Biosensors for characterizing the dynamics of rho family GTPases in living cells.
The biosensors developed in the authors' laboratory have been based on different designs, each imparting specific strengths and weaknesses. Here we describe detailed protocols for the application of three biosensors exemplifying different designs-first, a design in which an environmentally sensitive dye is used to report the activation of endogenous Cdc42, followed by two biosensors based on FRET, one using intramolecular and the other intermolecular FRET. The design differences lead to the need for different approaches in imaging and image analysis.
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Gruss-Lipper Biophotonics Center, Bronx, New York, USA.
Louis
L
Hodgson
Feimo
F
Shen
Klaus
K
Hahn
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002470:Cell Survival; D002477:Cells / Q000166:cytology* / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D051379:Mice; D057054:Molecular Imaging; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins