The Role(s) of Forked Proteins in Actin Fiber Bundle Formation

University Of Wyoming, Laramie WY

Investigators

Abstract

Regulation of the assembly of actin fibers is required for cell movement, changing and maintaining cell shape, and even for cell division. Actin binding proteins are essential in regulating these processes. In Drosophila melanogaster it is possible to use mutations to study the actin fiber bundles involved in bristle development during the pupal stage. These fiber bundles are morphologically very similar to actin fiber bundles present in vertebrate intestinal brush border and in stereocilia in the ear and kidney, but much larger. In the forked and singed mutants, actin fiber bundles are dramatically reduced in developing bristles. As a result, in the adult fly, the bristles are twisted and branched. The singed protein is homologous to sea urchin fascin and bundles actin fibers in vitro, indicating that it may play a role similar to fimbrin in intestinal brush boarder microvilli. The forked gene encodes six proteins with overlapping coding regions which differ primarily in their amino-terminal ends. The forked proteins are also actin binding proteins which are homologous to the vertebrate actin binding proteins, the espins. These were originally identified in rat testis ectoplasmic specializations but have now been also found in kidney and intestinal microvilli and in ear stereocilia. The predicted amino acid sequences of the espins are more similar to forked proteins than to other known actin binding proteins, and an antibody to espin specifically recognizes forked proteins on Western blots. This suggests that forked belongs to a new family of actin binding proteins which are involved in the assembly of specific types of tightly packed parallel actin bundles. The differences between these proteins may contribute to the differences in location and architecture of the bundles, while the similarities should reflect their common function(s). Dr. Petersen will continue to study the role(s) of forked proteins in actin fiber bundle formation in Drosophila bristles and in vertebrate cell lines. Her laboratory has shown that forked proteins are synthesized and concentrated in bristle tips just prior to the formation of actin bundles, and that they can induce actin bundle formation in transiently transfected vertebrate cells. This suggests that they play a critical role in initiating bundle formation. It is planned to use the transfection of vertebrate cells to further define the essential parts of the protein required for bundle formation, and to use affinity chromatography and the yeast two-hybrid system to identify the regions of forked which interact with other proteins. P-factor transformation with forked genes mutated in these regions will then be used to clarify the roles of each domain in actin bundle formation in bristles.