Supplementary MaterialsSupp Numbers1-S3: Figure S1

Supplementary MaterialsSupp Numbers1-S3: Figure S1. Measurement of protein secretion through SEAP assays. B. RNAi-mediated depletion of Sec31A does not affect secretion of SEAP. (i). Confirmation of Sec31A depletion through Western blotting analysis (ii). Measurement of SEAP ML133 hydrochloride secretion in cells depleted for Sec31A, Rabbit Polyclonal to Transglutaminase 2 treated with control (C) siRNA, or mock transfected in the absence of siRNA (?). Results in A and B are mean +/? SEM from three experiments. NIHMS733598-supplement-Supp_FigureS1-S3.pdf (424K) GUID:?49C97597-2219-44AF-85FD-C27C0F2DE055 SUMMARY is a food-borne pathogen that uses actinCdependent motility to spread between human cells. Cell-to-cell spread involves the formation by motile bacteria of plasma membrane-derived structures termed protrusions. In cultured enterocytes, the secreted protein InlC promotes protrusion formation by binding and inhibiting the human scaffolding protein Tuba. Here we demonstrate that protrusions are controlled by human COPII components that direct trafficking from the endoplasmic reticulum. Co-precipitation experiments indicated that the COPII proteins Sec31A and Sec13 interact directly with a Src Homology 3 domain in Tuba. This interaction was antagonized by InlC. Depletion of Sec31A or Sec13 restored normal protrusion formation to a mutant lacking spread otherwise imposed by COPII. Inhibition of Sec31A, Sec13, or Sar1 or brefeldin A treatment perturbed the framework of cell-cell junctions also. Collectively, these results demonstrate a significant part for COPII in managing spread. We suggest that COPII might act by delivering sponsor protein that generate tension at cell junctions. Intro Many intracellular bacterial pathogens possess evolved systems to actively pass on within human cells (Gouin spp., and it is a Gram-positive food-borne bacterium with the capacity of leading to gastroenteritis or intrusive disease culminating in meningitis or abortion (Vazquez-Boland is set up from the bacterial surface area proteins ActA, which induces the forming of F-actin comet tails (Domann proteins InlC acts together with ActA to market bacterial pass on (Rajabian mutant missing (inhibits Cdc42, a meeting crucial for bacterial protrusion development (Rigano spread isn’t completely understood, but may involve perturbation from the sponsor apical junction complicated- a framework consisting of limited junctions and root adherens junctions (Miyoshi and Takai, 2005). Tuba, N-WASP, and Cdc42 are each had a need to keep up with the linearity of apical junctions in epithelial cells (Otani induces slackening of apical junctions (Polle gene neglect to alter apical junctions, indicating that InlC is necessary because of this event. Collectively, these total outcomes resulted in the hypothesis that sponsor Tuba, N-WASP, and Cdc42 have the potential to restrict bacterial protrusions by generating cortical tension that opposes the outward force exerted by motile bacteria on the host plasma membrane (Ireton, 2013; Rajabian antagonizes Tuba and N-WASP, thereby relieving cortical tension and allowing efficient generation of bacterial protrusions. Despite recent advances on the control of protrusion formation, the physiological processes that affect cell junctions and bacterial spread remain unknown. Tuba and its effectors N-WASP and Cdc42 promote many events in mammalian ML133 hydrochloride cells, including actin ML133 hydrochloride polymerization, cell motility, endocytic and exocytic trafficking of vesicles, the formation and maintenance of cell junctions, centrosome organization, and cell polarity (Otani protrusions. Importantly, this ML133 hydrochloride Tuba ligand has an established function in the early secretory pathway, pointing to a host process that may regulate spread. We found that the carboxyl-terminal SH3 domain of Tuba, previously demonstrated to bind N-WASP (Salazar mutant strain, but not of wild-type bacteria. Thus the negative role of COPII in spread resembles that previously reported for Tuba, N-WASP, and Cdc42, suggesting that COPII may act with these other host proteins to control bacterial protrusions. Biochemical experiments demonstrate that InlC displaces Sec31A from the Tuba carboxyl-terminal SH3 domain, indicating that.