UCLA

Actin networks are dynamic components of the cytoskeleton, present in all eukaryotic cells and responsible for many important cellular processes, including cell motility and cell division. Improper regulation of actin can lead to detrimental effects in embryology and development. In Drosophila egg development, an actin mesh is assembled by the collaboration of two actin nucleators, Spire (Spir) and Cappuccino (Capu). The persistence of this mesh during mid-oogenesis contributes to slow cytoplasmic streaming, and its subsequent disappearance during late-oogenesis leads to fast streaming. This transition from slow to fast streaming is critical for establishing polarity and future embryo patterning. Besides Spir and Capu, the molecular machinery and mechanisms governing the regulation of the actin mesh remain unknown. To gain deeper insight into the regulation of the mesh as well as Spir and Capu, we used TurboID-based proximity labeling and co-immunoprecipitation combined with quantitative mass spectrometry to characterize the Spir/Capu interactome. These complementary methods not only validated well-established interactors but also uncovered many potential candidates, several of which we investigated. We focused specifically on Capu-associated proteins – Semaphorin-2a (Sema2a), Cut-up (Ctp), Homer, and TBC1D23.We discovered that Sema2a associates with Capu and plays a previously unidentified role in oogenesis. Notably, Sema2a-nulls contain considerably smaller or undetectable ovaries, with egg chambers that do not develop past mid-oogenesis. Sema2a-nulls and Sema2a/Capu trans-heterozygote mutants exhibit decreased mesh density and disruption of oskar (osk) mRNA localization. Furthermore, preliminary data from fluorescence anisotropy and cross-linking mass spectrometry (XL-MS) demonstrate that Ctp binds directly to the N-terminal half of Capu. Ctp mutants as well as Homer and TBC1D23 mutants display osk mRNA displacements that are sensitive to Capu protein levels. Together, these findings offer valuable insight into the Capu interaction network and extend beyond its well-known association with Spir. Further elucidation into the novel, high-confidence interactors will allow deeper understanding of the complex and dynamic processes underlying oogenesis and potentially translate to mammalian systems where the same set of actin nucleators are used to build a similar actin network.