The cytoskeleton plays a critical role in many developmental processes, from cell migration to asymmetric segregation of cellular components. Isolation of the C. elegans mutant
spe-15(
ok153) lacking a functional class VI myosin highlights the importance of motor proteins in development. Deletion of a myosin VI (SPE-15) results in defective asymmetric segregation of organelles and cytoskeletal components required for the formation of motile sperm that occurs during spermatogenesis (Kelleher et al. 2000. Curr. Biol. 10:1489-1496), and also causes a Spe phenotype (sterility that can be rescued by wild-type sperm). The potential role of SPE-15 in organelle transport was addressed by real-time imaging of organelle movements during spermatid budding. Secondary spermatocytes (SS) undergoing the process of spermatid budding were obtained either from adult wild-type or mutant males lacking SPE-15. Budding was observed by DIC microscopy over the course of 90 minutes. As the spermatids bud, organelles are moved into the growing spermatid and the remaining residual body (RB) appears transparent. However, in
spe-15(
ok153) SS, organelles appear to be segregated into the spermatids but are then released back into the RB. Subsequently, the spermatids undergo abnormal contractions. The overall time required for spermatid budding also appears to be decreased by about half in the mutant SS. These results suggest that SPE-15 is not required for the translocation of cellular components, but rather for the restriction of their localization to the budding spermatid. SPE-15 may also provide an opposing force to prevent premature budding and spermatid deformation during the final stages of budding. We are currently addressing this hypothesis by SPE-15 localization studies, including microparticle bombardment of a
spe-15::GFP construct and immunofluorescent staining of fixed spermatocytes with an anti-SPE-15 polyclonal antibody. An ongoing analysis of spermatogenesis will allow us to further define the role of myosin VI during this process.