Two coral fluorescent proteins of distinct colors for sharp visualization of cell-cycle progression

Abstract

We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright green fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided more reliable nuclear labeling for monitoring cell-cycle progression than the 1^st and 2^nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.Key words: fluorescent protein, cell cycle, time-lapse imaging, flow cytometry.

Keywords: cell cycle; flow cytometry; fluorescent protein; time-lapse imaging.

Fig. 1

Fucci probes with different ubiquitination domains of human Cdt1 (A) Fucci(SA) consists of an SCF^Skp2-sensitive hCdt1-based probe and an APC^Cdh1-sensitive hGem-based probe. Fucci(SA) corresponds to the original Fucci. A blue box in hCdt1(30/120) indicates the Cy motif. (B) Fucci(CA) consists of a CUL4^Ddb1-sensitive hCdt1-based probe and an APC^Cdh1-sensitive hGem-based probe. The dark red box and the gray box in hCdt1(1/100)Cy(–) indicates the PIP box and Cy(–): mutated Cy motif , respectively. (A, B) Domain structures (top) and cell-cycle phasing capabilities (bottom) are shown, assuming that the hCdt1- and hGem-based domains are fused to red- and green-emitting FPs. A theoretical temporal profile of the fluorescence intensity (F.I.) is shown below each domain structure. SCF, SCF^Skp2; CUL4, CUL4^Ddb1; APC, APC^Cdh1. Pink and black boxes in hGem(1/110) indicate the destruction box and nuclear localization signal, respectively. NEB: nuclear envelope breakdown. NER: re-formation of the nuclear envelope.

Fig. 2

Molecular and spectroscopic characterizations of AzaleaB5 (A) Montipora monasteriata. (B) Amino acid sequence (single-letter code) alignments of 1-41, Azalea, AzaleaB5, pporRFP, and DsRed. Residues whose side chains form the interior of the β-barrel are shaded. Residues responsible for chromophore synthesis are indicated by asterisks. In the sequences of Azalea and AzaleaB5, the substituted amino acids in comparison with 1-41 are indicated in magenta. In the sequence of AzaleaB5, the substituted amino acids in comparison with Azalea are indicated in cyan. Many GFP-like proteins from Anthozoa form tetrameric complexes and have two interfaces: AB and AC. The AC interface has a large hydrophobic surface to be more stable than the AB interface. (C) Absorption spectrum of 1-41. The spectrum is normalized by the peak at 260 nm. (D) Normalized excitation (dotted line) and emission (solid line) spectra of 1-41. F.I., fluorescence intensity. (E) Azalea. (F) Absorption spectrum of AzaleaB5. The spectrum is normalized by the peak at 280 nm. (G) Normalized excitation (dotted line) and emission (solid line) spectra of AzaleaB5. F.I., fluorescence intensity. (H) Emission spectrum of AzaleaB5 with excitation at 480 nm. F.I., fluorescence intensity. (I) pH dependence of the fluorescence of AzaleaB5. F.I., fluorescence intensity.

Fig. 3

Pseudo-native gel electrophoresis analysis EGFP and DsRed were used as size markers (monomer and tetramer, respectively). The gel was illuminated with UV light (365 nm) and imaged using a color CCD camera.

Fig. 4

Molecular and spectroscopic characterizations of h2-3 (A) Ricordea sp. (B) Amino acid sequence (single-letter code) alignments of h2-3, sarcGFP, and Aequorea victoria GFP (avGFP). Residues whose side chains form the interior of the β-barrel are shaded. Residues responsible for chromophore synthesis are indicated by asterisks. (C) Absorption spectrum of h2-3. The spectrum is normalized by the peak at 280 nm. (D) Normalized excitation (dotted line) and emission (solid line) spectra of h2-3. F.I., fluorescence intensity. (E) pH dependence of the fluorescence of h2-3. F.I., fluorescence intensity.

Fig. 5

Characterization of Fucci(SA)5 and Fucci(CA)5 for cell-cycle progression in HeLa cells (A) Fucci(SA)5 and its tandem Fucci variant, tFucci(SA)5. (B) Fucci(CA)5 and its tandem Fucci variant, tFucci(CA)5. (C, D) Time-lapse imaging of HeLa/Fucci(SA)5 (C) and HeLa/Fucci(CA)5 (D). Fucci fluorescence and DIC images were merged. These cells were in the exponentially growing phase. Images were taken every 17 min and each experiment spanned 48 h. top, Snapshot images. Cells in G1/S transition (C) and cells in G2 phase (D) are highlighted. bottom, Temporal profiles of fluorescence intensities (F.I.) of AzaleaB5 and h2-3 are indicated by red and green lines, respectively. Interphase separation by Fucci probes are labeled in blue. M, mitosis. Scale bar, 10 μm. (E, F) Flow cytometry analyses of HeLa/Fucci(SA)5 (E) and HeLa/Fucci(CA)5 (F). left, Cells showing red [AzaleaB5(+)h2-3(–)], yellow [AzaleaB5(+)h2-3(+)], and green [AzaleaB5(–)h2-3(+)] fluorescence were gated for quantification of their DNA contents by staining with Hoechst 33342. right, C values denote DNA content as a multiple of the normal haploid genome.

Fig. 6

Spectral properties of AzaleaB5 and h2-3 with laser wavelengths Normalized excitation (dotted line) and emission (solid line) spectra of AzaleaB5 (red) and h2-3 (green) are shown.