Profilin enhances Cdc42-induced nucleation of actin polymerization

We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline.Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.     

MARCKS-Related Protein Binds to Actin without Significantly Affecting Actin Polymerization or Network Structure

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.     

Pituicyte Stellation is Prevented by RhoA-or Cdc42-Dependent Actin Polymerization

Our aim was to shed light on different steps leading from metabotropic receptor activation to changes in cell shape, such as those that characterize the morphological plasticity of neurohypophysial astrocytes (pituicytes). Using explant cultures of adult rat pituicytes, we have previously established that adenosine A1 receptor activation induces stellation via inhibition of RhoA monomeric GTPase and subsequent disruption of actin stress fibers. Here, we rule out RhoA phosphorylation as a mechanism for that inhibition. Rather, our results are more consistent with involvement of a GTPase-activating protein (GAP). siRNA and pull-down experiments suggest that a step downstream of RhoA might involve Cdc42, another GTPase of the Rho family. However, RhoA activation, e.g., in the presence of serum, induces stress fibers, whereas direct Cdc42 activation appears to confine actin within a submembrane—i.e., cortical—network, which also prevents stellation. Therefore, we propose that RhoA may activate Cdc42 in parallel with an effector, such as p160Rho-kinase, that induces and maintains actin stress fibers in a dominant fashion. Rac1 is not involved in the stellation process per se but appears to induce a dendritogenic effect. Ultimately, it may be stated that pituicyte stellation is inducible upon mere actin depolymerization, and preventable upon actin organization, be it in the form of stress fibers or in a cortical configuration.     

A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast

The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.     

爪蟾p21活化激酶2参与卵母细胞胞质分裂过程不依赖于Cdc42

研究p21活化激酶2(p21-activated kinase2,PAK2)在卵母细胞成熟过程中的作用.以爪蟾卵母细胞为模型,分别向爪蟾卵母细胞显微注射PAK2-N端(PAK2-N-terminal,PAK2-NT)和PAK2-N端突变体(PAK2-N-terminal mutation,PAK2-NTm)mRNA,荧光显微镜下观察胚泡破裂发生.采用共聚焦显微镜,时间延迟摄影法观察正常卵母细胞、PAK2-NTmRNA注射组和PAK2-NTm mRNA注射组卵母细胞胞质分裂、极体形成及与Cdc42活性的关系.结果表明,PAK2-NTmRNA和PAK2-NTm mRNA注射组的卵母细胞与正常卵母细胞胚泡破裂发生相似,但PAK2-NTmRNA和PAK2-NTm mRNA注射组未见胞质分裂和极体形成.结果提示,PAK2参与卵母细胞胞质分裂和极体形成可能不依赖于Cdc42的调节过程.     

Regulation of neurogenesis by Fgf8a requires Cdc42 signaling and a novel Cdc42 effector protein

Fibroblast growth factor (FGF) signaling is required for numerous aspects of neural development, including neural induction, CNS patterning and neurogenesis. The ability of FGFs to activate Ras/MAPK signaling is thought to be critical for these functions. However, it is unlikely that MAPK signaling can fully explain the diversity of responses to FGFs. We have characterized a Cdc42-dependent signaling pathway operating downstream of the Fgf8a splice isoform. We show that a Cdc42 effector 4-like protein (Cdc42ep4-l or Cep4l) has robust neuronal-inducing activity in Xenopus embryos. Furthermore, we find that Cep4l and Cdc42 itself are necessary and sufficient for sensory neurogenesis in vivo. Furthermore, both proteins are involved in Fgf8a-induced neuronal induction, and Cdc42/Cep4l association is promoted specifically by the Fgf8a isoform of Fgf8, but not by Fgf8b, which lacks neuronal inducing activity. Overall, these data suggest a novel role for Cdc42 in an Fgf8a-specific signaling pathway essential for vertebrate neuronal development.     

Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway

Glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present at the surface of living cells in cholesterol dependent nanoscale clusters. These clusters appear to act as sorting signals for the selective endocytosis of GPI-APs via a Cdc42-regulated, dynamin and clathrin-independent pinocytic pathway called the GPI-AP-enriched early endosomal compartments (GEECs) pathway. Here we show that endocytosis via the GEECs pathway is inhibited by mild depletion of cholesterol, perturbation of actin polymerization or overexpression of the Cdc42/Rac-interactive-binding (CRIB) motif of neural Wiskott-Aldrich syndrome protein (N-WASP). Consistent with the involvement of Cdc42-based actin nanomachinery, nascent endocytic vesicles containing cargo for the GEEC pathway co-localize with fluorescent protein-tagged isoforms of Cdc42, CRIB domain, N-WASP and actin; high-resolution electron microscopy on plasma membrane sheets reveals Cdc42-labelled regions rich in green fluorescent protein-GPI. Using total internal reflection fluorescence microscopy at the single-molecule scale, we find that mild cholesterol depletion alters the dynamics of actin polymerization at the cell surface by inhibiting Cdc42 activation and consequently its stabilization at the cell surface. These results suggest that endocytosis into GEECs occurs through a cholesterol-sensitive, Cdc42-based recruitment of the actin polymerization machinery.     

Cdc42 and its BORG2 and BORG3 effectors control the subcellular localization of septins between actin stress fibers and microtubules

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Rac1, RhoA, and Cdc42 participate in HeLa cell invasion by group B streptococcus

The group B streptococcus (GBS) is an important human pathogen with the ability to cause invasive disease. To do so, the bacteria must invade host cells. It has been well documented that GBS are able to invade a variety of nonphagocytic host cell types, and this process is thought to involve a number of pathogen-host cell interactions. While some of the molecular aspects of the GBS-host cell invasion process have been characterized, many events still remain unclear. The objective of this investigation was to evaluate the role of the Rho-family GTPases Rac, Rho, and Cdc42 in GBS invasion into epithelial cells. The epithelial cell invasion process was modeled using HeLa 229 cell culture. Treatment of HeLa cells with 10 microM compactin, a pan-GTPase inhibitor, abolished GBS internalization, suggesting that GTPases are involved in the GBS invasion process. The addition of Toxin B or exoenzyme C3 to HeLa cells before GBS infection reduced invasion by 50%, further suggesting that the Rho-family GTPases are involved in GBS entry. Examining invasion of GBS into HeLa cells with altered genetic backgrounds was used to confirm these findings; GBS invasion into HeLa cells transiently transfected with dominant negative Rac1, Cdc42, or RhoA reduced invasion by 75%, 51%, and 42%, respectively. Results of this study suggest that the Rho-family GTPases are required for efficient invasion of HeLa cells by GBS.     

Cdc42 and Tks5: A minimal and universal molecular signature for functional invadosomes

Invadosomes are actin-based structures involved in extracellular-matrix degradation. Invadosomes, either known as podosomes or invadopodia, are found in an increasing number of cell types. Moreover, their overall organization and molecular composition may vary from one cell type to the other. Some are constitutive such as podosomes in hematopoietic cells whereas others are inducible. However, they share the same feature, their ability to interact and to degrade the extracellular matrix. Based on the literature and our own experiments, the aim of this study was to establish a minimal molecular definition of active invadosomes. We first highlighted that Cdc42 is the key RhoGTPase involved in invadosome formation in all described models. Using different cellular models, such as NIH-3T3, HeLa, and endothelial cells, we demonstrated that overexpression of an active form of Cdc42 is sufficient to form invadosome actin cores. Therefore, active Cdc42 must be considered not only as an inducer of filopodia, but also as an inducer of invadosomes. Depending on the expression level of Tks5, these Cdc42-dependent actin cores were endowed or not with a proteolytic activity. In fact, Tks5 overexpression rescued this activity in Tks5 low expressing cells. We thus described the adaptor protein Tks5 as a major actor of the invadosome degradation function. Surprisingly, we found that Src kinases are not always required for invadosome formation and function. These data suggest that even if Src family members are the principal kinases involved in the majority of invadosomes, it cannot be considered as a common element for all invadosome structures. We thus define a minimal and universal molecular signature of invadosome that includes Cdc42 activity and Tks5 presence in order to drive the actin machinery and the proteolytic activity of these invasive structures.     

Cdc42-driven podosome formation in endothelial cells

Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.     

NMR assignment of Cdc42(T35A), an active Switch I mutant of Cdc42

Cdc42(T35A) is an active construct of Cdc42, a Ras GTPase involved in signal transduction, containing a single-point mutation in an important effector-binding region. We determined the backbone and side chain resonance assignments of ¹³C,¹⁵N-labelled Cdc42(T35A) from E. coli.     

Cdc42 expression in keratinocytes is required for the maintenance of the basement membrane in skin

Cdc42 is a small GTPase, which acts as a molecular switch to regulate a wide variety of cellular functions, such as actin cytoskeleton organization, cell proliferation, apoptosis, cell migration and in particular, cell polarity. Formation and maintenance of the basement membrane is a polarized process, which requires directed secretion, deposition and organization of basement membrane components at the basal side of epithelial cells. In the current study, we analyzed the maintenance of skin basement membrane in mice with a keratinocyte-restricted deletion of the Cdc42 gene. In the absence of Cdc42, basement membrane components became aberrantly deposited and the processing of laminin 5 was impaired in parts of the dermal-epidermal junction. These impairments became more severe with age and corresponded to local defects of the basement membrane in 4.5-month-old mutant mice. However, both, structure and number of hemidesomosomes were not significantly changed in the Cdc42 mutant skin compared with the control mice and no blister formation was observed in mutant skin. These data indicate that Cdc42 in keratinocytes is important for maintenance of the basement membrane of skin.     

Signaling role of Cdc42 in regulating mammalian physiology

Cdc42 is a member of the Rho GTPase family of intracellular molecular switches regulating multiple signaling pathways involved in actomyosin organization and cell proliferation. Knowledge of its signaling function in mammalian cells came mostly from studies using the dominant-negative or constitutively active mutant overexpression approach in the past 2 decades. Such an approach imposes a number of experimental limitations related to specificity, dosage, and/or clonal variability. Recent studies by conditional gene targeting of cdc42 in mice have revealed its tissue- and cell type-specific role and provide definitive information of the physiological signaling functions of Cdc42 in vivo.     

Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization

The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization. Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms. To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis. The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins. Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells. Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p. The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins. Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.     

Rac1、Cdc42单分子探针在活细胞中表达差异的比较

利用构建的Rac1、Cdc42单分子探针,探索其在NIH3T3、Hela、COS7等活细胞中的最佳表达效果.结果表明在胞浆中表达的探针其荧光值在诱导5～10 min达到最大值,随着时间的推移,其荧光强度逐渐减弱;在细胞膜上表达的探针也是相似的.DNA/转染试剂为1:3时的表达效果最佳;每毫升转染培养基的质粒DNA含量为4或6 μg时,其转染表达可达到最佳;在不同种类的细胞中表达时其差异不明显;在细胞的不同部位表达差异不明显.     

Identification and characterization of the Cdc42-binding site of IQGAP1

IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP1 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1.     

Cdc42 is crucial for the maturation of primordial cell junctions in keratinocytes independent of Rac1

Cell-cell contacts are crucial for the integrity of all tissues. Contrasting reports have been published about the role of Cdc42 in epithelial cell-cell contacts in vitro. In keratinocytes, it was suggested that Rac1 and not Cdc42 is crucial for the formation of mature epithelial junctions, based on dominant negative inhibition experiments. Deletion of the Cdc42 gene in keratinocytes in vivo slowly impaired the maintenance of cell-cell contacts by an increased degradation of β-catenin. Whether Cdc42 is required for the formation of mature junctions was not tested.We show now that Cdc42-deficient immortalized and primary keratinocytes form only punctate primordial cell contacts in vitro, which cannot mature into belt-like junctions. This defect was independent of enhanced degradation of β-catenin, but correlated to an impaired activation and localization of aPKCζ in the Cdc42-null keratinocytes. Inhibition of aPKCζ by the inhibitor Gö6983 reproduced the phenotype, suggesting that decreased activation of aPKCζ was sufficient to explain the defective junctional maturation. In the absence of Cdc42, Rac1 activation was strongly decreased, indicating that Cdc42 is upstream of Rac1 activation. These data reveal that Cdc42 is crucial for the formation of mature epithelial cell junctions between keratinocytes by regulating activation of aPKCζ.     

Group I and II mammalian PAKs have different modes of activation by Cdc42

p21‐activated kinases (PAKs) are Cdc42 effectors found in metazoans, fungi and protozoa. They are subdivided into PAK1‐like (group I) or PAK4‐like (group II) kinases. Human PAK4 is widely expressed and its regulatory mechanism is unknown. We show that PAK4 is strongly inhibited by a newly identified auto‐inhibitory domain (AID) formed by amino acids 20 to 68, which is evolutionarily related to that of other PAKs. In contrast to group I kinases, PAK4 is constitutively phosphorylated on Ser 474 in the activation loop, but held in an inactive state until Cdc42 binding. Thus, group II PAKs are regulated through conformational changes in the AID rather than A‐loop phosphorylation.