The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin⅐4. Determining how cells regulate actin assembly is vital for understanding motility. Resting cells contain high concentrations of unpolymerized actin, typically 200 -400 M, or about 600 -1200 times greater than the critical concentration for assembly of pure actin (1, 2). Actin-sequestering proteins prevent spontaneous assembly of monomeric actin, and nonmuscle cells contain two such sequestering proteins: the 15-kDa protein profilin and the 5-kDa thymosin-␤4 (3, 4). These proteins were mainly thought to bind actin monomers with a one-to-one stoichiometry and to sequester actin monomers, and both have a higher affinity toward Actin⅐ATP as compared with Actin⅐ADP. The current view is that profilin and thymosin-␤4 are likely to play complementary roles in the cell. Thymosin-␤4 has a single mode of action (5-8), namely binding actin monomers to create a sequestered pool of monomers to supply Actin⅐ATP during periods of active filament growth. The mode of action of profilin, however, is more complex and in many respects has remained controversial and elusive. Although the Profilin⅐Actin complex was initially also thought to be strictly a monomersequestering protein (9 -11), Pollard and Cooper (12) later confirmed the inference that it adds to the barbed ends of actin filaments (13). Initial rate and steady-state assembly measurements showed that profilin can efficiently bind actin monomers (K d Ͻ 5 M) and can weakly interact with filament barbed ends (K capping ϳ 100 M). Profilin catalyzes exchange of free ATP with actin-bound ADP to form actin-bound ATP and free ADP (14 -16). Profilin also exhibits affinity toward oligoproline modules (17), another unique property that facilitates transfer of actin monomers to the polymerization zone (16) lying immediately behind motile Listeria (18), Shigella (19), and vaccinia (20). By concentrating Profilin⅐Actin complex in regions of active filament assembly, explosive rates of filament growth (Ն500 monomers s Ϫ1 ) are readily catalyzed. By contrast, thymosin-␤4 does not bind to oligoproline and fails to concentrate in the polymerization zone.Highly motile cells contain high concentrations of both profilin and thymosin-␤4, and these proteins probably act in concert to promote rapid filament assembly. Pantaloni and Carlier (21) investigated the role of profilin in the absence and presence of thymosin-␤4. They suggested that profilin reduces the critical monomer concentration needed for filament assembly, and they suggested that this is accomplished by accelerating the irreversible hydrolysis of polymer-bound Profilin⅐Actin⅐ATP complex. However, their experimental design was compromised in several significant ways: 1) they used pyrenyl-actin polymerization and had to correct the observed data for the low affinity of profilin for pyrenyl-actin (9,22,23); 2) all of their determinations of the steady-state extent of polymerization rested on th...

Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.

To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-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 intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the 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 vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1–3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 ± 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878– 21885) and to vasodilator-stimulated phosphoprotein (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 model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.