In vitro expression of Drosophila melanogaster formins

Abstract

Actin cytoskeletal regulation, a crucial feature of cellular remodeling, is a dynamic process and has been extensively investigated. Formins, a class of actin nucleators, have been demonstrated to actively regulate actin nucleation and are implicated in many essential functions such as cell division, migration, adhesion, etc. Given the variety of functions that are performed by formins and the fact that multiple homologues are present in eukaryotes, it becomes important to understand the isoform specific mechanisms of regulating actin nucleation and the redundant roles they play. In silico analyses of Drosophila melanogaster genome have identified six FH2 domain containing putative formin proteins. Three formins: Diaphanous, Cappuccino and Disheveled associated activator of morphogenesis (DAAM) have been characterized in vitro and are shown to have the ability to nucleate actin while the other three: Fhos, Formin3 and CG32138 remain uncharacterized. To investigate their actin nucleation capability in vitro, we have attempted to clone and over-express the FH1-FH2 fragments of these novel formins, and will use these constructs in future to see how they regulate actin polymerization through in vitro pyrene actin assays.