Drak is a potential mechanotransduction partner of Filamin

ABSTRACT Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development. Summary statement We show that Drak binds to mechanically open Filamin in vitro, while developmental evidence suggests brief, context-dependent interaction Competing Interest Statement The authors have declared no competing interest. Footnotes We have tuned down the language about the interpretation of our results and modified the title accordingly. We have included all yeast two hybrid screen results in the supplemental material. We have added p-values for Pearson correlation of the co-localization analysis. We now explain more clearly the use of Spaghetti Squash-mCherry and Filamin-mCherry as markers of the developing myotubes and tendon cells, respectively, during the time-lapse analysis of IFM development in pupal stage. We now discus the limitations of the Drak-GFP allele, including the failure to detect with anti-GFP by immunoblotting. https://doi.org/10.23729/1899cb2a-1647-4b2f-b20c-aed491ce551b