Activation on the EGF pathway alters parts of the cytoskeleton involved with actin organization, focal adhesion formation and reso lution, likewise as cell cell adhesion. We had previ ously identified that pre remedy of INT 407 cells with PD168393 and erlotinib, that are particular inhibitors of EGF receptor tyrosine phosphorylation, appreciably inhibited C. jejuni mediated host cell membrane ruffling and invasion. We had also uncovered that the EGF re ceptor gets phosphorylated at web pages Tyr 845 and Tyr 1068 upon infection with C. jejuni, which can be consist ent together with the idea the EGF receptor is stimulated through integrin signaling. Noteworthy is that pre remedy of INT 407 cells with EGF rescues the invasiveness of the non invasive C. jejuni mutant. Here we report that infection of HeLa cells with C.
jejuni induces EGF recep tor activation, as evidenced by a rise in straight from the source the level of phosphorylated EGF receptor when when compared with unin fected cells. We have now also identified that pre treatment method of HeLa cells with MBCD disrupts C. jejuni dependent EGF receptor phosphorylation. Our findings are consist ent with reports exhibiting that the EGF receptor is nearby ized in lipid rafts, but not in caveolae. To dissect the romantic relationship between Rac1 action as well as the involvement of actin in C. jejuni internalization, HeLa cells had been handled with cytochalasin D just before in fection. Cytochalasin D depolymerizes actin filaments by binding towards the finish of F actin, thereby blocking the addition of G actin subunits to these internet sites. We found the therapy of HeLa cells with cytochalasin D had no have an effect on on Cia protein delivery or Rac1 activation, but prevented C.
jejuni induced mem brane ruffling. This finding is consist ent with preceding operate demonstrating the involvement of microfilaments in C. jejuni internalization and our model through which we propose that C. jejuni host cell make contact with and also the delivery of selleck chemicals pd173074 the Cia proteins towards the host cell cytosol promotes the activation of focal com plex elements and Rac1. Although earlier scientific studies have shown that C. jejuni internalization is sensitive to microtubule inhibitors, the position of microtubules in C. jejuni inva sion hasn’t been clear. To achieve insight into the poten tial function of microtubules in C. jejuni invasion, we handled HeLa cells with nocodazole. This drug binds B tubulin, preventing tubulin polymerization. We observed that the treatment method of HeLa cells with nocodazole had no have an effect on on Cia protein delivery, but prevented both Rac1 activation and C. jejuni induced membrane ruffling. 1 doable purpose that nocodazole therapy decreases C. jejuni internalization is the fact that MTs and MFs act cooperatively in recycling integrin receptors.