The objective of this application for an Independent Scientist Award is to provide the PI with the opportunity to have a period of intensive research concentration that will enable him to expand his potential to make significant contributions to our understanding of the molecular organization of the Sertoli cell ectoplasmic specialization (ES) and its roles in spermatogenesis and fertility. The ES is thought to anchor and position spermatids within the seminiferous epithelium throughout much of spermiogenesis and may also contribute to the blood-testis barrier between Sertoli cells. These activities are believed to depend on the ES junctional plaque, which characterized by a layer of parallel actin bundles sandwiched between the Sertoli cell plasma membrane and a cistern of endoplasmic reticulum. The PI and his coworkers have recently identified espin, a novel approximately 110-kDa protein that is a major chemical constituent of the ES (approximately 5 million copies/ES) localized to the parallel actin bundles of the junctional plaque. The localization, stoichiometry and tissue distribution of espin, together with the actin-binding/bundling properties displayed by its fragments, suggest that espin is a major actin-bundling protein responsible for the formation of the parallel actin bundles of the ES junctional plaque. The PI proposes: (1) to test the hypothesis that espin is an actin-bundling protein by examining the actin-binding/ bundling properties of full-length espin in vitro and in vivo in transiently transfected or microinjected cell models; (2) to provide new information about the molecular organization of the ES and to test the hypothesis that espin is involved in the formation of the actin bundles of the junctional plaque by localizing espin in relation to other ES proteins and ultrastructural landmarks during the assembly and disassembly of ESs that accompanies spermatogenesis; (3) to test the hypothesis that espin and the ES play critical roles in spermatogenesis and male fertility by examining the consequences of eliminating espin expression by gene targeting. Receipt of this K02 Award would allow the PI to capitalize on this key discovery of a novel protein component of the ES and permit him to bring his considerable cell biological expertise to bear on the development of a world-class research program centered on this critically important, yet grossly understudied, intercellular junction of Sertoli cells.
