Disruption of the Diaphanous-related formin Drf1 gene encoding mDia1 reveals a role for Drf3 as an effector for Cdc42.

Mammalian Diaphanous-related formins (Drfs) act as Rho small GTPase effectors during growth factor-induced cytoskeletal remodeling and cell division. While both p140 mDia1 (herein called Drf1) and p134 mDia2 (Drf3) have been shown to bind in vitro to activated RhoA-C, and Drf3 has also been shown to bind to Cdc42, little is known about the cellular function of these GTPase effector pairs. Thus, we have begun targeting the murine Drf genes to address their various contributions to small GTPase signaling in cytoskeletal remodeling and development. Drf1 +/+, +/-, and -/- cell lines were derived from embryonic stem cells. While some Drf1 +/- lines had fewer actin stress fibers, several Drf1 +/- and -/- cells were more motile and had more abundant lamella and filopodia. Because the apparent "gain-of-function" corresponded with elevated levels of Drf3 protein expression, we hypothesized that the effects on the actin cytoskeleton were due to Cdc42 utilization of Drf3 as an effector. In this study, we found that inactive Drf3 variants and microinjected Drf3 antibodies interfered with Cdc42-induced filopodia. In addition, we observed that Drf3 contains a previously unidentified CRIB-like motif within its GTPase binding domain (GBD). By fluorescent resonance energy transfer (FRET) analysis, we demonstrate that this motif is required for Cdc42 binding and Drf3 recruitment to the leading edge and, surprisingly, to the microtubule organizing center (MTOC) of migrating fibroblasts. Our observations extend the role of the mammalian Drfs in cell signaling and demonstrate that Cdc42 not only activates Drf3, but guides the effector to sites at the cell cortex where it remodels the actin cytoskeleton.