Translational Patterns During Spermiogenesis in Marsilea

University Of Maryland, College Park, College Park MD

Investigators

Abstract

Marsilea vestita is a water fern that makes motile spermatozoids. Each of these gametes possesses ~140 cilia and a complex cytoskeleton. The gametes are formed in a rapid process that is initiated by placing dry microspores (meiotic products) into water, in a process known as imbibition. Spermiogenesis is synchronous in populations of microspores, and a distinctive feature of gamete formation in this organism is the de novo formation of basal bodies in cells that lack preexisting centrioles. In each spermatocyte, a discrete particle known as a blepharoplast forms approximately 4 h after imbibition, and during the next two hours the blepharoplast splits and functions as a centrosome for the last mitotic division in the gametophyte, and then it matures. Each blepharoplast produces approximately 140 basal bodies. Dry microspores contain large quantities of stored protein and stored mRNA. The translation of specific mRNAs at particular stages of spermiogenesis is necessary for gamete maturation, including formation of the blepharoplast and basal bodies. The translation of centrin is essential for blepharoplast formation in these gametophytes, and centrin and b-tubulin can be detected immunocytochemically in blepharoplasts (where they serve as marker proteins for the organelle), which form 4 h after imbibition. This project will focus on aspects of spermiogenesis in M. vestita that extend ongoing work and utilize probes and procedures this lab has developed in the past several years. Studies on essential proteins that participate in the de novo formation of basal bodies will be expanded. Reverse-genetics with RNAi will be used to perform a series of translational knockouts to see how Xgrip109, RanBPM and other members of the g-tubulin ring complex participate in the basal body formation. Then, ongoing studies with mago nashi (a polarity gene in embryogenesis in a variety of organisms) will be extended to examine its role in the early segregation of cytoplasm in the gametophyte into spermatogenous and sterile domains as the single-cell gametophyte develops and forms its two distinct types of progeny - sterile somatic jacket cells and spermatogenous cells. The movements of stored mRNA and proteins will be monitored, and actomyosin as a driving force for some of these events will be examined. This project will potentially provide new insights into the roles played by various proteins as cells assemble new basal bodies, the site in all eukaryotic cells where the growth of cilia and flagella initiates. Also, new perspectives will be provided into how cells differentiate their cytoplasm into functionally distinct regions, a process of central importance in the growth and development of all cells.