Rapidly Fixes For the Mdm2 Troubles

We showed that SopE and ARNO were both required for generating Salmonella-induced macropinosomes and that they facilitated pathogen invasion via the same route, supporting their dual role in WRC activation. Indeed, we showed that WRC recruitment to the membrane, its activation, and subsequent actin polymerization was enhanced when SopE and ARNO cooperated, a synergy that was also evident when SopE and ARNO were coexpressed in cells. Our data provide a mechanism in which cooperating GEFs trigger coincident activation http://www.selleckchem.com/products/Cyclopamine.html of Arf and Rac at specific locations to activate the WRC and initiate actin polymerization (illustrated in our model in Figure?S5B). We propose that Salmonella employs this mechanism by recruiting the host GEF ARNO and delivering the bacterial GEF SopE to the plasma membrane beneath the invading bacterium, which triggers WRC-dependent membrane ruffling and consequent pathogen macropinocytosis. Wild-type Salmonella enterica serovar Typhimurium SL1344 (gift from Jean Guard-Petter), isogenic ��sopE��sopE2 (kind gift from Wolf-Dietrich Hardt), ��invG, and ��sopB were constructed as described ( Humphreys et?al., 2009). For visualizing Salmonella by fluorescence microscopy, bacteria were washed with phosphate-buffered saline, conjugated Mdm2 to either Alexa Fluor 350 or Alexa Fluor 594 carboxylic acid succinimidyl ester (15?min, 37��C), washed in Tris (pH 7.4)-buffered saline, then used to infect HeLa cells (moi of 50). Infected HeLa cells (15?min, unless stated otherwise) were used either to quantify invasion by gentamicin protection ( Humphreys et?al., 2009) or to quantify Salmonella-induced membrane ruffling by determining the proportion of adherent Salmonella associated with actin-rich foci using fluorescence microscopy (?150 cells per experiment). When appropriate, HeLa cells were incubated with 25?��M SecinH3 (Merck). Immunofluorescence microscopy and images were assembled as described ( Humphreys et?al., 2009). All experiments were performed at least three times. Geometric means were calculated and significance determined by one-way analysis of variance (ANOVA) followed by a post hoc Dunnett's comparison. p?Sotrastaurin motility-mix (extract) was prepared on ice in the following order; 40?��l brain extract, 3?��l 20�� energy mix (300?mM creatine phosphate, 40?mM MgCl2, 40?mM ATP), 3?��l G-actin/rhodamine actin (140?��M, prepared as described) (McGhie et?al., 2004), 6?��l 10�� salt buffer (600?mM KCL, 200?mM 3-phosphoglycerate), 6?��l 50?mM BAPTA (Merck) and 1?��l 300?mM DTT (Merck) and, when appropriate, 1?��l 30?mM GTP��S (Roche). Actin-motility assays were initiated by adding 0.1 vol phospholipids-coated beads to 10?��l motility mix, then1?��l was applied to a microscope slide.