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. 2022 Jun 29:10:893468.
doi: 10.3389/fcell.2022.893468. eCollection 2022.

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

Lin Ning  1 , Yang Geng  1   2 , Matthew Lovett-Barron  3 , Xiaoman Niu  2 , Mengying Deng  4 , Liang Wang  4 , Niloufar Ataie  5 , Alex Sens  5 , Ho-Leung Ng  5 , Shoudeng Chen  6 , Karl Deisseroth  3 , Michael Z Lin  1   3 , Jun Chu  4
Affiliations

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

Lin Ning et al. Front Cell Dev Biol. .

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.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Characterization of Crimson in vitro and neurons. (A) Fluorescence spectra of Crimson. Inset, purified Crimson in visible light. (B) Absorption (left) and emission (right) of RFPs with excitation maxima at 580–590 nm. Absorbance spectra are scaled to peak extinction coefficient. Emission spectra are scaled so that areas under the curves are proportional to peak brightness (product of peak extinction coefficient and quantum yield). (C) Photobleaching kinetics of purified RFPs under arc lamp illumination with a 568/20-nm excitation filter. Time is scaled so that emission is normalized to 1000 photons per s. Imaging interval = 1 s. Each curve is the mean of two independent experiments with the error bars denoting SD (standard deviation, n ≥ 6). (D) Photobleaching of RFPs in transfected neurons under 585-nm laser illumination. Fluorescent intensity of each time frame is subtracted against the background and normalized to time point 0. Imaging interval = 10 s. Each curve is the mean with the error bars denoting SD (n = 3).
FIGURE 2
FIGURE 2
Structural basis of blue-shifting for Asn41, His28, and Ser11 in Crimson. (A) Alignment of Crimson (magenta), Neptune (PDB entry 3IP2, light blue) and mKate (PDB entry 3BXC, pink) chromophores. The chromophore rings are more coplanar in Crimson or Neptune than in mKate. The chromophore acylimine oxygen is indicated by the dashed box. (B) Hydrogen-bond interactions between the chromophore acylimine oxygen and its surrounding residues in Crimson, mKate and Neptune. In mKate, the acylimine oxygen does not engage in any hydrogen bond interactions. In Neptune, a water molecule donates a hydrogen bond to the acylimine oxygen and accepts a hydrogen bond from Ser-28. In Crimson, a hydrogen bond between Ser-11 and His-28 precludes one between His-28 and the acylimine oxygen. In addition, the side chain of Asn-41 is too short to hydrogen bond to the acylimine oxygen.
FIGURE 3
FIGURE 3
Cytosolic Crimson is brighter and less cytotoxic than other RFPs in neurons. (A) Comparison of RFPs in rat hippocampal neurons imaged with confocal microscopy 3 days post-transfection at 12 DIV. Top, representative images of transfected neurons acquired and displayed with identical settings are shown. Scale bar = 20 µm. Bottom, representative images with brightness adjusted to similar levels for display. Cell bodies are enlarged in the insets within the upper panels. Dendritic segments indicated by the dashed rectangle are enlarged in the lower panels. Scale bar = 5 µm. (B) Quantification of RFP brightness 3 days post-transfection at 12 DIV. Error bars = SEM (Standard Error of the Mean). Overall p < 0.0001 by one-factor ANOVA. (C) Quantification of the percentage of neurons with aggregates among total transfected neurons 3 days post-transfection at 12 DIV. (D) Quantification of the percentage of healthy neurons among total transfected neurons 9 days post-transfection at 18 DIV. Error bars = SEM. Overall p < 0.0001 by one-factor ANOVA.
FIGURE 4
FIGURE 4
Crimson-CAAX labels the cytoplasmic membrane well in neurons. (A) Brightness comparison of RFP-CAAX fusions in rat hippocampal neurons 3 days post-transfection at 12 DIV. Representative confocal images of transfected neurons acquired and displayed with identical settings are shown. Scale bar = 20 µm. (B) RFP-CAAX fusions visualized with confocal microscopy 3 days post-transfection at 12 DIV, with image brightness adjusted to similar levels for display. Cell bodies are enlarged in the insets within the upper panels. Dendritic segments indicated by the dashed rectangle are enlarged in the lower panels. Scale bar = 20 µm (upper panels) or 5 µm (lower panels). (C) Quantification of brightness of RFP-CAAX constructs 3 days post-transfection at 12 DIV. Error bars = SEM. Overall p < 0.001 by one-factor ANOVA. (D) Quantification of the percentage of healthy neurons among total transfected neurons 6 days post-transfection at 15 DIV. Crimson-CAAX-expressing neurons demonstrated the highest viability. Error bars = SEM. Overall p < 0.0001 by one-factor ANOVA.
FIGURE 5
FIGURE 5
Membrane-bound Crimson improves the detection of small processes. (A,B) Visualization of cultured rat hippocampal neurons with an epifluorescent microscope (A) and a confocal microscope (B). Neurons were co-transfected with cytosolic mTurquoise2 and Crimson-CAAX at 9 DIV and imaged at 14 DIV. Lower panels show dendrites from the same culture at higher magnification. Arrows indicate thin spines and filopodia visible with Crimson-CAAX but not with cytosolic mTurquoise2. Scale bars = 10 µm (upper panels) and 5 µm (lower panels). (C) Visualization of zebrafish trigeminal ganglion by two-photon microscopy in vivo. Zebrafish was injected with DNA at one-cell stage and imaged at 7-day post-fertilization. Scale bar = 20 µm.
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