VCAM-1 and/or fibronectin (56). Chemokines and their Gαi-protein-coupled receptors are key regulators of lymphocyte trafficking (32). For instance, CCL19 and CCL21 are constitutively produced by HEVs and by fibroblastic reticular cells of T-cell areas of LNs (21, 28, 29). These two chemokines share the receptor CCR7, expressed by naïve T cells and a fraction of memory T cells (47). They play a major role in lymphocyte homing to LNs, in steady state as well as under conditions of inflammation, and may control T-cell positioning within defined functional compartments (1, 17, 18, 47). CXCR4 and its ligand CXCL12/SDF-1 also contribute to T-cell entry into LNs (5, 23, 40). In addition, effector and memory T cells express a broad range of receptors binding inflammatory chemokines, such as the CCR5 ligands CCL3 (MIP1α), CCL4 (MIP1β), and CCL5 (Rantes).Efficient accomplishment of lymphocyte migration and immune functions requires tight regulation of the cellular cytoskeleton (59). This is mediated by the small GTPases of the Rho subfamily, such as Rho, Rac, and Cdc42 (11, 58). They activate specific actin filament assembly factors to generate sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli) (6). These structures have different functions. Lamellipodia and ruffles are formed during crawling cell motility and spreading. Filopodia protrude from the leading edges of many motile cells. They appear to perform sensory and exploratory functions to steer cells, depending on cues from the environment (42). Moreover, filopodia, or other thin structures called tunneling nanotubes, have been shown to form intercellular bridges, allowing viruses to spread through remote contacts between infected cells and targets (44, 48, 49, 52).HIV-1 hijacks cytoskeleton dynamics in order to ensure viral entry and transport within and egress from target cells (34; reviewed in reference 13). In particular, the viral protein Nef modifies actin remodeling in various cell systems. In T cells, Nef alters actin rearrangements triggered by activation of T-cell (TCR) or chemokine receptors (22, 54). Nef inhibits immunological synapse formation, a dynamic process involving rapid actin modifications (57). Nef also affects plasma membrane plasticity, inducing secretion of microvesicle clusters (33). In macrophages, Nef induces the extension of long intercellular conduits allowing its own transfer to B cells (61). A number of studies have reported that Nef affects T-cell chemotaxis (generally to CXCL12) through the modulation of Rho-GTPase-regulated signaling pathways (7, 24, 39, 54). Migration studies have generally been performed using Nef-expressing cells, and rarely in the context of HIV-1 infection (54). From a molecular standpoint, it has recently been proposed that Nef acts in part by deregulating cofilin, an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (54).In the present study, our goal was to gain further insights into the effect of HIV-1 infection on cytoskeleton dynamics. We used a panel of innovative techniques allowing analysis of cell shape, adhesion, and motility. We report that in HIV-infected lymphocytes, Nef promotes filopodium-like formation while it inhibits membrane ruffling. Nef impairs cell adhesion on the extracellular matrix and decreases intrinsic cell motility. Lymphocyte migration toward various chemokines (CXCL12, CCL3, and CCL19) is also inhibited. Our results suggest that Nef may facilitate viral spread and contribute to AIDS pathogenesis by manipulating the migration of lymphocytes.