Investigating mechanisms of coordinating actinstructure across multiple levels oforder - PROJECT SUMMARY: An organism's capacity to develop and grow is heavily dependent on its ability to organize various aspects of structure, ranging from G-actin to F-actin, cross-linked filaments, F-actin networks, and multicellular networks—in short, coordinate structure at multiple levels of order. Despite advances in characterizing proteins that affect actin at different levels of the structural hierarchy, we have limited knowledge of how regulation of actin is integrated across these hierarchical levels. Bitesize, the only Synaptotagmin-like protein in flies, is a membrane-bound protein that has recently been demonstrated to also directly bundle filamentous actin; preliminary data has further demonstrated that Bitesize promotes F-actin assembly. However, the mechanisms driving these actin-organizing behaviors of Bitesize have not been determined; moreover, the manner in which such mechanisms combine to produce a net effect in vivo is unknown. The long-term goal of this work is to elucidate how mechanisms that control different hierarchical levels of actin organization are integrated to facilitate morphogenesis. The overall objective of this proposal is to determine the mechanisms underlying Bitesize's organization of actin and their combined effect on Drosophila embryo development. My rationale for the proposed work is that Bitesize demonstrates direct interaction with actin, and therefore offers a unique opportunity to study the direct regulation of actin's structural hierarchy at multiples levels through the probing of a single protein. My hypothesis is that Bitesize integrates the regulation of actin assembly and actin bundling via distinct domains along its length. My aims are to: 1) determine the mechanism used by Bitesize to bundle actin, and 2) determine whether Bitesize separately regulates assembly and nucleation of actin. This approach is innovative in its coupling of structure-function analysis of Bitesize’s interaction partners with the in vivo study of btsz gene function in an animal tissue using precise gene edits. My proposed research is significant because it will determine means of coordinating hierarchical structures of actin, and because it is expected to demonstrate new mechanisms of regulating actin, such as stimulation of Dia via formin elongation effector domains, that have not been demonstrated in animals in vivo. This research will be accompanied by training in biochemistry as well as Drosophila embryology and genetics, which will take place in the Martin lab and Schwartz lab in the Department of Biology and Cryo-EM Facility at MIT, as well as in the Goode lab at Brandeis University. Together, this research and training will prepare me to establish an independent lab to investigate fundamental questions related to the formation of functional, hierarchical structures in cells and tissues.
