FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs

Bryce T Bajar; Xinmeng Guan; Amy Lam; Michael Z Lin; Ryohei Yasuda; Tal Laviv; Jun Chu

doi:10.1007/978-1-0716-2035-9_2

FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs

Methods Mol Biol. 2022:2438:31-43. doi: 10.1007/978-1-0716-2035-9_2.

Authors

Bryce T Bajar^#¹, Xinmeng Guan^#², Amy Lam^#³, Michael Z Lin³, Ryohei Yasuda⁴, Tal Laviv^{5

6}, Jun Chu⁷

Affiliations

¹ Department of Biological Chemistry, Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
² Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology & Center for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
³ Departments of Neurobiology and Bioengineering, Stanford University, Stanford, CA, USA.
⁴ Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA.
⁵ Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA. lavivtal@tauex.tau.ac.il.
⁶ Department of Physiology and Pharmacology, Sackler School of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel. lavivtal@tauex.tau.ac.il.
⁷ Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology & Center for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China. jun.chu@siat.ac.cn.

^# Contributed equally.

Abstract

With the development of fluorescent proteins (FPs) and advanced optical microscopy techniques, Förster or fluorescence resonance energy transfer (FRET) has become a powerful tool for real-time noninvasive visualization of a variety of biological processes, including kinase activities, with high spatiotemporal resolution in living cells and organisms. FRET can be detected in appropriately configured microscopes as changes in fluorescence intensity, lifetime, and anisotropy. Here, we describe the preparation of samples expressing FP-based FRET sensors for RhoA kinase, intensity- and lifetime-based FRET imaging, and postimaging data analysis.

Keywords: FLIM-FRET; FRET; Fluorescence lifetime; Fluorescent protein; Rho GTPase; RhoA; Sensitized emission FRET.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Fluorescence Resonance Energy Transfer* / methods
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
Luminescent Proteins / genetics
Luminescent Proteins / metabolism
Microscopy, Fluorescence / methods
Red Fluorescent Protein
rho GTP-Binding Proteins* / genetics
rho GTP-Binding Proteins* / metabolism

Substances

Luminescent Proteins
Green Fluorescent Proteins
rho GTP-Binding Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding