Brad Wallar - Research Interests

  • Protein interactions involved in the regulation of the Diaphanous-related formins (DRFs).
  • The role of full length DRF proteins in cytoskeletal regulation and cell morphology.

Formins are multidomain proteins that govern dynamic remodeling of the cytoskeleton. Conserved in a wide range of species, including humans, mice, fruit flies, roundworms, plants, budding yeast, filamentous yeast, and slime mold, these proteins participate in the assembly of both the actin and microtubule cytoskeletons in multiple contexts, including cytokinesis, cell migration, intracellular trafficking, and development. Common to all formins is the formin homology-2 (FH2) domain that has multiple activities in vitro, including the nucleation of actin filaments from monomers, competition with barbed-end capping proteins, actin filament severing, and processive elongation by moving on the barbed end of actin filaments.

Figure 1:Typical domain structure of the Diaphonous-related formins (specifically modeled after mDia1-3). The domains not listed below are abbreviated as follows: GTPase-binding domain (GBD), dimerization domain (DD), coiled-coil region (CC), formin homology domain 1 and 2 (FH1, FH2).

Members of a subfamily of formins, the Diaphanous-related formins (DRFs, see Figure 1) have the ability to be activated by Rho GTPases. As depicted in Figure 2, the model of DRF regulation involves a mechanism of autoinhibition, where the binding of Diaphanous-autoregulatory domain (DAD) to the Diaphanous inhibitory domain (DID) normally keeps the DRF inactive. After the appropriate cellular signal, an activated Rho GTPase binds to the GTPase Binding Domain (GBD) and releases the DAD from DID, thereby opening and activating the full length DRF protein. Autoinhibition was recently demonstrated to be mediated by DID, as it acts as a high-affinity anchor for the DAD region, thereby allowing the DID-DAD interaction to keep the DRF protein inactive until signaled to open by a small Rho GTPase.

Since the DID-DAD interaction is crucial to the autoinhibition mechanism of the DRFs, numerous studies have been done to identify and characterize the specific protein interactions involved in the DID-DAD complex.

Figure 3: Structure of the mDia1 DID-DAD binding surface based on the pdb coordinates (2F31) from Nezami, et al. (2006). Using the raw coordinates, the DID(blue)-DAD(yellow) interface was modeled (by Rachel Powers, GVSU) using PyMol. Residues known to abolish DID-DAD binding are listed in bold type. Residues in DID that are potentially important to binding are italicized.

More recently, the crystal structure has been determined for the DAD segment of mDia1 in complex with the DID domain. Both DID-DAD structures clearly revealed that the DAD core segment forms an amphipathic helix that binds to a conserved, concave surface on the DID domain (Figure 3).

 

The long term goal of our laboratory is to fully understand the molecular regulation of multiple members of the DRF family of proteins. The regulation of DRFs involves a mechanism of autoinhibition, where the intramolecular binding of two domains Diaphanous-autoregulatory domain (DAD) and Diaphanous inhibitory domain (DID)] serves to keep the DRF inactive. Building on published work with undergraduate students that examined the regulatory interactions in the mouse DRF, mDia2, our laboratory hopes to identify and characterize the intramolecular interactions (DID-DAD binding) in four different DRF proteins. We also seek to discover specific mutations of amino acid residues that abolish DID-DAD binding. In the context of the full length DRF, the disruption of the regulatory interactions would render a constitutively activated version of the protein. While most research on activated DRFs has used truncated versions of the proteins, the ability to observe the cellular localization and cytoskeletal effects of active full length DRFs would provide a more accurate picture of DRF function and regulation. Through a combination of cell microinjection, immunofluorescence, site-directed mutagenesis, molecular biology, protein biochemistry, and fluorescence anisotropy, our laboratory hopes to contribute to the understanding of DRF protein regulation in the context of cytoskeletal remodeling.

Page last modified May 12, 2011