UCLA

Spire, an actin nucleator, is critical for proper oocyte development in D. melanogaster via the establishment of an actin mesh network. Knowledge of its interaction with Drosophila Rab GTPases, a group of master regulators of membrane trafficking dynamics, would contribute to our understanding of Spire’s role in the developing oocyte. Drosophila Spire has a putative Rab-binding sequence, the Spir-box, located in the C-terminal half of the protein. Flies lacking the Spir-box have about a 50% decrease in fertility, which supports the idea that the interaction between Drosophila Spire and Drosophila Rabs is functionally significant. However, the specific Rab(s) involved in the interaction remains unknown. Thus, we set out to identify which specific Drosophila Rabs interact with Drosophila Spire and whether it is through direct or indirect interactions. Based on previous data showing binding interaction between mammalian Spire1 and mammalian Rabs 6 and 11 as well as Drosophila Rab expression patterns in the oocyte, we chose to start with Drosophila Rabs 5, 6, and 11. However, we did not detect a direct interaction in GST pulldown assays with GST tagged Rab 5, 6 and 11 and C-terminal constructs of Spire. This raises the possibility of indirect interactions or the requirement of additional components to stabilize the complex of Drosophila Rab(s) and Drosophila Spire. Co-IP experiments from ovary lysate will be performed to probe for indirect binding between Spire and Rab 5, 6, and 11. Once specific Rabs have been identified, the complex will be further characterized biochemically with the long-term goal of modifying the binding sites in vitro and determining the functional consequences of these changes in vivo.