The long-term goal of this work is to understand how intercellular junctions called ring canals contribute to instances of syncytial development. Ring canals originate as arrested mitotic cleavage furrows that become stabilized by a rim of cytoskeletal protein. They are prevalent in developing germ cells throughout the animal kingdom and have also been found in a number of somatic cells. However, the functional significance of ring canals is poorly understood in most of these cases. Disruptions in the coordination of cellular events as a result of faulty ring canal development are likely to cause severe aberrations in cell behavior such as loss of growth control. The experimental system is Drosophila oogenesis, during which nurse cells feed growing oocytes maternal components by transferring cytoplasm to the oocyte through ring canals. By studying mutant fly strains in which defective transport of cytoplasm leads to female sterility, several ring canal components have been identified. The work proposed in this grant application is focused on functional analysis of these proteins.
The specific aims of the proposal are: (1) identification of proteins that interact with filamin (encoded by cheerio) during ring canal morphogenesis; (2) functional analysis of proteins encoded by the hts locus and studies on hts protein processing; (3) analysis of kelch protein function and identification of kelch interacting proteins. The ring canals of Drosophila egg chambers have an actin-based cytoskeleton that maintains ring canal integrity and also allows ring canals to grow over 10-fold in diameter during oncogenes. The mechanism for linking the cytoskeleton to the plasma membrane of ring canals is unknown. Filamin is a good candidate for providing such a linkage, and studies are proposed to identify proteins that interact with filamin in egg chambers. This should provide information on the transition from cleavage furrow to ring canal. The hts locus is required in both follicle cells and germline cells. In the germline, hts products are found in both ring canals and the fusome. Studies in aim 2 are focused on characterizing the link between fusome behavior and ring canal assembly. Finally, the actin cytoskeleton of ring canals is unusual because the filaments are arrange in a circle. The kelch protein is important for maintaining the packing organization of actin during the growth of ring canals.
In aim 3, work is proposed to define the biochemical role of kelch. The work will include high resolution studies of actin organization in wild type and kelch mutant egg chambers using cryoelectron microscopy.
