Drosophila fascin mutants are rescued by overexpression of the villin-like protein, quail.

Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells requires two actin bundling proteins, fascin encoded by the singed gene and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. We used P-element mediated germline transformation to overexpress quail in singed mutants and test whether these proteins have redundant functions in vivo. Overexpression of quail protein in a sterile singed background restores actin bundle formation in egg chambers. The degree of rescue by quail depends on the level of quail protein overexpression, as well as residual levels of fascin function. In nurse cells that contain excess quail but no fascin, the cytoplasmic actin network initially appears wild type but then becomes disorganized in the final stages of nurse cell cytoplasm transport. The ability of quail overexpression to compensate for the absence of fascin demonstrates that fascin is partially redundant with quail in the Drosophila germline. Quail appears to function as a bundle initiator while fascin provides bundle organization.

Single amino acid mutations in Drosophila fascin disrupt actin bundling function in vivo.

Fascins bundle actin filaments into large, tightly packed hexagonal arrays that support diverse cellular processes including microvillar projections and filopodial extensions. In Drosophila, fascin is encoded by the singed locus. Severe singed mutants have gnarled bristles and are female sterile due to a defect in rapid cytoplasm transport during oogenesis. In this paper, we report the results of a large EMS mutagenesis screen to generate new singed alleles. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of fascin in vivo; singedG409E mutants have kinked bristles and are fertile with a mild nurse cell cytoplasm transport defect. This mutation is in a small conserved domain near the C-terminus of fascin. A mutation that changes serine 289 to asparagine almost completely inactivates fascin in vivo; singedS289N mutants have gnarled bristles and are sterile due to a severe defect in nurse cell cytoplasm transport caused by the absence of nurse cell cytoplasmic actin bundles. A subsequent EMS mutagenesis screen for dominant suppressors of singedS289N sterility revealed an intragenic suppressor mutation that changes serine 251 to phenylalanine and restores much of fascin's function. These two mutations, S289N and S251F, draw attention to a central domain in fascin.

Morphogenesis of Drosophila ovarian ring canals.

We analyzed the structure of cytoplasmic bridges called ring canals in Drosophila egg chambers. Two mutations, hu-li tai shao (hts) and kelch, disrupt normal ring canal development. We raised antibodies against the carboxy-terminal tail of hts and found that they recognize a protein that localizes specifically to ring canals very early in ring canal assembly. Accumulation of filamentous actin on ring canals coincides with the appearance of the hts protein. kelch, which is localized to the ring canals hours after hts and actin, is necessary for maintaining a highly ordered ring canal rim since kelch mutant egg chambers have ring canals that are obstructed by disordered actin and hts. Anti-phosphotyrosine antibodies immunostain ring canals beginning early in the germarium before hts and actin and throughout egg chamber development. The use of antibody reagents to analyze the structure of wild-type and mutant ring canals has shown that ring canal development is a dynamic process of cytoskeletal protein assembly, possibly regulated by tyrosine phosphorylation of some ring canal components.

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension.

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.