Precise positioning of myosin VI on endocytic vesicles in vivo.
Myosin VI has been studied in both a monomeric and a dimeric form in vitro. Because the functional characteristics of the motor are dramatically different for these two forms, it is important to understand whether myosin VI heavy chains are brought together on endocytic vesicles. We have used fluorescence anisotropy measurements to detect fluorescence resonance energy transfer between identical fluorophores (homoFRET) resulting from myosin VI heavy chains being brought into close proximity. We observed that, when associated with clathrin-mediated endocytic vesicles, myosin VI heavy chains are precisely positioned to bring their tail domains in close proximity. Our data show that on endocytic vesicles, myosin VI heavy chains are brought together in an orientation that previous in vitro studies have shown causes dimerization of the motor. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function.
Department of Biochemistry, Stanford University Medical Center, Stanford, California, United States of America.
David
D
Altman
Debanjan
D
Goswami
Tama
T
Hasson
James A
JA
Spudich
Satyajit
S
Mayor
D000818:Animals; D016880:Anisotropy; D002460:Cell Line; D019281:Dimerization; D004705:Endocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism*
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D014168:Transferrin; C089785:myosin VI; D018995:Myosin Heavy Chains