Chapter title |
Using Fluorescence Resonance Energy Transfer-Based Biosensors to Probe Rho GTPase Activation During Phagocytosis.
|
---|---|
Chapter number | 9 |
Book title |
Phagocytosis and Phagosomes
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6581-6_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6579-3, 978-1-4939-6581-6
|
Authors |
Veronika Miskolci, Louis Hodgson Ph.D., Dianne Cox Ph.D., Louis Hodgson, Dianne Cox |
Editors |
Roberto Botelho |
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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