Identification of the BORG2/Cdc42EP3 actin binding element

The Binder of Rho GTPase protein family (BORG), also known as Cdc42 Effector Proteins, consists of five members (BORG1-5). Each member of this family is defined by a Cdc42/Rac Interactive Binding motif (CRIB) and BORG Homology domains, BH1, BH2, and BH3. BORG proteins are known to be the first regulators of the septin cytoskeleton in non-dividing mammal cells. Recent studies in CancerAssociated Fibroblasts (CAFs) have shown that BORG2 works with the septin cytoskeleton to stabilize actin filaments in stress fibers. Although, it is established that the BH3 domain of BORG proteins interacts with septin complexes, the region responsible for actin interaction is not clearly established. Here, we have designed an N-terminal and C-terminal series of BORG2 truncations based on conserved regions in the sequence. By employing Tetramethylrhodamine (TMR) maleimide labeling at a unique cysteine, we have developed a fluorescence anisotropy assay for actin binding. Using this assay, we have established the region necessary and sufficient for actin binding. Notably, this region differs from a previously proposed actin binding site and exists in other BORG proteins, suggesting that the actin interaction may be a widespread and previously unrecognized feature of the BORG protein family.