In order for cells to perform their various functions, they must assemble and continually regulate specialized actin-based structures and networks. Remodeling of actin networks is critical to allow cells to respond to external stimuli and carry out important processes during development. In the Drosophila germline, ring canals (RCs) are specialized actin-based structures that function as intercellular bridges and connect germline cells to support growth of the oocyte. RCs grow from 1 µm to 10 µm in diameter during development of the egg chamber, and their growth is driven by a robust and dynamic actin cytoskeleton. We previously found that mutations in – or inhibition of – components of a Cullin3-RING E3 ubiquitin ligase complex (CRL3Kelch) caused aberrant accumulation of F-actin in the lumen of the RC – referred to as a kelch-like phenotype. Inhibition of the proteasome in the female germline also caused a kelch-like RC phenotype, and loss of one copy of kelch (the substrate recognition subunit of the ubiquitin ligase complex) enhanced the kelch-like phenotype, suggesting that Kelch and the proteasome function in a common pathway. We hypothesize that CRL3Kelch functions to target a ring canal-specific substrate for ubiquitination and proteasomal degradation, a process required for proper regulation and remodeling of the RC actin cytoskeleton during development. We are currently investigating HtsRC, a novel ring canal protein, as the substrate based on the following genetic evidence: (1) overexpression of HtsRC induces kelch-like RCs; (2) downregulation of HtsRC suppresses the kelch-like phenotype; and (3) HtsRC protein levels are dependent on Kelch protein levels. We are currently performing biochemical assays to validate HtsRC as the substrate. Specifically, we are testing if HtsRC is ubiquitinated, and if its ubiquitination is dependent on CRL3Kelch. Further, we have made a non-degradable HtsRC by mutating all 18 lysine residues (sites of ubiquitination) to arginine, and we are testing if expression of non-degradable HtsRC induces kelch-like RCs. Lastly, we are performing co-IP binding assays to see if HtsRC and Kelch physically interact. Given the role HtsRC may be playing in the crucial remodeling and organization of the RC actin cytoskeleton during development, we are now interested in identifying its binding partners. To this end, we fused HtsRC to the APEX enzyme to achieve proximity-dependent biotinylation of HtsRC-interacting proteins in egg chambers. These biotinylated proteins can be captured by streptavidin beads and identified by mass spectrometry (MS). Excitingly, HtsRC::APEX MS hits include known ring canal proteins and are unique compared to Pav::APEX (another RC-specific fusion) hits, indicating that this technique can be used to identify interacting proteins with high spatial specificity.