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In this article it is shown that EGF stimulation leads to rapid activation of RhoG through Vav GEFs and the GEF PLEKHG6. Importantly, different cellular responses induced by EGF are determined by the available GEFs. Furthermore, this article presents results showing that EGF-stimulated cell migration and EGFR internalization are regulated by RhoG., RhoG is a member of the Rac-like subgroup of Rho GTPases and has been linked to a variety of different cellular functions. Nevertheless, many aspects of RhoG upstream and downstream signaling remain unclear; in particular, few extracellular stimuli that modulate RhoG activity have been identified. Here, we describe that stimulation of epithelial cells with epidermal growth factor leads to strong and rapid activation of RhoG. Importantly, this rapid activation was not observed with other growth factors tested. The kinetics of RhoG activation after epidermal growth factor (EGF) stimulation parallel the previously described Rac1 activation. However, we show that both GTPases are activated independently of one another. Kinase inhibition studies indicate that the rapid activation of RhoG and Rac1 after EGF treatment requires the activity of the EGF receptor kinase, but neither phosphatidylinositol 3-kinase nor Src kinases. By using nucleotide-free RhoG pull-down assays and small interfering RNA-mediated knockdown studies, we further show that guanine-nucleotide exchange factors (GEFs) of the Vav family mediate EGF-induced rapid activation of RhoG. In addition, we found that in certain cell types the recently described RhoG GEF PLEKHG6 can also contribute to the rapid activation of RhoG after EGF stimulation. Finally, we present results that show that RhoG has functions in EGF-stimulated cell migration and in regulating EGF receptor internalization.

Resident dendritic cells (DC) within the T cell area of the lymph node take up soluble antigens that enter via the afferent lymphatics before antigen carrying DC arrive from the periphery. The reticular network within the lymph node is a conduit system forming the infrastructure for the fast delivery of soluble substances from the afferent lymph to the lumen of high endothelial venules (HEVs). Using high-resolution light microscopy and 3D reconstruction, we show here that these conduits are unique basement membrane-like structures ensheathed by fibroblastic reticular cells with occasional resident DC embedded within this cell layer. Conduit-associated DC are capable of taking up and processing soluble antigens transported within the conduits, whereas immigrated mature DC occur remote from the reticular fibers. The conduit system is, therefore, not a closed compartment that shuttles substances through the lymph node but represents the morphological equivalent to the filtering function of the lymph node.

Supplemental Data Supplemental Figure S1 Characterization of WT and aPKC-deficient podocytes. A–C: Genomic DNA isolated from deficient and control cell lines was tested for the presence of Cre recombinase (A), floxed and WT alleles of PKCλ/ι (B), or WT and knockout alleles of PKCζ (C). As controls, genomic DNA samples of tail biopsies were used. D: Differentiated deficient or control cells were stained with antibodies against synaptopodin or WT-1. All used cell lines were positive for the tested podocyte markers. Scale bars = 50 μm. Download Supplemental Figure S2 Relative mRNA and protein expression of PKCλ/ι, PKCζ, and Def-6 in deficient and control podocytes. A–C: Real-time PCR measurements and Western blot analysis of PKCλ/ι- and PKCζ-deficient cells in comparison with control cells. A: PKCλ/ι mRNA and protein are reduced in the PKCλ/ι −/− cells. B: PKCζ mRNA and protein are reduced in the PKCζ −/− cells. C: Def-6 mRNA is up-regulated in the PKCλ/ι −/− cells but not in PKCζ −/− cells. mRNA level is normalized for HPRT-1. Def-6 protein expression is not changed. ∗∗P < 0.01. Download Supplemental Table S1 Download Supplemental Table S2 Download Supplemental Table S3 Download Supplemental Table S4 Download Supplemental Table S5 Download Supplemental Table S6 Download Supplemental Table S7 Download Supplemental Table S8 Download Supplemental Data Supplemental material for this article can be found at . The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes.

The passage of leukocytes across the endothelium and into arterial walls is a critical step in the development of atherosclerosis. Previously, we showed in vitro that the RhoG guanine nucleotide exchange factor SGEF (Arhgef26) contributes to the formation of ICAM-1-induced endothelial docking structures that facilitate leukocyte transendothelial migration. To further explore the in vivo role of this protein during inflammation, we generated SGEF-deficient mice. When crossed with ApoE null mice and fed a Western diet, mice lacking SGEF showed a significant decrease in the formation of atherosclerosis in multiple aortic areas. A fluorescent biosensor revealed local activation of RhoG around bead-clustered ICAM-1 in mouse aortic endothelial cells. Notably, this activation was decreased in cells from SGEF-deficient aortas compared to wild type. In addition, scanning electron microscopy of intimal surfaces of SGEF(-/-) mouse aortas revealed reduced docking structures around beads that were coated with ICAM-1 antibody. Similarly, under conditions of flow, these beads adhered less stably to the luminal surface of carotid arteries from SGEF(-/-) mice. Taken together, these results show for the first time that a Rho-GEF, namely SGEF, contributes to the formation of atherosclerosis by promoting endothelial docking structures and thereby retention of leukocytes at athero-prone sites of inflammation experiencing high shear flow. SGEF may therefore provide a novel therapeutic target for inhibiting the development of atherosclerosis.

Integrin alpha7beta1 is the major laminin binding integrin receptor of muscle cells. The alpha7 chain occurs in several splice isoforms, of which alpha7A and alpha7B differ in their intracellular domains only. The fact that the expression of alpha7A and alpha7B is tightly regulated during skeletal muscle development suggests different and distinct roles for both isoforms. However, so far, functional properties and interacting proteins were described for the alpha7B chain only. Using a yeast two-hybrid screen, we have found that Def-6, a guanine nucleotide exchange factor for Rac1, binds to the intracellular domain of the alpha7A subunit. The specificity of the Def-6-alpha7A interaction has been shown by direct yeast two-hybrid binding assays and coprecipitation experiments. This is the first description of an alpha7A-specific and -exclusive interaction, because Def-6 did not bind to any other tested integrin cytoplasmic domain. Interestingly, the binding of Def-6 to alpha7A was abolished, when cells were cotransfected with an Src-related kinase, which is known to phosphorylate Def-6 and stimulate its exchange activity. We found expression of Def-6 was not only restricted to T-lymphocytes as described thus far but in a more widespread manner, including different muscle tissues. In cells, Def-6 is seen in newly forming cell protrusions and focal adhesions, and its localization partially overlaps with the alpha7A integrin receptor. C2C12 myoblasts overexpressing Def-6 show a delay of Rac1 inactivation during myogenic differentiation and abnormal myotube formation. Thus, our data suggest a role for Def-6 in the fine regulation of Rac1 during myogenesis with the integrin alpha7A chain guiding this regulation in a spatio-temporal manner.

FHL1, FHL2, and FHL3 are members of the four and one-half LIM domain protein subclass that are expressed in striated muscles. Here we show that FHL2 and FHL3 are novel alpha(7)beta(1) integrin-interacting proteins. They bind both the alpha- and the beta-subunit as well as different splice isoforms. The minimal binding sites for FHL2 and FHL3 on beta(1A)-chain overlap, whereas on alpha(7A) and alpha(7B) subunits they are situated adjacent. Determining the binding sites for integrins on FHL2 or FHL3 revealed that the suprastructure of the whole molecule is important for these associations, rather than any single LIM domain. Immunofluorescence studies with cells expressing full-length FHL proteins or their deletion mutants showed that FHL2 and FHL3 but not FHL1 colocalize with integrins at cell adhesion sites. Further, their recruitment to the membrane results from binding to either the alpha- or the beta-chain of the integrin receptor. The association of FHL2 or FHL3 with integrin receptors neither influences attachment of cells to different substrates nor changes their migration capacity. However, in cardiac and skeletal muscles, FHL2 and FHL3, respectively, are colocalized with alpha(7)beta(1) integrin receptor at the periphery of Z-discs, suggesting a role in mechanical stabilization of muscle cells.