FUNCTIONAL ANALYSIS OF TROPOMYOSIN ISOFORMS IN VIVO

[摘要] Precise regulation of a dynamic actin cytoskeleton is an essential function of animal cells without which vesicle trafficking, cytokinesis, cell adhesion and cell movement would be impossible. Therefore, understanding the mechanisms underlying actin dynamics is fundamental for understanding basic cellular biology as well as gaining insight into mechanisms of embryonic development, tissue homeostasis and regeneration, and pathological processes such as inflammation and tumor metastasis. Actin filaments are a major source of the protrusive and contractile forces that drive many cellular behaviors. Contractile forces require the action of non-muscle myosin II, which assembles onto actin filaments to form acto-myosin. The interaction between actin and myosin can occur spontaneously in vitro, but in cells it is regulated by accessory proteins including Tropomyosins (Tms). A complete understanding of Tm function has been elusive due in part to the large number of isoforms: 44 predicted isoforms from 4 genes in humans.The goal of this study was to decipher the functional roles of different Tm isoforms at the molecular, cellular and tissue levels in vivo. Drosophila egg chambers are a genetically tractable system that expresses far fewer Tm isoforms than mammalian cells. We identified three tropomyosin isoforms expressed in follicle cells, including one previously annotated as muscle-specific. We generated and characterized isoform-specific antibodies, RNAi lines, and mutant alleles, and discovered that they function non-redundantly in the two cell types we studied: border cells, a well-studied example of collective migration, and epithelial follicle cells, which develop contractile stress fibers that shape the egg chamber. We also found that Tm mutations interact genetically with Psidin, a conserved but poorly characterized protein that we found regulates lamellipodia dynamics in both Drosophila and mammalian cells. Although strong loss-of-function mutations in either Tm or Psidin inhibit border cell migration individually, mutation of Tm suppresses the Psidin mutant phenotype. Furthermore, in a biochemical assay, we showed that Psidin inhibited Tm protein binding to actin filaments. Taken together this work has generated reagents that will be generally useful in deciphering Tm isoform functions in vivo in Drosophila, has revealed isoform-specific functions, and has revealed a novel genetic and biochemical interaction.

[发布机构] Johns Hopkins University

[效力级别] Tropomyosin

[关键词] Actin dynamics;Tropomyosin;Drosophila egg chamber;Biology