This revised application from Dr. Lynn Cooley outlines experiments to study proteins involved in the structure and function of specialized intracellular bridges called ring canals, which are found in the egg chambers of Drosophila females. Ring canal-like structures are also found in the germlines of other organisms, including humans. In Drosophila oogenesis, the ring canals are important conduits for the flow of cytoplasmic materials from the nurse cells to the oocyte. Their formation involves at least four proteins: a phosphotyrosine protein, filamentous actin, the hu-li tai shao (hts) protein, and the kelch protein. It is thought that actin filaments provide the pathway for movement through the ring canals. Dr. Cooley hypothesizes that these filaments are stabilized around the rim of the canal by the hts protein and that they are grouped into bundles by the kelch protein. In addition, she believes that the product of another gene, cheerio, is involved in ring canal formation. The first part of Dr. Cooley's plan is to study the kelch protein.
One aim i s to use germline transformation constructs to test the requirements for products of the two ORFs of the kelch gene. These ORFs are separated by a single stop codon which is evidentally subject to translational suppression. The kelch gene therefore makes two polypeptides, one corresponding to the first ORF, the other to a combination of the two ORFs; both polypeptides are found in ring canals. Dr. Cooley will investigate the functions of these polypeptides by making transformation constructs in which the second ORF is deleted, or in which the stop codon between the ORFs is mutated or deleted. These constructs will be driven by a promoter from either the ovarian tumor (otu) or chickadee genes, and once inserted into the genome, will be tested for their ability to rescue sterilizing kelch mutations. Antibody staining of fully or partially rescued animals will be used to study the cellular localization of the transgenic kelch proteins and to assess the integrity of their ring canals. Similar transformation experiments will be carried out to analyze domains within the kelch proteins. These experiments will be supplemented with in vitro tests to determine if fragments of the kelch proteins can bind, bundle, cap and nucleate actin filaments. The proteins for these tests will be synthesized in E. coli or in insect cells infected with a baculovirus expression construct. Dr. Cooley will also use an epitope tagging procedure to determine if kelch proteins dimerize in vivo. DNA sequence analysis of EMS-induced kelch mutations and cellular localization experiments with antibodies to the ORF2 portion of the larger kelch polypeptide are also planned. The second part of Dr. Cooley's project is to study the hts protein. This protein contains an N-terminal domain homologous to vertebrate adducin. The C-terminal domain is novel and appears to be found exclusively in ring canals. Dr. Cooley hypothesizes that it is produced by proteolytic cleavage of a precursor protein that is made from an alternately spliced, ovary-specific RNA. Germline transformation experiments will be used to determine if the ovary-specific RNA does, in fact, produce the 60 kd hts ring canal protein. Then various constructs will be made to investigate the functional significance of the last three exons of the hts gene. The main question is whether or not the C-terminal part of the hts protein can rescue the hts mutant phenotype. In addition, purified hts protein will be used in actin binding, bundling, capping and nucleating assays, as already described for the kelch proteins. The third part of Dr. Cooley's plan is to clone and characterize the cheerio gene. Preliminary work has shown that cheerio lies in the interval between 89F and 90A on chromosome III (not in 66A, as previously thought). Dr. Cooley will use a transposon hopping scheme to jump P element insertions in 89E11-12 or 90B3-4 into the cheerio locus. Jumps into this gene will be identified by screening for a female sterile phenotype when a chromosome with a jumped element is put over a known cheerio allele. Standard procedures will then be used to isolate and clone the gene.
