SAXS TO FOLLOW F-ACTIN DEPOLYMERIZATION BY GELSOLIN

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Physiological functions that involve cellular shape rearrangement and/or cellular motility require spatial and temporal changes in the assembly and disassembly of filamentous (F-) actin from pools of monomeric (globular, G-) actin within the cell's cytoskeleton. This process is tightly regulated by various actin-binding proteins, such as gelsolin, which generally act in response to specific cell signals, such as an increase in cellular Ca2+ levels. Gelsolin regulation of actin is under the control of Ca2+ and poly-phosphoinositides and includes severing, capping, and nucleation of filaments. Work from several research groups has shown that Ca2+ regulation of gelsolin is complex and that certain functions are activated in the 1 micromolar range whereas others are activated at higher concentrations (10 ? 1000 micromolar). The main aim of our proposed SAXS experiment is to follow the structural changes during depolymerization of F-actin when in the presence of gelsolin and as a function of Ca2+ concentration. We propose to collect SAXS data on samples of filamentous F-actin and Factin: gelsolin complexes at varying molar ratios (from 1:20 to 1:1) of gelsolin:actin and at 5 levels of calcium concentrations (0, 0.1, 1.0, 100 and 1000 micromolar). These data will be analyzed by typical SAXS analysis tools such as Porod and Guinier approximation, Indirect Fourier Transform methods and molecular modeling. Results will aid in the elucidation of a mechanism for actin de-polymerization by gelsolin, providing a structural basis for understanding how the continuous reorganization of actin filaments is controlled by specific protein-protein interactions.