Comparative molecular physiology of mammalian formins

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DESCRIPTION (provided by applicant): The diversity of cellular actin-containing structures necessitates multiple mechanisms for actin filament formation in a context-specific manner. Since purified actin monomers polymerize slowly in vitro, cells must possess factors that accelerate actin polymerization. Recent evidence from yeast shows that formin proteins accelerate actin polymerization in vitro, and is necessary for generating specific cellular actin-containing structures, such as cables and the cytokinetic cleavage furrow. Although at least 12 mammalian formins exist, their cellular roles and biochemical activities are not well defined. The overall goal of this grant is to determine how biochemical activities of mammalian formins influence their cellular function, with a particular focus on assembly of microvilli in lymphocytes. Circulating lymphocytes express three formins at high levels, one diaphanous formin (DRF1) and two highly homologous non-diaphanous formins (FRLa and a novel ORF termed mouse4). Both DRF1 and FRLa accelerate actin polymerization, but the specifics of these effects suggest different mechanisms. These proteins possess several interesting biochemical properties, including: multimerization; actin filament binding; and actin monomer binding. At least one formin (DRF1) localizes to lymphocyte microvilli. In Aim 1, mechanisms by which lymphocyte formins accelerate actin polymerization will be examined using biochemical and biophysical techniques. Amino acid sequences controlling lymphocyte forming biochemical properties will be mapped by mutagenesis. In addition, the role of profilin in formin-mediated actin polymerization acceleration will be examined. Aim 2 will elucidate regulatory mechanisms controlling formin-mediated polymerization acceleration, by determining effects of intramolecular interactions and interactions with other proteins. In Aim 3, the function of formins in lymphocytes will be examined, including cellular formin localization, effects of RNAi-induced protein suppression on lymphocyte actin structures, and effects on lymphocyte morphology of mutations disrupting formin biochemical properties. Parallel studies using Swiss 3T3 cells will examine roles of formins on structures not found in lymphocytes, such as lamellipodia and stress fibers. Biochemical results from Aims 1 and 2 will provide the basis for the cellular studies in Aim 3.