HYDROSTATIC-PRESSURE SHOWS THAT LAMELLIPODIAL MOTILITY IN ASCARIS SPERM REQUIRES MEMBRANE-ASSOCIATED MAJOR SPERM PROTEIN FILAMENT NUCLEATION AND ELONGATION

Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in p lace of an actin cytoskeleton to drive their ameboid locomotion. Motil ity is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been re constituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle f orward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, an d C.A. Fontana, 1996. Cell. 84:105-114). We used changes in hydrostati c pressure within a microscope optical chamber to investigate the mech anism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vit ro system to show that filament assembly occurs in the immediate vicin ity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, norm al fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number-of filaments assembled at the vesicle (proportional to fib er optical density and filament nucleation rate), and their rate of as sembly (proportional to the rates of fiber growth and filament elongat ion). Thus, fiber growth is not simply because of the addition of subu nits onto the ends of existing filaments, but rather is regulated by p ressure-sensitive factors at or near the vesicle surface. Once a filam ent is incorporated into a fiber, its rates of addition and loss of su bunits are very slow and disassembly occurs by pathways distinct from assembly, The effects of pressure on fiber assembly are sensitive to d ilution of the extract but largely independent of MSP concentration, i ndicating that a cytosolic component other than MSP is required for ve sicle-association filament nucleation and elongation. Based on these d ata we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading ed ge.