A Bright, Nontoxic, and Non-aggregating red Fluorescent Protein for Long-Term Labeling of Fine Structures in Neurons

Lin Ning; Yang Geng; Matthew Lovett-Barron; Xiaoman Niu; Mengying Deng; Liang Wang; Niloufar Ataie; Alex Sens; Ho-Leung Ng; Shoudeng Chen; Karl Deisseroth; Michael Z Lin; Jun Chu

doi:10.3389/fcell.2022.893468

A Bright, Nontoxic, and Non-aggregating red Fluorescent Protein for Long-Term Labeling of Fine Structures in Neurons

Front Cell Dev Biol. 2022 Jun 29:10:893468. doi: 10.3389/fcell.2022.893468. eCollection 2022.

Authors

Lin Ning¹, Yang Geng^{1

2}, Matthew Lovett-Barron³, Xiaoman Niu², Mengying Deng⁴, Liang Wang⁴, Niloufar Ataie⁵, Alex Sens⁵, Ho-Leung Ng⁵, Shoudeng Chen⁶, Karl Deisseroth³, Michael Z Lin^{1

3}, Jun Chu⁴

Affiliations

¹ Department of Neurobiology, Stanford University, Stanford, CA, United States.
² Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China University of Chinese Academy of Sciences, Beijing, China.
³ Department of Bioengineering, Stanford University, Stanford, CA, United States.
⁴ Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology and Center for Biomedical Optics and Molecular Imaging, and CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
⁵ Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, United States.
⁶ Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China.

Abstract

Red fluorescent proteins are useful as morphological markers in neurons, often complementing green fluorescent protein-based probes of neuronal activity. However, commonly used red fluorescent proteins show aggregation and toxicity in neurons or are dim. We report the engineering of a bright red fluorescent protein, Crimson, that enables long-term morphological labeling of neurons without aggregation or toxicity. Crimson is similar to mCherry and mKate2 in fluorescence spectra but is 100 and 28% greater in molecular brightness, respectively. We used a membrane-localized Crimson-CAAX to label thin neurites, dendritic spines and filopodia, enhancing detection of these small structures compared to cytosolic markers.

Keywords: RFP; crimson; label; long-term; neuron; non-aggregating; red fluorescent protein.