The basic region of the diaphanous-autoregulatory domain (DAD) is required for autoregulatory interactions with the diaphanous-related formin inhibitory domain

(M) under bar ammalian (dia) under bar phanous-related (mDia) formins act as Rho GTPase effectors during cytoskeletal remodeling. Rho binding to mDia amino-terminal (G) under bar TPase-(b) under bar inding (b) under bar omains (GBDs) causes the adjacent (D) under bar ia-(i) under bar nhibitory (d) under bar omain (DID) to release the carboxyl-terminal (D) under bar ia-(a) under bar utoregulatory (DAD) domain that flanks the formin (h) under bar omology-2 (FH2) domain. The release of DAD allows the FH2 domain to then nucleate and elongate nonbranched actin filaments. DAD, initially discovered as a region of homology shared between a phylogenetically divergent set of formin proteins, is comprised of a core motif, MDXLLXL, and an adjacent region is comprised of numerous basic residues, typically RRKR in the mDia family. Here, we show that these specific amino acids within the basic region of DAD contribute to the binding of DID and therefore the maintenance of the mDia autoregulatory mechanism. In addition, expression of full-length versions of mDia2 containing amino acid substitutions in either the DAD core or basic regions causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge. These studies further refine our understanding of the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions.