The long term objective of this application is to gain a molecular understanding of the cell cycle-regulated cytoskeletal rearrangements that accompany mitosis. The specific process that will be examined is mitotic actin filament reorganization during the rapid nuclear divisions that initiate embryogenesis in Drosophila melanogaster. The primary experimental approach will be based on molecular, cytological, and biochemical characterization of the scrambled locus. The scrambled mutation disrupts mitotic actin organization, but does not block cell cycle progression or severely affect interphase actin structure or function. The scrambled gene product, therefore, plays a central role in triggering actin reorganization in response to mitotic signals. Initial experiments will focus on isolating genomic and cDNA clones of scrambled. The scrambled mutation was induced by a P element transposon insertion, and genomic sequences flanking this transposon have been recovered. Using these sequences, additional scrambled genomic region clones will be isolated. To localize scrambled coding regions within these clones, northern blots of mutant and wild type RNAs will be probed with genomic sub-fragments. Sequences encoding RNAs that are altered by the scrambled mutation will then be used to isolate corresponding cDNAs. To determine if candidate cDNAs encode functional scrambled protein, complementary mRNAs will be synthesized in vitro, microinjected into mutant embryos, and scored for complementation of the scrambled phenotype. Alternatively, genomic sequences encoding candidate mRNAs will be reintroduced into the germline by P element transformation and then tested for scrambled complementation. Scrambled cDNAs will be sequenced, and the resulting primary sequence information used to search for scrambled homologues. Monospecific antibodies to bacterial fusion proteins will be used to localize scrambled in situ, and to biochemically assay for scrambled protein. The scrambled polypeptide will be functionally dissected using a combination of in vitro mutagenesis and in vivo cytological techniques. The results of these studies will be correlated with biochemical analyses of scrambled protein function. As a complement to these studies, a classical genetic approach will be used to define the pathway controlling mitotic action reorganization during early embryogenesis. Embryos derived from females homozygous for maternal effect lethal mutations will be screened for nuclear division defects similar to those produced by the actin disrupting drug cytochalasin, and the mutations thus identified will be examined for cell cycle-specific actin defects using time-lapse confocal microscopy and immunocytochemical techniques. Only 80 to 90 maternal-effect loci in the Drosophila genome produce development defects during very early embryogenesis, and are therefore candidates for analysis in this screen. Maternal control of the embryonic actin cytoskeleton will thus be systematically analyzed through the examination of a small number of mutations. To facilitate molecular characterization of additional genes controlling mitotic actin function, P element-induced mutations will also be examined in this screen.
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