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2006 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Recent studies have shown that these specific amino acids within the basic region of DAD (highlighted in blue in Fig. 2) contribute to the binding of DID and therefore to the maintenance of the mDia autoregulatory mechanism. Expression of full-length versions of mDia2, previously shown to have amino acid substitutions in either the DAD core or the basic region, causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge (Fig. 3). These studies further refine the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions. The importance of the observations is severalfold. They further our understanding of how these conserved actin-nucleating proteins are controlled in cells. Since there are numerous drugs that target the cytoskeleton and several (such as paclitaxel [Taxol]) that are effective anti-cancer drugs, the information gleaned from the DAD-DID binding studies will further our efforts to develop drugs that target this family of proteins. Finally, the observation that mDia proteins have a role in filopodia assembly should impact multiple fields beyond that of cancer cell motility. Filopodia and similar structures are important, for example, for the ability of neuronal cells to change shape and form neurites. Neurites are the structures that nerve cells use to contact and communicate with other nerve cells. Figure 2. 8 Figure 2. Formins can be deregulated by disrupting autoinhibition via mutations in DAD that block its ability to interact with DID. The amino acid sequence alignment from Dia-autoregulatory domains from human, mouse, fruit fly, and fungal formins shows the conserved residues in red; similar residues are shown in green. The basic region in DAD is highlighted in blue. Alanine substitutions, such as M1041A in mDia2, disrupt the autoregulatory DID-DAD interaction, thereby leading to a constitutively active protein. Figure 3. Figure 3. Constitutively active mDia2-M1041A localizes to the tips of filopodia. YFP-mDia2-M1041A, co-expressed with an interfering form of the GTPase Rac1, causes cells to form extensions (filopodia) resulting from the elongation of F-actin within the filopodia. The result also shows that Rac activity is not involved in mDia2-assembled actin structures.

VARI | 2006 External Collaborators Kathy Siminovitch, University of Toronto, Canada Philippe Chavrier, Institut Curie, Paris, France 9 Recent Publications From left: Chen, Holman, DeWard, Eisenmann, Kitchen, Alberts, Hildebrand Wallar, Bradley J., Brittany N. Stropich, Jessica A. Schoenherr, Holly A. Holman, Susan M. Kitchen, and Arthur S. Alberts. 2006. The basic region of the diaphanous-autoregulatory domain (DAD) is required for autoregulatory interactions with the Diaphanous-related formin inhibitory domain. Journal of Biological Chemistry 281(7): 4300–4307. Alberts, Arthur S., Huajun Qin, Heather S. Carr, and Jeffery A. Frost. 2005. PAK1 negatively regulates the activity of the Rho exchange factor NET1. Journal of Biological Chemistry 280(13): 12152–12161. Colucci-Guyon, Emma, Florence Niedergang, Bradley J. Wallar, Jun Peng, Arthur S. Alberts, and Philippe Chavrier. 2005. A role for mammalian Diaphanous-related formins in complement receptor (CR-3)-mediated phagocytosis in macrophages. Current Biology 15: 2007–2012. Eisenmann, Kathryn M., Jun Peng, Bradley J. Wallar, and Arthur S. Alberts. 2005. Rho GTPase-formin pairs in cytoskeletal remodeling. In Signalling Networks in Cell Shape and Motility, Novartis Foundation Symp. series, Vol. 269. London, U.K.: Novartis Foundation, pp. 206–230.

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