Versatile protein tagging in cells with split fluorescent protein

Abstract

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.

Figure 1. Cellular protein labelling with FP11-tag.

(a) Schematic diagram for FP11-Tag, illustrated on the crystal structure of sfCherry, and the split schemes. (b) Images of HeLa cells co-expressing GFP1-10 and GFP11::β-actin or sfCherry 1-10 and sfCherry 11::β-actin. (c) Average fluorescence intensity of whole cells expressing β-actin labelled with full-length sfGFP, sfCherry or the corresponding FP11 tags. n=6–18. Error bars are s.e.m. (d) Fluorescence images of Drosophila S2 cells expressing GFP11-tagged of β-tubulin and calreticulin, and HeLa cells expressing GFP11-tagged clathrin light chain, histone H2B, zyxin and β₂AR. (e) An image of mixed S2 cells expressing either CFP1-10+GFP11::β-actin or YFP1-10+GFP11::β-actin. The weak fluorescence of the CFP1-10-expressing cell in the YFP channel is due to the bleed-through of CFP emission. All the scale bars indicate 5 μm.

Figure 2. Labelling endogenous proteins using GFP11-tag.

(a) GFP11 knock-in efficiencies by co-transfection with Cas9/sgRNA expression plasmids and donor templates, quantified by the combined TaqMan PCR/ddPCR assay (see Supplementary Fig. 2). (b) GFP fluorescence and immunofluorescence images of knock-in cells. All the error bars are s.e.m. All the scale bars indicate 5 μm. neg, negative.

Figure 3. Amplification of fluorescence signal by tandem FP11.

(a) Schematic of fluorescence signal amplification by tandem FP11 (b) Images of Drosophila S2 cells expressing GFP11_{x1, x3} or _x7::mCherry::β-tubulin together with overexpressed GFP1-10. The three panel sets were acquired and displayed with identical settings. (c) Quantification of GFP to mCherry fluorescence intensity ratio with different number of GFP11 repeats and different linker lengths between the repeats. Fifty cells were analysed in each case. Error bars are s.e.m. (d) Images of HeLa cells expressing sfCherry11_x1 or _x4::β-actin together with overexpressed sfCherry1-10. (e) Average whole-cell fluorescence intensity with sfCherry11_x1 or _x4::β-actin. n as indicated in the figure. Error bars are s.e.m. (f) Two-colour imaging of GFP11-labelled clathrin light chain and sfCherry11_x4 labelled β-actin. All the scale bars indicate 5 μm.

Figure 4. Live cell imaging using tandem FP11 tag.

(a) Tracking of IFT particles in mouse IMCD3 cells expressing either IFT20::GFP or IFT20::GFP11_x7+GFP1-10. Kymographs show both retrograde and anterograde transport (see Supplementary Fig. 3 and Supplementary Movie 1). (b) Comparison of IFT20 movement speed for IFT20::GFP and IFT20::GFP11_x7. The number of particles that were analysed for each case is indicated in the figure. Error bars are standard deviations. (c) Comparison of the photobleaching rate in imaging β-tubulin labelled with GFP, GFP11 and GFP11_x7, showing snapshots of S2 cells expressing the three different constructs at the beginning and the end of a 400 s movie. GFP11_x7 samples were imaged with one-seventh the excitation laser power. Scale bars indicate 5 μm. (d) Fluorescence photobleaching time traces. Five cells were averaged in each condition. The error bars are standard deviations.

Figure 5. Controlled protein multimerization using GFP11-tag.

(a) Schematic of CXCR4 gene activation by either dCas9::VP64 or dCas9::GFP11_x7+GFP1-10::VP64. (b) Plot of CXCR4 fluorescence signals measured by flow cytometry. Only green fluorescence positive cells were analysed, which indicated the expression of both dCas9::GFP11_x7 and GFP1-10::VP64. Error bars are s.e.m. (c) Images of K256 cells infected by lentivirus expressing the two dCas9 constructs with either a targeting or a non-targeting sgRNA. The cells were stained with a fluorescently labelled anti-CXCR4 antibody as an indicator of CXCR4 expression. All the scale bars indicate 5 μm.