Rho GTPases: Secretion and actin dynamics in permeabilized mast cells

This chapter describes the Rho guanosine 5'-triphosphates (GTPases) secretion and actin dynamics in permeabilized mast cells. Permeabilized mast cells proved to be a valuable tool for studying the function of Rho-related GTPases. Rho, Rac, and Cdc42 were shown to be crucial for regulation of the late steps of exocytosis. The chapter discusses the use of primary mast cells—permeabilized by streptolysin O (SLO)—for studying Rho family GTPase. The chapter discusses the preparation of recombinant GTPases, an assay for GTP-binding activity, and subsequent loading of recombinant proteins with GTP. The chapter describes various procedures for introducing GTPase into mast cells and evaluation of their effects on the cytoskeleton and exocytosis. The chapter also discusses the isolation, permeabilization, and stimulation of rat peritoneal mast cells. The chapter concludes with a discussion of the analysis of F-actin content and morphology.