Multiplex Imaging of Rho GTPase Activities in Living Cells.

Autor: Bhalla RM; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA., Hülsemann M; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA., Verkhusha PV; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA., Walker MG; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA., Shcherbakova DM; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA., Hodgson L; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA. louis.hodgson@einsteinmed.org.; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA. louis.hodgson@einsteinmed.org.
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2350, pp. 43-68.
DOI: 10.1007/978-1-0716-1593-5_4
Abstrakt: Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.
(© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE