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

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 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.


Introduction
In recent years, there have been remarkable improvements in our ability to comprehensively unravel the fine details of cellular events.This is owing to the development of the green fluorescent protein from the jellyfish Aequorea victoria (avGFP), its spectral variants, such as cyan-and yellow-emitting variants (CFP and YFP, respectively), and GFP-like proteins including redemitting fluorescent proteins (RFPs) from other organisms.
These fluorescent proteins (FPs) can be incorporated into proteins by genetic fusion to develop genetically encoded probes for a variety of cellular functions (Rodriguez et al., 2017).
Fucci is an FP-based probe for visualizing cell-cycle progression (Sakaue- Sawano et al., 2008).The technology harnesses the cell-cycle -dependent proteolysis of Cdt1 and Geminin (Fig. 1).Over the course of the cell cycle, SCF Skp2 and APC Cdh1 E3 ligase activities oscillate reciprocally and the protein levels of their direct substrates oscillate accordingly.Geminin, the inhibitor of Cdt1, is degraded under the control of APC Cdh1 E3 ligase.The original Fucci-S/G2/M probe had GFP or YFP fused Cell Structure and Function 48: 135-144 (2023) https://doi.org/10.1247/csf.23028to the APC Cdh1 -mediated ubiquitination domain (1-110) of human Geminin (hGem(1/110)); this chimeric protein is the direct substrate of APC Cdh1 E3 ligase.On the other hand, the original Fucci-G1 probe had RFP fused to residues 30-120 of human Cdt1 (hCdt1(30/120)), which can serve as the direct substrate of SCF Skp2 E3 ligase.Thus, the original Fucci probe can be called Fucci(SA) because it monitors the balance between SCF Skp2 and APC Cdh1 E3 ligase activities.Fucci(SA) effectively highlights the transition process from G1 phase to S phase (Fig. 1A).
However, visualizing cell-cycle transitions other than G1/S is just as important.Thus, we next engineered the hCdt1-based RFP-containing probe to make it sensitive to CUL4 Ddb1 instead of SCF Skp2 .By combining the resultant probe with the hGem(1/110)containing green or yellow probe sensitive to APC Cdh1 , we developed Fucci(CA), which monitors the balance between CUL4 Ddb1 and APC Cdh1 (Sakaue- Sawano et al., 2017).Fucci(CA) distinguishes clearly interphase boundaries between G1, S, and G2 phases (Fig. 1B).
In the present study, we developed practically useful GFP and RFP named h2-3 and AzaleaB5, respectively, from corals, and substituted this GFP/RFP pair for the mVenus/mCherry pair in both Fucci(SA)2 and Fucci(CA)2 probes.The resultant probes: Fucci(SA)5 and Fucci(CA)5, respectively, would become more disseminatable than before.

Materials and Methods cDNA cloning
The soft coral Ricordia sp. and the stony coral Montipora monasteriata were purchased from an aquarium shop.For each coral, whole tissue was frozen and ground down with a MultiBeads Shocker (Yasui Kikai), and total RNA was isolated by TRIzol Reagent (Thermo Fisher Scientific).mRNAs were purified using an Oligotex-dT30<Super> (JSR).cDNA was synthesized with a SalI site at the 5' end and a NotI site at the 3' end by using a SuperScript TM Plasmid System with Gateway ® Technology for cDNA Synthesis and Cloning (Thermo Fisher).Ligation of the cDNAs into a SalI/NotI-cleaved pRSET-FastBac plasmid (Ando et al., 2002) produced a directional cDNA library in a prokaryotic expression vector.The libraries were transformed into the E. coli strain JM109 (DE3).Colonies were screened for fluorescence by using a UV illuminator (365 nm) and a LED transilluminator (green).

Mutagenesis
Site-directed and semi-random mutations were introduced according to our protocols as described previously (Sawano and Miyawaki, 2000).Error-prone mutagenesis was based on PCR using GoTaq DNA polymerase (Promega) supplemented with 1 mM MnCl 2 .

Establishment of stable cell lines
For the generation of HeLa cell lines stably expressing tFucci probes, the PiggyBac transposon system was employed (Aoki et al., 2013).The pPBbsr-based tFucci probes and pCMV-mPBase (neo-) encoding the piggyBac transposase were co-transfected into HeLa cells using PEI (Polyethylenimine) at a ratio of 3:1.
tFucci-expressing single cell clones were further isolated by limited dilution.

Flow cytometry
Hoechst 33342 solution (56 μl of 1 mg/ml stock) (DOJINDO, Kumamoto, Japan) was added to a 10-cm dish containing HeLa/ Fucci cells.After incubation for 30 min, cells were harvested and analyzed using a FACSAria II (BD Bioscience, San Jose, CA).h2-3 was excited by a 488-nm laser line (laser diode) and its emission was collected through 530/30BP; AzaleaB5 was excited by a 561-nm laser line and its emission was collected through 610/20 BP.Hoechst 33342 was excited by a UV Laser at 355 nm, and its emission was collected through 450/50 BP.
The data were analyzed using FlowJo software (Tree Star).See

Long-term time-lapse imaging
Cells were grown on 35-mm glass-bottom dishes in phenol red-free DMEM containing 10% FBS.Cells were subjected to long-term, time-lapse imaging using a computer-assisted fluorescence microscope (Olympus, LCV100) equipped with an objective lens (Olympus, UAPO 40×/340 N.A. = 0.90), a halogen lamp, a red LED (620 nm), a CMOS camera (Hamamatsu Photonics, ORCA-Flash4.0),differential interference contrast (DIC) optical components, and interference filters.The halogen lamp was used with BrightLine® single-band filter set (Semrock): "FITC-2024B" for observing the h2-3 fluorescence, and "mCherry-C" for observing the AxaleaB5 fluorescence.For DIC imaging, the red LED was used with a filter cube containing an analyzer.Image acquisition and analysis were performed using MetaMorph 6.37 and 7.10 software (Molecular Devices), respectively.See Table II for details.

Manual cell tracking
Image processing was performed manually using the "Journal" functions implemented in MetaMorph (Molecular Devices).First, fluorescence images of AzaleaB5 and h2-3 were merged.In addition, DIC images acquired at slightly different focal planes were merged for delineating individual cell nuclei.This morphology observation was particularly useful for marking mitotic events.Time sequence data of tracked cells are saved in "TrackRef" files.The mean fluorescence intensities of tracked nuclei were calculated using the "Region measurements" function.

Results and Discussion
We screened approximately 100,000 bacterial colonies containing a cDNA library prepared from Montipora monasteriata (Fig. 2A) for fluorescence.One clone was selected that appeared to encode an RFP, and temporarily referred to as 1-41.
Based on an amino acid sequence alignment (Fig. 2B), 1-41 was supposed to have a similar β-can fold to other common FPs.The closest homologue was pporRFP, an RFP cloned from Porites porites (Poritiina, Poritidae) (Alieva et al., 2008), which shared 80.8% identity.Transformation of the cDNA into Escherichia coli generated dim red fluorescent colonies.The addition of a histidine 6 tag at the N-terminus of the protein allowed purification by metal affinity chromatography for spectroscopic and biochemical characterizations.The absorption spectrum of 1-41 at pH 7.4 displayed a major peak at 573 nm (Fig. 2C) and a slight shoulder at 537 nm; a small peak at 503 nm was indicative of a green-emitting byproduct (Miyawaki et al., 2012).Excitation at around 540 nm produced weak fluorescence peaking at 592 nm (Fig. 2D).Pseudo-native gel electrophoresis analysis revealed that 1-41 formed a highly multimeric complex (Fig. 3).We adopted semi-random mutagenesis to transform 1-41 into a useful RFP.We performed site-directed mutagenesis to break the multimeric structure, followed by random mutagenesis to rescue the red fluorescence (Campbell et al., 2002;Ando et al., 2004).We first introduced 14 mutations: S8T, H67C, T108D, A109V, N121I, R133L, D146E, R151V, Q159M, D162L, H164D, M165I, K190E, and Q219G into #1-41 (Fig. 2B).Among them, T108D was introduced into the AB interface, and D146E, R151V, D162L, and H164D were introduced into the AC interface.We found that M165I was effective in increasing the photostability of the red fluorescence (Fig. S1).The resultant RFP was practically bright and named "Azalea" after Wako City's designated flower (Fig. 2E).Next, we used Azalea as the parental FP to develop several better mutants.One of them was AzaleaB5, which was generated by adding I85L and T176M into Azalea.Apparently, these two mutations further improved both brightness and folding efficiency.We also introduced silent base changes to optimize the coding sequence based on human codon-usage preferences.The absorption spectrum of AzaleaB5 at pH 7.4 displayed a major absorption maximum at 574 nm (ε = 104,000 M -1 • cm -1 ) with a slight shoulder around 542 nm (Fig. 2F).
Excitation and emission spectra were analyzed to characterize the red-emitting component (Fig. 2G); the fluorescence quantum yield (QY) was 0.58.The spectral characteristics of AzaleaB5 are summarized in Table I.Excitation at 480 nm gave a negligible green emission compared with the red one (Fig. 2H), indicating that AzaleaB5 was free from contamination by the greenemitting component.The red fluorescence was stable at pH 6-8, but decreased with increasing acidity and alkalinity (Fig. 2I).
Such alkaline sensitivity seemed to be unique to AzaleaB5; most conventional RFPs were stable in an alkaline as well as a neutral pH region.In pseudo-native gel electrophoresis, AzaleaB5 appeared to behave as a monomer (Fig. 3).
We also cloned a cDNA that encoded a bright green-emitting FP from Ricordea sp. (Fig. 4A).The FP was temporarily referred to as 2-3.We also generated a mutated cDNA that encoded 2-3 with human codon-usage preferences.The resultant FP was named h2-3.Sequence analysis revealed that its nearest homologue was sarcGFP from Sarcophyton sp.(Octocorallia, Alcyoniidae) (Fig. 4B) (Alieva et al., 2008).The absorption spectrum of h2-3 at pH 7.4 displayed a major absorption maximum at 506 nm (ε = 130,000 M -1 • cm -1 ) with a slight shoulder around 479 nm (Fig. 4C).The protein exhibited an emission spectrum peaking at 516 nm (Fig. 4D), which was sensitive to acidity with a pK a of 4.6 (Fig. 4E).The spectral characteristics of h2-3 are summarized in Table I.It was shown by pseudo-native gel electrophoresis analysis that h2-3 formed an obligate dimeric complex (Fig. 3).

Fig. 1
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.

RFig. 2
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
Fig. 3 Pseudo-native gel electrophoresis analysisEGFP 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.
Table II for details.