Coiled-coil-mediated dimerization is not required for myosin VI to stabilize actin during spermatid individualization in Drosophila melanogaster.
Myosin VI is a pointed-end-directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.
Noguchi
Tatsuhiko
T
Center for Developmental Biology, Riken, Kobe, Japan.
Frank
Deborah J
DJ
Isaji
Mamiko
M
Miller
Kathryn G
KG
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019281:Dimerization; D004331:Drosophila melanogaster / Q000166:cytology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013087:Spermatids / Q000378:metabolism* / Q000648:ultrastructure; D013737:Testis / Q000378:metabolism / Q000648:ultrastructure; D019076:Transgenes
D000199:Actins; D011993:Recombinant Fusion Proteins; C089785:myosin VI; D018995:Myosin Heavy Chains