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 variety of genetic, biochemical and localization studies. However, it has yet to be determined whether myosin-V functions as an organelle motor. To further investigate this possibility, we conducted a biochemical and functional analysis of organelle-associated brain myosin-V. Using the initial fractionation steps of an established protocol for the purification of brain myosin-V, we isolated a population of brain microsomes that is approx. fivefold enriched for myosin-V, and is similarly enriched for synaptic vesicle proteins. As demonstrated by immunoelectron microscopy, myosin-V associates with 30-40% of the vesicles in this population. Although a majority of myosin-V-associated vesicles also label 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 vesicles that label only with SV2 antibodies (60+/-30 nm). To determine if these vesicles are capable of actin-based transport, we used an in vitro actin filament motility assay in which vesicles were adsorbed to motility 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 vesicle-associated. Finally, function-blocking antibodies against the myosin-V motor domain completely inhibited motility on these substrates. These 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.
Evans
L L
LL
Department of Anatomy and Neurobiology, Washington University, St Louis, MO 63110, USA. lisa.evans@yale.edu.
Lee
A J
AJ
Bridgman
P C
PC
Mooseker
M S
MS
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D002645:Chickens; D007074:Immunoglobulin G; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure; D018994:Myosin Light Chains / Q000378:metabolism; D024701:Myosin Type V; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure
D000199:Actins; D002148:Calmodulin-Binding Proteins; D007074:Immunoglobulin G; D018994:Myosin Light Chains; D009419:Nerve Tissue Proteins; C078829:chicken brain myosin-V p190; D024701:Myosin Type V