Because many critical vents in organismal development require the proper functioning of the actin cytoskeleton, functional mutations in proteins required for these processes are lethal. However, many disorders of early development might arise through abnormal modulation of signal transduction pathways which alter the temporal or spatial regulation of these events, leading to less severe phenotypic variants which survive. Many different actin binding proteins are involved in the organization of the actin cytoskeleton and its dynamic reorganization in response to environmental cues. Among these are the ubiquitous F-actin binding/severing and monomer sequestering proteins of the ADF/cofilin family. These proteins are enriched in ruffling membranes and neuronal growth cones, regions of high actin assembly dynamics. Functional mutations in the ADF/cofilin gene are lethal in yeast, fruit fly and worm. Interactions of ADF/cofilin with actin are modulated by phosphatidylinositides, pH and direct phosphorylation of a single serine residue. ADF cofilin can limit the amount of cytoplasmic actin available for assembly by transporting it to the nucleus. Many intracellular events coupled to cytoskeletal reorganization are triggered by transmembrane signals involving pH, phosphatidylinositol (PI) metabolism, or cAMP production. A direct activation (dephosphorylation) of ADF/cofilin by a cAMP-dependent mechanism occurs rapidly in many cell types undergoing a change in morphology. Thus, the ADF/cofilin family could be a common target of all these pathways and link transmembrane signalling to enhanced actin dynamics. Studies are proposed here to determine how the diverse modes of ADF/cofilin regulation are utilized by cells to alter their behavior. Using cultured animal cells, each cell type selected to best answer important questions about ADF regulation and function in differentiated cells, we propose: 1) to study the behavior of cells expressing a non- phosphorylatable site-directed mutant of ADF, and to determine the effect of its expression upon actin synthesis; 2) to elucidate the signal transduction pathway(s) leading to ADF phosphorylation/dephosphorylation by studying the dynamics of ADF, and to determine the effect of its expression upon actin synthesis; 2) to elucidate the signal transduction pathway(s) leading to ADF phosphorylation/dephosphorylation by studying the dynamics of ADF phosphorylation in cells treated with activators and inhibitors of membrane ruffling; 3) to investigate the role of intracellular pH in regulating ADF activity; 4) to quantify ADF/cofilin binding to PIs in cell membranes and the effect of binding on PI turnover or ADF release following activation of PI-3-kinase and PLCgamma; and 5) to determine the mechanism by which ADF expression is down regulated in cells expressing a mutant form of actin. Because actin has such a vast number of interdependent roles in normal cell function, it is not surprising that multiple signal transduction pathways can impact its organization. It would make sense for a regulator of actin assembly to be a common target for many, if not all, of these pathways.
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