This proposal is to continue the exploration of oocyte development in Drosophila. The long-term goal is to fully comprehend the mechanism and regulation of intercellular cytoplasm transport, and the underlying cytoskeletal dynamics in germline cells. These studies are broadly relevant to achieving full understanding Of cellular processes that underly disease progression. A major example is metastasis, which requires the modulation of cytoskeletal function for the acquisition of migratory ability in transformed cells, so that they can escape their location and colonize a new one, The specific aims of this proposal include both forward and reverse geneticsappmaches to characterizing genes involved with intercellular cytoplasm transporting Drosophila egg chambers. Previous work from the lab has established the importance of actin cytoskeleton regulation to carry out cytoplasm transport and several actimbinding proteins have been characterized in detail.
Aim 1 is to clone and characterize two essential genes that affect cytoplasm transport in genetically mosaic egg chambers where the nurse cells are mutant. The phenotype of one gene suggests a role in the cell cortex, possibly in anchoring actin filaments to the membrane. The second gene displays an oocyte specific phenotype in which oocyte shape is aberrant. The Cooley lab is carrying out a screen for randomly generated GFP-fusion protein lines (protein traps) to identify new genes involved with oocyte development.
Aim 2 proposes to continue the protein trapping screen using an improved protocol.
Aim 3 focuses on the characterization of genes encoding GFP-fusion proteins that localize to the actin cytoskeleton. The first one identified in the screen, CG2556, resides on the X chromosome and has not been previously studied. The availability of a large number of protein trap lines provides the opportunity to study the transport kinetics of a variety of maternal proteins into the oocyte.
Aim 4 will categorize maternal proteins according to their transport schedules and analyze these categories using bioinformatics, gene Ucs and cell biology. Candidate motifs for controlling transport will be tested by site-directed mutagenesis. Genetic analyses will be done on the small class of maternal proteins that becomes enriched at the posterior pole of oocytes and are likely to encode proteins involved with anterior/posterior axis determination or germline development in the embryo.
Showing the most recent 10 out of 33 publications
