Fascins: Identification of actin-binding domains and analysis of cellular function

Julia Dianne Wulfkuhle, Purdue University

Abstract

The fascins are a widely distributed family of actin-bundling proteins with homologs in echinoderms, Drosophila, Xenopus, mice, and humans. The purpose of this work was to study various members of the fascin family and analyze their cellular function, functional homology and identify domains involved in F-actin binding. The Drosophila singed (sn) gene product is a member of the fascin family and is required for the maintenance of F-actin structures involved in bristle development and oogenesis. Analysis of the distribution of F-actin during bristle development in wild type and $sn\sp3$ mutant flies showed that the sn gene product was required for maintaining the F-actin structures found at the late stages of bristle development, but it was not required for maintaining the F-actin structures found early in bristle development. It was determined that another putative actin-associated protein, the forked (f) gene product was a component of the F-actin structures found in early bristle development and f mutant analysis demonstrated that the f gene product was required for the proper formation of the F-actin structures found in early bristle development. These observations suggest that the f and sn gene products must work sequentially to maintain the F-actin structures required for bristle development. Drosophila germline transformants carrying exogenous copies of the wild type sn cDNA or the cDNA encoding sea urchin fascin were generated to determine if the sea urchin fascin protein could functionally substitute for the sn protein in bristle development and rescue the $sn\sp3$ bristle phenotype. Expression analysis demonstrated that none of the control transformant lines carrying the $sn\sp+$ cDNA expressed sufficient levels of sn protein to rescue the $sn\sp3$ mutant phenotype. F-actin pelleting experiments with tryptic digests of recombinant mouse fascin identified two unique fragments of fascin that bound F-actin. One fragment represented the C-terminal portion of fascin from Gln277. The second fragment represented from Ser133 to approximately Lys247 and was the first evidence that a region from the N-terminal half of fascin contains an F-actin binding domain. These two fragments contain a conserved motif of 20 amino acids and also have regions of similarity with the actin binding sequence of ABP-120.

Degree

Ph.D.

Advisors

Otto, Purdue University.

Subject Area

Molecular biology|Biochemistry

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