Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility

ELMO1 has an essential role in the internalization of Salmonella Typhimurium into enteric macrophages that impacts disease outcome.

Backgrounds and aims4-6 million people die of enteric infections each year. After invading intestinal epithelial cells, enteric bacteria encounter phagocytes. However, little is known about how phagocytes internalize the bacteria to generate host responses. Previously, we have shown that BAI1 (Brain Angiogenesis Inhibitor 1) binds and internalizes Gram-negative bacteria through an ELMO1 (Engulfment and cell Motility protein 1)/Rac1-dependent mechanism. Here we delineate the role of ELMO1 in host inflammatory responses following enteric infection.MethodsELMO1-depleted murine macrophage cell lines, intestinal macrophages and ELMO1 deficient mice (total or myeloid-cell specific) was infected with Salmonella enterica serovar Typhimurium. The bacterial load, inflammatory cytokines and histopathology was evaluated in the ileum, cecum and spleen. The ELMO1 dependent host cytokines were detected by a cytokine array. ELMO1 mediated Rac1 activity was measured by pulldown assay.ResultsThe cytokine array showed reduced release of pro-inflammatory cytokines, including TNF-α and MCP-1, by ELMO1-depleted macrophages. Inhibition of ELMO1 expression in macrophages decreased Rac1 activation (~6 fold) and reduced internalization of Salmonella. ELMO1-dependent internalization was indispensable for TNF-α and MCP-1. Simultaneous inhibition of ELMO1 and Rac function virtually abrogated TNF-α responses to infection. Further, activation of NF-κB, ERK1/2 and p38 MAP kinases were impaired in ELMO1-depleted cells. Strikingly, bacterial internalization by intestinal macrophages was completely dependent on ELMO1. Salmonella infection of ELMO1-deficient mice resulted in a 90% reduction in bacterial burden and attenuated inflammatory responses in the ileum, spleen and cecum.ConclusionThese findings suggest a novel role for ELMO1 in facilitating intracellular bacterial sensing and the induction of inflammatory responses following infection with Salmonella

The Arf6 GEF GEP100/BRAG2 Regulates Cell Adhesion by Controlling Endocytosis of β1 Integrins

SummaryThe small GTPase Arf6 has been shown to regulate the post-endocytic trafficking of a subset of membrane proteins, including β1 integrins, and inhibition of Arf6 function impairs both cell adhesion and motility [1]. The activity of Arf GTPases is regulated by a large family of guanine nucleotide exchange factors (GEFs) [2]. Arf-GEP100/BRAG2 is a GEF with reported specificity for Arf6 in vitro [3], but it is otherwise poorly characterized. Here we report that BRAG2 exists in two ubiquitously expressed isoforms, which we call BRAG2a and BRAG2b, both of which can activate Arf6 in vivo. Depletion of endogenous BRAG2 by siRNA leads to dramatic effects in the cell periphery; one such effect is an accumulation of β1 integrin on the cell surface and a corresponding enhancement of cell attachment and spreading on fibronectin-coated substrates. In contrast, depletion of Arf6 leads to intracellular accumulation of β1 integrin and reduced adhesion and spreading. These findings suggest that Arf6 regulates both endocytosis and recycling of β1 integrins and that BRAG2 functions selectively to activate Arf6 during integrin internalization

Distribution of lutzomyia longipalpis chemotype populations in Sao Paulo state, Brazil

American visceral leishmaniasis (AVL) is an emerging disease in the state of Sao Paulo, Brazil. Its geographical expansion and the increase in the number of human cases has been linked to dispersion of Lutzomyia longipalpis into urban areas. To produce more accurate risk maps we investigated the geographic distribution and routes of expansion of the disease as well as chemotype populations of the vector. A database, containing the annual records of municipalities which had notified human and canine AVL cases as well as the presence of the vector, was compiled. The chemotypes of L. longipalpis populations from municipalities in different regions of Sao Paulo State were determined by Coupled Gas Chromatography - Mass Spectrometry. From 1997 to June 2014, L. longipalpis has been reported in 166 municipalities, 148 of them in the Western region. A total of 106 municipalities were identified with transmission and 99 were located in the Western region, where all 2,204 autochthonous human cases occurred. Both the vector and the occurrence of human cases have expanded in a South-easterly direction, from the Western to central region, and from there, a further expansion to the North and the South. The (S)-9-methylgermacrene-B population of L. longipalpis is widely distributed in the Western region and the cembrene-1 population is restricted to the Eastern region. The maps in the present study show that there are two distinct epidemiological patterns of AVL in Sao Paulo State and that the expansion of human and canine AVL cases through the Western region has followed the same dispersion route of only one of the two species of the L. longipalpis complex, (S)-9-methylgermacrene-B. Entomological vigilance based on the routes of dispersion and identification of the chemotype population could be used to identify at-risk areas and consequently define the priorities for control measures93COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESSuperintendencia de Controle de Endemias (SUCEN); Wellcome Trus

Rab4 Orchestrates a Small GTPase Cascade for Recruitment of Adaptor Proteins to Early Endosomes

SummaryBackgroundEarly, sorting endosomes are a major crossroad of membrane traffic, at the intersection of the endocytic and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations is mediated by a number of distinct coat protein complexes, including adaptor protein 1 (AP-1), AP-3, and Golgi-localized, gamma adaptin ear-containing, Arf-binding (GGAs) protein. Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal membrane, but the mechanisms of coat recruitment and assembly at this site remain poorly understood.ResultsHere we report that the endosomal Rab protein Rab4 orchestrates a GTPase cascade that results in the sequential recruitment of the ADP-ribosylation factor (Arf)-like protein Arl1; the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2; and the class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1 and BIG2 activity with brefeldin A results in the loss of AP-1, AP-3, and GGA-3, but not Arl1, from endosomal membranes and the formation of elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal membranes, inhibits the formation of tubular subdomains, and blocks recruitment of BIG1 and BIG2, Arfs, and adaptor protein complexes to the endosome.ConclusionsTogether these findings indicate that Arl1 links Rab4-dependent formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that constitute the endosomal sorting machinery

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

BACKGROUND: Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. RESULTS: Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. CONCLUSIONS: Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor

PERP, a host tetraspanning membrane protein, is required for Salmonella-induced inflammation

Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast-two-hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP-22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth

ARF GTPases and their GEFs and GAPs: concepts and challenges

Detailed structural, biochemical, cell biological, and genetic studies of any gene/protein are required to develop models of its actions in cells. Studying a protein family in the aggregate yields additional information, as one can include analyses of their coevolution, acquisition or loss of functionalities, structural pliability, and the emergence of shared or variations in molecular mechanisms. An even richer understanding of cell biology can be achieved through evaluating functionally linked protein families. In this review, we summarize current knowledge of three protein families: the ARF GTPases, the guanine nucleotide exchange factors (ARF GEFs) that activate them, and the GTPase-activating proteins (ARF GAPs) that have the ability to both propagate and terminate signaling. However, despite decades of scrutiny, our understanding of how these essential proteins function in cells remains fragmentary. We believe that the inherent complexity of ARF signaling and its regulation by GEFs and GAPs will require the concerted effort of many laboratories working together, ideally within a consortium to optimally pool information and resources. The collaborative study of these three functionally connected families ( \u3e /=70 mammalian genes) will yield transformative insights into regulation of cell signaling