Genetic and biochemical characterization of Drosophila singed, a homolog of the actin bundling protein fascin

Kelly Cant, Yale University.

This is an Open Access Thesis


Drosophila singed mutants were named for their gnarled bristle phenotype, however, severe alleles are also female sterile due to a defect in nurse cell cytoplasm transport. Recently, singed protein was shown to have 35% peptide identity with the actin bundling protein fascin. I present evidence that singed is a homolog of fascin. In vivo, singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis and in the cellular extension that forms a bristle. In vitro, purified bacterially-expressed singed bundles actin filaments with the same 12 nm periodicity and stoichiometry reported for sea urchin fascin.While many homologs of fascin have been described, functional domain organization is not understood. I generated 15 new sn alleles by EMS mutagenesis; surprisingly only 2 of these expressed fascin. $singed\sp{G409E}$ mutants have multiply bent bristles and are fertile, although nurse cell cytoplasmic actin bundles appear disorganized and cytoplasm transport is partially incomplete. $singed\sp{S289N}$ mutants have gnarled bristles and are sterile. I used the $singed\sp{S289N}$ allele in a dominant suppressor of sterility screen and identified an intragenic mutation S251F that appears to restore much of fascin's function. These mutations, G409E, S289N and S251F, draw attention to critical amino aclds in fascin.At wild type protein levels, both fascin and quail, a villin-like protein, are required for cytoplasmic actin bundle formation. I looked for genetic interaction between singed and quail. Mutations in quail enhance the intermediate egg chamber phenotype of $singed\sp{G409E}$. Furthermore, overexpression of quail protein can rescue the cytoplasmic actin defect and sterility of $singed\sp{S289N}$ and of the null allele, $singed\sp{28}$. In the absence of fascin, overexpression of quail is sufficient for the formation of actin bundles.