Fluorescence resonance energy transfer (FRET) imaging can measure the spatial and temporal distributions of activated Rho GTPases within living cells. This information is essential for understanding how signaling networks influence Rho-GTPase switching and for elucidating the mechanisms of Rho GTPase control of the cytoskeleton. This chapter describes FRET microscopy methods to image the distribution of GTP-bound Rac and Cdc42 during the well-defined morphological transitions of phagocytosis by macrophages. Specifically, we describe the use of FRET microscopy to detect the binding of genetically encoded fluorescent protein fusions to Rac1 or Cdc42 with a fluorescent protein fusion to a p21-binding domain (PBD) that recognizes their GTP-bound states. We focus on quantifying the kinetics and activation levels of Rac and Cdc42 during Fc receptor-mediated phagocytosis by macrophages. This process is a Rac1, Cdc42, and actin-dependent process, by which macrophages engulf particles ranging in size from 0.5 to 20 μm and is an ideal model system for studying the spatial and temporal control of these GTPases. Quantitative FRET analysis for measuring the fractions of activated GTPase to allow comparison between cells, independent of the relative expression levels of the fluorescent fusions is also discussed.
