A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

Benjamin B Kim; Haodi Wu; Yukun A Hao; Michael Pan; Mariya Chavarha; Yufeng Zhao; Michael Westberg; François St-Pierre; Joseph C Wu; Michael Z Lin

doi:10.1038/s41598-022-07313-1

A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

Sci Rep. 2022 Mar 7;12(1):3678. doi: 10.1038/s41598-022-07313-1.

Authors

Benjamin B Kim¹, Haodi Wu^{2

3}, Yukun A Hao¹, Michael Pan⁴, Mariya Chavarha¹, Yufeng Zhao⁴, Michael Westberg^{4

5}, François St-Pierre⁶, Joseph C Wu², Michael Z Lin^{7

8}

Affiliations

¹ Department of Bioengineering, Stanford University, Stanford, CA, USA.
² Stanford Cardiovascular Institute, Stanford University, Stanford, USA.
³ Department of Medicine, Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA.
⁴ Department of Neurobiology, Stanford University, Stanford, CA, USA.
⁵ Department of Chemistry, Aarhus University, Aarhus, Denmark.
⁶ Baylor College of Medicine, Houston, TX, USA.
⁷ Department of Bioengineering, Stanford University, Stanford, CA, USA. mzlin@stanford.edu.
⁸ Department of Neurobiology, Stanford University, Stanford, CA, USA. mzlin@stanford.edu.

Abstract

A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in the presence of sample motion. We performed combinatorial multi-site mutagenesis on a cyan-excitable red fluorescent protein to create the bright and monomeric mCyRFP3, which proved to be uniquely non-perturbing when fused to the GEVI ASAP3. The green/red ratio from ASAP3-mCyRFP3 (ASAP3-R3) reported voltage while correcting for motion artifacts, allowing the visualization of membrane voltage changes in contracting cardiomyocytes and throughout the cell cycle of motile cells.

Publication types

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

MeSH terms

Diagnostic Imaging*
Luminescent Proteins / genetics
Luminescent Proteins / metabolism
Mutagenesis
Neurons* / metabolism
Red Fluorescent Protein

Substances

Luminescent Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding