ADF was discovered by Bamburg at the start of his career and he has focused on it, almost exclusively. ADF is a small monomer that typifies a class of actin-binding proteins by having a curious mixture of actin-binding activities including severing actin filaments and binding actin monomers. These proteins are found in all eukaryotes and are essential in all of several studied cases, including yeast and Dictyostelium. Cells contain a large amount of them, at a molar ratio of about 1:10 relative to actin. They are regulated by phosphoinositides, pH and phosphorylation. Most notably, Bamburg has found that phosphorylation regulates the actin-binding activity of ADF in vitro, and that phosphorylation occurs in developing muscle cells in a manner predicted from the actin assembly that occurs there. ADF is therefore the best case of regulation of a protein that regulates actin assembly. Bamburg has identified the phosphorylation site as a Serine and prepared substitution mutants that constitutively mimic the on and off states (Glu and Ala, respectively). In preliminary work for this application, Bamburg has isolated and analyzed cDNA's and Ab's for Xenopus ADF along with preparing recombinant ADF that is fully functional in vitro. He finds developmental changes in the phosphorylation that controls actin-binding activity. Ab localization studies show ADF in the same places as filamentous actin.
Aim 1 is to determine whether ADF is important for actin reorganization during oogenesis.
Aim 2 is to determine the nature of the phosphatase that activates ADF by dephosphorylating it after fertilization. The strategy will be to increase the concentration of various second messengers that lie upstream of individual phosphatases. Also, the phosphatase will be purified biochemically from fertilized eggs.
Aim 3 is to determine if ADF is important in actin reorganization events that follow fertilization, using injection of inhibitory Abs, and active or inactive ADF mutants.
Aim 4 is to determine how ADF influences actin filaments during cytokinesis, which has been found in new preliminary data. The approach will be similar to that in Aim 3.
Aim 5 is to determine if the small G-proteins rho, rac1 and cdc42 are important for cortical rotation and cytokinesis and if so, if their effects are mediated through ADF. Again, dominant negative and constitutively active forms will be microinjected, with observation of changes in the biological phenomena. Effects on phosphorylation of ADF will be examined, along with the ability of inhibitory anti-ADF Abs to block these effects.
