Using Fluorescence Resonance Energy Transfer-Based Biosensors to Probe Rho GTPase Activation During Phagocytosis.
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.
Departments of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.
Veronika
V
Miskolci
Departments of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA. louis.hodgson@einstein.yu.edu.|Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA. louis.hodgson@einstein.yu.edu.
Louis
L
Hodgson
Departments of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA. dianne.cox@einstein.yu.edu.|Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA. dianne.cox@einstein.yu.edu.|Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, USA. dianne.cox@einstein.yu.edu.
Dianne
D
Cox
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D021621:Imaging, Three-Dimensional; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010587:Phagocytosis; D000067996:RAW 264.7 Cells; D013223:Statistics as Topic; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins
Biosensors; Deconvolution; FRET; Macrophages; Phagosome; Ratiometric imaging; Z-stack