Abstract
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.
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Acknowledgements
This work was supported by National Institutes of Health grants T32GM007491 to VM, GM071828 to DC, and GM093121 to LH.
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Miskolci, V., Hodgson, L., Cox, D. (2017). Using Fluorescence Resonance Energy Transfer-Based Biosensors to Probe Rho GTPase Activation During Phagocytosis. In: Botelho, R. (eds) Phagocytosis and Phagosomes. Methods in Molecular Biology, vol 1519. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6581-6_9
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DOI: https://doi.org/10.1007/978-1-4939-6581-6_9
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