Far-red fluorescent tags for protein imaging in living tissues

Abstract

A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

Figure 1. Alignment of the amino acid sequences for the selected fluorescent proteins

Structurally important regions are highlighted in grey, β-strands are shown by arrows, and α-helixes are shown by ribbons. Buried residues are shaded. Amino acid residues responsible for the far-red fluorescence of the cis-chromophore of Katushka, mKate and mKate2 are underlined. Random mutations generated in the course of mKate optimization are shown white on black, targeted mutations are shown bold. Overall alignment numbering follows that of avGFP.

Figure 2. Spectral characteristics of mKate2 in comparison with selected fluorescent proteins

(a) Normalized mKate2 absorption (solid line), fluorescence excitation (– –) and emission (- - -) spectra. (b) pH stability of mKate2 (solid line) and mKate (broken line) fluorescence. (c) Emission spectra of mCherry, far-red monomeric fluorescent proteins and tdKatushka2 relative to their calculated brightness (Table 1). Scaling was applied to the area of the peak. The favourable `optical window' is shaded grey. (d) Normalized photobleaching curves for mCherry, far-red monomeric fluorescent proteins and tdKatushka2 using laser-scanning confocal microscopy. (e) Normalized photobleaching curves, widefield fluorescence microscopy under metal halide illumination.

Figure 3. Fluorescence imaging of mKate2 fusion vectors

(a)–(g) C-terminal mKate2 fusion constructs: (a) mKate2–β-actin-7 (human); (b) mKate2–endosomes-14 (human RhoB GTPase and c-Myc epitope tag); (c) mKate2–lamin B1–10 (human); (d) mKate2–clathrin light chain-15 (human); (e) mKate2–peroxisomes-2 (peroxisomal targeting signal 1; PTS1); (f) mKate2–membrane-5 (20-amino-acid farnesylation signal from c-Ha-Ras); (g) mKate2–α-tubulin-6 (human); (h) mKate2–VASP-5 (mouse vasodilator-stimulated phosphoprotein); (i) mKate2–annexin (A4)-12 (human). (j)–(t) N-terminal fusion constructs; (j) mKate2–paxillin-22 (chicken); (k) mKate2–α-actinin-19 (human non-muscle); (l) mKate2–vimentin-7 (human); (m) mKate2–Golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (n) mKate2–EB3-7 (human microtubule-associated protein; RP/EB family); (o) mKate2–keratin-17 (human cytokeratin 18); (p) mKate2–zyxin-7 (human); (q) mKate2–Cx43-7 (rat α-1 connexin 43); (r) mKate2–H2B-6 (human; prophase); (s) mKate2–mitochondria-7 (human cytochrome c oxidase subunit VIII); (t) mKate2–lysosomes-20 (human lysosomal membrane glycoprotein 1). The cell line used for expressing mKate2 fusion vectors was grey fox lung fibroblast cells (FoLu) in panels (a), (g) and (n), and human cervical adenocarcinoma cells (HeLa; ATCC, CCL2) in all other panels. A different strain of HeLa (ATCC, S3) was used for mitosis studies in (r). Scale bars, 10 μm. For each fusion protein, the linker amino acid length is indicated after the name of the targeted organelle or fusion protein.

Figure 4. Imaging of mKate2 in X. laevis embryos

(A) Expression of mKate2 under the Xanf1 promoter in the transgenic embryos at stage 28 is specifically localized in the forehead region, including eyes, the forebrain and nasal placodes. The embryo is shown from the right-hand side, with the dorsal side at the top and left. (B) Control images, non-transgenic embryo at the same stage. (C) Upper image: expression of mKate2–zyxin under CMV promoter in the transgenic embryo of stage 35 (2 days). Despite quite intensive and ubiquitous expression of mKate2–zyxin, the embryo appears normal and healthy. Lower image: control embryo of the same stage. The embryos are shown from the right-hand side, with the dorsal side at the top.

Figure 5. Fluorescence imaging of tdKatushka2 fusion vectors

(a)–(g) C-terminal tdKatushka2 fusion constructs: (a) tdKatushka2–clathrin light chain-15 (human); (b) tdKatushka2–β-actin-7 (human); (c) tdKatushka2–VASP-5 (mouse vasodilator-stimulated phosphoprotein); (d) tdKatushka2–lamin B1–10 (human); (e) tdKatushka2–membrane-5 (20-amino acid farnesylation signal from c-Ha-Ras); (f) tdKatushka2–fibrillarin-7 (human); (g) tdKatushka2–peroxisomes-2 (peroxisomal targeting signal 1; PTS1). (h)–(p) N-terminal tdKatushka fusion constructs: (h) tdKatushka2–EB3-7 (human microtubule-associated protein; RP/EB family); (i) tdKatushka2–Golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (j) tdKatushka2–zyxin-7 (human); (k) tdKatushka2–Cx26-7 (rat β2 connexin 26); (l) tdKatushka2–paxillin-22 (chicken); (m) tdKatushka2–lysosomes-20 (human lysosomal membrane glycoprotein 1); (n) tdKatushka2–VE-cadherin (human vascular epithelial cadherin); (o) tdKatushka–VSVG-12 (vesicular stomatitis virus glycoprotein; membrane); (p) tdKatushka–CENPB-22 (human CENP-B DNA-binding domain). The cell line used for expressing tdKatushka2 fusion vectors was grey fox lung fibroblast cells (FoLu) in panel (h) and human cervical adenocarcinoma cells (HeLa; ATCC, CCL2) in all other panels. Scale bars, 10 μm. For each fusion protein, the linker amino acid length is indicated after the name of the targeted organelle or fusion protein.