UCLA

The Drosophila oocyte has long served as a model for understanding oogenesis, the process of egg development. An essential structure in Drosophila oocytes is a cytoplasmic actin meshwork that persists during mid-oogenesis. This complex actin network is built by the collaboration of actin nucleators: Spire (Spir) and Cappuccino (Capu). Removal of the actin mesh, and the concurrent decrease in Spir and Capu expression, coincides with the onset of fast cytoplasmic streaming, mixing cytoplasmic contents and reinforcing the establishment of polarity. Analogous actin meshes, built by Spir and Capu, have been characterized in other systems. The function of these networks appears to differ based on the localization of Capu while the role of the Drosophila actin mesh, outside of restricting cytoplasmic streaming, remains uncertain. This is in part, due to the current limitations of imaging the egg chamber ex vivo during mesh removal and the requirement of mesh components during earlier stages of development. To better characterize what the actin mesh does in oogenesis, we set out to generate improved tools for studying Spir and Capu. We established a gene-specific driver for Capu, capu-Gal4, that improved the rescue of capu null to 90% fertility. Using this driver, we uncovered evidence of additional roles for Capu in development outside of the actin mesh. Our attempts to improve transgene rescue of Spir were unsuccessful. In addition, we performed genome editing to endogenously tag Spir and Capu in Drosophila. With these tools, we confirmed the expression patterns of Spir and Capu and revealed previously undescribed localization in somatic cells. In addition, we employed knock-down screens to identify other genes that regulate the actin mesh or interact with Spir. Lastly, we have made progress in adapting long term live imaging methods to visualize the removal of the actin mesh. In sum, this work contributes new insights into Drosophila oogenesis and establishes the groundwork for further developing tools for the Drosophila research community. More specifically, it demonstrates that subtle changes in the interaction of actin nucleators leads to the formation of actin-based structures that play distinct cellular roles and exemplifies the need for scrupulous genetic investigations.