Functional characterization of BAR domain proteins in actomyosin network organization during Drosophila embryogenesis

Abstract

Membrane remodelling is coupled to cytoskeletal dynamics in cell migration and cleavage furrow formation during cytokinesis. Metazoan embryogenesis serves as an interesting context to study the function of membrane remodelling proteins containing a BAR domain on the actomyosin network during cell formation and division. We used Drosophila embryogenesis to perform a screen for elucidating the role of the BAR domain containing proteins in plasma membrane associated actin remodelling in the blastoderm embryo. Depletion of several BAR domains containing proteins gave defects in actin remodelling during early morphogenesis and cell division. We further characterized the role of a multi-domain protein GRAF in regulating cytokinetic furrow formation in Drosophila cellularization. GRAF contains a BAR, PH, RhoGAP and SH3 domain and is present in multicellular organisms. RhoGTPase exchange factors (RhoGEF) and RhoGTPase activating proteins (RhoGAP) together regulate the levels of Rho-GTP to drive actomyosin ring constriction in cleavage furrow formation. We found that a CRISPR-Cas9 induced null mutant of GRAF showed ring hyper constriction due to increased Rho-GTP and Myosin II levels in cleavage furrow formation in a RhoGAP domain dependent manner. RhoGEF2 depletion and Myosin II inactivation in Rho Kinase suppressed the hyper constriction defect in Graf mutants. GRAF was enriched at the cleavage furrow during the early stages of cleavage furrow formation. BAR and SH3 domains were required for cleavage furrow recruitment whereas PH and RhoGAP domains played a role in its dissociation from the furrow. In addition to Myosin II, GRAF also regulated the distribution of key actin regulatory proteins at the cleavage furrow. In summary, we found that the spatiotemporal recruitment of GRAF to the cleavage furrow fine-tuned Rho-GTP levels and regulated actomyosin ring constriction during cleavage furrow formation in Drosophila cellularization