VESICLE-ASSOCIATED BRAIN MYOSIN-V CAN BE ACTIVATED TO CATALYZE ACTIN-BASED TRANSPORT

Myosin-V has been linked to actin-based organelle transport by a varie ty of genetic, biochemical and localization studies, However, it has y et to be determined whether myosin-V functions as an organelle motor. To further investigate this possibility, we conducted biochemical and functional analysis of organelle-associated brain myosin-V. Using the initial fractionation steps of an established protocol for the purific ation of brain myosin-V, we isolated a population of brain microsomes that is approx, fivefold enriched for myosin-V, and is similarly enric hed for synaptic vesicle proteins, As demonstrated by immunoelectron m icroscopy, myosin-V associates with 30-40% of the vesicles in this pop ulation. Although a majority of myosin-V-associated vesicles also labe l with the synaptic vesicle marker protein, SV2, less than half of the total SV2-positive vesicles label with myosin-V, The average size of myosin-V/SV2 double-labeled vesicles (90+/-45 nm) is larger than vesic les that label only with SV2 antibodies (60+/-30 nm), To determine if these vesicles are capable of actin-based transport, we used an in vit ro actin filament motility assay in which vesicles were adsorbed to mo tility assay substrates. As isolated, the myosin-V-associated vesicle fraction was nonmotile, However, vesicles pre-treated with ice-cold 0. 1% Triton X-100 supported actin filament motility at rates comparable to those on purified myosin-V, This dilute detergent treatment did not disrupt vesicle integrity. Furthermore, while this treatment removed over 80% of the total vesicle proteins, myosin-V remained tightly vesi cle-associated. Finally, function-blocking antibodies against the myos in-V motor domain completely inhibited motility on these substrates. T hese studies provide direct evidence that vesicle-associated myosin-V is capable of actin transport, and suggest that the activity of myosin -V may be regulated by proteins or lipids on the vesicle surface.