Using Fluorescence Resonance Energy Transfer-Based Biosensors to Probe Rho GTPase Activation During Phagocytosis
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The p21-family members of Rho GTPases are important for the control of actin cytoskeleton dynamics, and are critical regulators of phagocytosis. The three-dimensional structure of phagosomes and the highly compartmentalized nature of the signaling mechanisms during phagocytosis require high-resolution imaging using ratiometric biosensors to decipher Rho GTPase activities regulating phagosome formation and function. Here we describe methods for the expression and ratiometric imaging of FRET-based Rho GTPase biosensors in macrophages during phagocytosis. As an example, we show Cdc42 activity at the phagosome over Z-serial planes. In addition, we demonstrate the usage of a new, fast, and user-friendly deconvolution package that delivers significant improvements in the attainable details of Rho GTPase activity in phagosome structures.
Key wordsMacrophages Phagosome Ratiometric imaging FRET Biosensors Z-stack Deconvolution
This work was supported by National Institutes of Health grants T32GM007491 to VM, GM071828 to DC, and GM093121 to LH.
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