The Critical Concentration of Actin and Actin-Binding Protein Function

University Of Florida, Gainesville FL

Investigators

Abstract

The actin cytoskeleton is a dynamic network of filamentous structures that provides a "skeleton" for cells and also regulates aspects of cell shape, motility, adhesion, signaling, and differentiation. In much the same way that a child plays toy blocks, a cell constantly reorganizes the actin cytoskeleton by assembling and disassembling filaments composed of the protein, actin. Reorganization is rapid and tightly controlled, resulting in multiple intricate cellular behaviors such as cell crawling and cellular division. Regulation of this complex process is accomplished by more than one hundred actin-binding proteins. This project takes advantage of a novel application of fluorescence anisotropy to develop a quantitative technique which can be used to follow the molecular events involved in the assembly and disassembly of actin filament networks. The technique, which is relatively easy to perform, works by measuring the speed of molecular rotation of actin-binding proteins. When actin regulatory proteins bind to actin, fluorescence anisotropy detects this binding event as a change in the protein's speed of rotation. The mechanisms of specific actin-binding proteins previously implicated in actin cytoskeletal dynamics are investigated in detail in the project. Another goal of the research is to validate the technique for more general use by other investigators studying the cytoskeleton. This project provides a tool that can be used by many investigators to study the molecular regulation of actin networks. The organization of actin networks underlies crucial developmental and physiological processes, such as the formation of inner ear structures, wound healing and metastasis. Because so many cellular events depend on actin filament dynamics, it is not surprising that defects in actin regulation are associated with developmental abnormalities and various diseases. The project will also provide training in research with a strong quantitative foundation for undergraduate and graduate students.