VINCULIN PROTEOLYSIS UNMASKS AN ACTA HOMOLOG FOR ACTIN-BASED SHIGELLAMOTILITY

To generate the forces needed for motility, the plasma membranes of no nmuscle cells adopt an activated state that dynamically reorganizes th e actin cytoskeleton. By usurping components from focal contacts and t he actin cytoskeleton, the intracellular pathogens Shigella flexneri a nd Listeria monocytogenes use molecular mimicry to create their own ac tin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited i ntracellular locomotion upon microinjection of Shigella-infected cells , and (c) cross-reacted with the proteolytically derived 90-kD human v inculin head fragment that contains the Vinc-1 oligoproiine sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculi n, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteo lysis. Immunofluoresence staining with a monoclonal antibody against t he head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS -1 antibody), indicating that motile bacteria attract a form of vincul in containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arreste d Shigella intracellular motility, underscoring the functional importa nce of this sequence. Western blots revealed that Shigella infection i nduces vinculin proteolysis in PtK2 cells and generates p90 head fragm ent over the same 1-3 h time frame when intracellular bacteria move wi thin the host cell cytoplasm. We also discovered that microinjected p9 0, but not full-length vinculin, accelerates rates of pathogen motilit y by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These expe riments suggest that vinculin p90 is a rate-limiting component in acti n-based Shigella motility, and that supplementing cells with p90 stimu lates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Bi ol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotei n (VASP) (Brindle, N.P.J., M.R. Hold, J.E. Davies, C.J. Price, and D.R . Critchley. 1996. Biochem. J. 318:753-757). We now offer a working mo del in which proteolysis unmasks vinculin's ActA-like oligoproline seq uence. Unmasking of this site serves as a molecular switch that initia tes assembly of an actin-based motility complex containing VASP and pr ofilin.