Remodeling the Actin Cytoskeleton by Ubiquitin‐Dependent Proteolysis

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. Ubiquitin‐mediated protein degradation is emerging as a new regulatory mode of cytoskeletal organization and remodeling. The Drosophila ring canal provides a useful model system to study how ubiquitination affects the actin cytoskeleton. Ring canals are intercellular bridges that grow during development from 1 μm to 10 μm in diameter as a result of a dynamic and robust actin cytoskeleton. Interestingly, mutation of kelch results in aberrant accumulation of F‐actin that completely occludes the lumen of ring canals (referred to as the kelch ‐like phenotype). We have recently characterized Kelch function as the substrate adaptor component of a Cullin3‐based ubiquitin ligase complex (notated as CRL3 Kelch ). We showed that the CRL3 Kelch ubiquitin ligase functions to target a ring canal substrate for ubiquitination and proteasomal degradation – a process essential for the proper regulation of the ring canal actin cytoskeleton. 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. Preliminary data from cultured Drosophila S2 cells suggests that HtsRC is ubiquitinated and degraded by the proteasome, and its ubiquitination may be dependent on CRL3 Kelch . Additionally, we are performing co‐IP binding assays to map the HtsRC‐Kelch binding interaction. 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 specific interacting proteins with high spatial specificity in living tissue. Support or Funding Information This work was funded by NIH RO1 GM043301 grant to L.C. K.M.M. was supported in part by the National Institute of General Medical Sciences NIH training grant T32 GM007223.