Bitesize bundles F-actin and influences actin remodeling in syncytial Drosophila embryo development.

Actin networks undergo rearrangements that influence cell and tissue shape. Actin network assembly and organization is regulated in space and time by a host of actin binding proteins. The Drosophila Synaptotagmin-like protein, Bitesize (Btsz), is known to organize actin at epithelial cell apical junctions in a manner that depends on its interaction with the actin-binding protein, Moesin. Here, we showed that Btsz functions in actin reorganization at earlier, syncytial stages of Drosophila embryo development. Btsz was required for the formation of stable metaphase pseudocleavage furrows that prevented spindle collisions and nuclear fallout prior to cellularization. While previous studies focused on Btsz isoforms containing the Moesin Binding Domain (MBD), we found that isoforms lacking the MBD also function in actin remodeling. Consistent with this, we found that the C-terminal half of BtszB cooperatively binds to and bundles F-actin, suggesting a direct mechanism for Synaptotagmin-like proteins regulating actin organization during animal development.

Tissue-specific tagging of endogenous loci in Drosophila melanogaster.

Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissue-specific tagging of endogenous proteins) that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway) and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex) in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues.