Screening for Rho GTPase Modulators in Actin-Dependent Processes Exemplified by Phagocytosis

Rho GTPases, a family of molecular switches, are essential for the assembly and rearrangement of the cellular actin network. Actin remodeling is a central component of many important biological phenomena including chemotaxis, immunological synapse formation, and phagocytosis. Proper execution of these processes requires careful modulation of Rho GTPase activity in space and time. This is accomplished by delicate coordination of Rho GTPase activation and inactivation by Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase-activating proteins (RhoGAPs), respectively. Elucidating the function of these Rho GTPase modulators is complicated by their diversity, varied expression across different tissues, and multiplicity of substrates. To overcome some of these hurdles, we describe here a systematic and unbiased screening approach consisting of three sequential steps: (1) monitoring the subcellular localization of a library of Rho GTPase modulators; (2) assessing endogenous levels of expression of the suitably localized candidates in the cell type of interest; and (3) validating the functional relevance of the identified candidates by siRNA, followed by determining the effects of gene silencing on Rho GTPase activity and actin polymerization. To this end, we describe the expression and visualization of fluorescent Rho GTPase modulators, and the use of genetically encoded biosensors for active Rac/Cdc42 and of fluorescent probes of polymerized actin. Phagocytosis by macrophages is used in this chapter as an experimental paradigm, but the methods described herein can be easily extended to other cells and actin-dependent processes.