Recent studies have shown unconventional myosin V to be a widely expressed actin-based molecular motor involved in vesicular movement. We recently cloned the full-length cDNA of the Drosophila isoform (Bonafe and Sellers, J. Muscl. Res. Cell Motil., in press) and showed that its heavy chain is encoded by a single gene (43C-D) in flies while it is represented by two different genes in vertebrates and yeast. The transcripts are expressed throughout development from embryonic stages to adult flies. These findings imply that Drosophila is a particularly suitable model for studying the general features of myosin V function. The primary sequence of the protein indicates structural characteristics of a typical myosin V member, linking more closely to the vertebrate isoforms (Va and Vb) and C. elegans hum-2 than to those of yeast. However, it branches with neither mammalian Va nor Vb, suggesting that it diverged before the two vertebrate isoform diverged. Polyclonal antibodies specific to the unique tail domain were generated, recognizing a single protein of 207 kDa in western blots of extracts from larvae and adult heads. The embryonic-derived Drosophila Schneider's 2 cell line was used to examine further the cellular distribution of myosin V. Cellular fractionation demonstrated a tight association of the molecule with the membrane compartment while immunocytochemistry studies on these cells revealed a characteristic punctated cytoplasmic distribution. Interestingly, immunolocalization of myosin V in development egg chambers (in collaboration with Dr. Tom Hays, St. Paul, MN) showed accumulation to the oocyte. In addition, preliminary analyses of the subcellular localization within the oocyte distinguished a cortical localization. We are planning to further look at the distribution of the protein in a variety of mutants presenting defects throughout the cytoskeleton rearrangements (actin, dynein...). At the molecular level, it has been reported that the avian myosin V heavy chain has both calmodulin and an essential light chain (ELC) bound to the neck region. In order to determine whether this is a feature of Drosophila myosin V, we have first expressed its regulatory domain as a GST fusion protein in E. coli and, by 125-I-CAM blot overlays, evidence was obtained for binding of calmodulin by at least some of the 6 IQ motifs, which was reduced in the presence of calcium. Then we have cloned out of a cDNA library and successfully expressed in E. coli the unique Drosophila ELC and are pursuing the binding assays. In addition, it has been reported that a low molecular weight protein binds to the tail domain of the avian myosin V and that this polypeptide is identical to a cytoplasmic dynein light chain. Therefore, we have cloned out of a cDNA library the Drosophila cytoplasmic dynein light chain (ddlc1) and expressed it in E. coli along with the tail domain of the Drosophila myosin V. We are currently testing them in binding assays as well. Finally, taking advantage of the fly genetics, we searched for a mutant fly that has altered expression of myosin V. We contacted Dr. Kim Kaiser (Glasgow, Scotland) who has a large collection of lines with recessive lethal insertions of a P[lacW] transposon on their second chromosome. Using our myosin V probe, he isolated a P-element-inserted rescued plasmid corresponding to an insertion in the myosin V gene (43CD). The insertion was determined to occur 5' to the coding sequence in the regulatory region. We have recently received the corresponding balancer line from Dr. Kiss (Szged, Hungary) and are in the process of determining the extent to which the P-element disrupts the expression of myosin V, hoping to obtain insight into myosin V function in Drosophila.
| Mermall, Valerie; Bonafe, Nathalie; Jones, Lynn et al. (2005) Drosophila myosin V is required for larval development and spermatid individualization. Dev Biol 286:238-55 |
| Toth, Judit; Kovacs, Mihaly; Wang, Fei et al. (2005) Myosin V from Drosophila reveals diversity of motor mechanisms within the myosin V family. J Biol Chem 280:30594-603 |
