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Samantha Seaberg ACF Abstract FY10

Characterizing the Cellular Regulation of the Diaphanous-related Formin, mDia3, by Expression of the Constitutively Active Full-length Protein

Conference:  American Society for Cell Biology (ASCB) 49th Annual Meeting

A family of proteins known as Diaphanous-related Formins (DRFs) are important in the regulation of the cellular cytoskeleton. DRFs are regulated by autoinhibition, a mechanism which involves maintaining the DRF protein in an inactive state by the intramolecular binding of the Diaphanous-inhibitory domain (DID) to the Diaphanous-autoregulatory domain (DAD). Upon binding of an activated Rho GTPase to the DRF GTPase binding domain (GBD), the DID-DAD interaction is released, thereby activating the DRF protein. Possessing a very similar sequence homology to the well characterized mDia1 and mDia2 proteins, mDia3 (mouse) / hDia2 (human) is among the least studied DRF family members. While a past study has shown that mDia3 interacts with Cdc42 to regulate microtubule attachment to kinetochores1, the autoregulation and cellular localization of activated mDia3 has not been widely characterized. Therefore, our laboratory has been probing the similarities and/or differences in the regulation and cellular localization between mDia3 and other DRF proteins. Here, we show that M1053 in the DAD region of mDia3, much like the M1041 in mDia2 and the M1182 in mDia1, is involved in regulation by DID-DAD binding. By engineering full-length, constitutively active mDia3, we have been able to express mDia3 in three different cell lines (NIH3T3/mouse fibroblast, PC12/rat pheochromocytomas, N1E-115/mouse neuroblastomas). Constitutively activated mDia3 results in dramatically increased numbers of filopodia-like extensions in which mDia3 is significantly localized at the tips of the filopodia. This is similar to the expression pattern of mDia2, yet different from DAAM1, another DRF family member, which has been shown to be localized throughout the entire filopodia. Fluorescence anisotropy confirms that the M1053A mutation in DAD results in the complete inability to bind to the DID region of mDia3. In summary, these results demonstrate the critical contribution of M1053 to mDia3 autoregulation, as well as shed some light on the cellular effects and localization of full-length constitutively activated mDia3.