Fluorescent indicators for simultaneous reporting of all four cell cycle phases

Abstract

A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

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Figure 1: Development of a four-color imaging method and an S–G2 transition reporter.
Figure 2: Fucci4, a reporter system for visualizing all four cell cycle stages.

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GenBank/EMBL/DDBJ

Referenced accessions

NCBI Reference Sequence

Protein Data Bank

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Acknowledgements

We thank A. Moskaleva and J. Ferrell (Stanford University) for plasmids, cell lines, and advice on cell cycle experiments; A. Straight (Stanford University) for HeLa-Flp-In-TREX cells; M. Davidson (Florida State University) for plasmids; and other members of the Lin laboratory for assistance with experiments. This work was supported by a Siebel Scholar Award (A.J.L.), a Stanford Graduate Fellowship (X.X.Z.), a Howard Hughes Medical Institute International Student Research Fellowship (X.X.Z.), NIH Molecular Biophysics Predoctoral Research Training grant 5T32GM008294 (A.L.Y.), Stanford Research Experience for Undergraduates grants (K.K. and J.J.T.), NIH grant R01GM118377 (T.M.), NIH grant P50GM107615 to the Stanford Center for Systems Biology (M.C., T.M., and M.Z.L.), a Burroughs Wellcome Foundation Career Award for Medical Scientists (M.Z.L.), a Rita Allen Foundation Scholar Award (M.Z.L.), and Pioneer Award 5DP1GM111003 (M.Z.L.).

Author information

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Authors

Contributions

A.J.L., B.T.B., and M.Z.L. designed and analyzed experiments. A.J.L., Y.-H.O., J.C., X.X.Z., A.L.Y., B.F.C., K.K., and J.J.T. developed and characterized Maroon variants. B.B.K. performed further characterization. B.T.B. created the S–G2 sensor. B.T.B., R.K.B., N.K., and M.C. characterized the S–G2 sensor and analyzed the data. T.M., X.-D.S., and M.Z.L. provided advice. B.T.B., A.J.L., and M.Z.L. wrote the manuscript.

Corresponding author

Correspondence to Michael Z Lin.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Novel red-shift mechanism in Maroon0.1.

(a) Excitation and emission spectra of mNeptune2 T60P M160W (Maroon0.1), compared to parental mNeptune2 and the related mCardinal. (b) A π-π stacking interaction between a Tyr residue and the top surface of the chromophore (when protein termini are oriented upwards) is responsible for red-shifting of the absorbance spectrum of YFP (left, rendering of PDB file 1HUY). A similar π-π stacking interaction presumably occurs in E2-Crimson and TagRFP657 as well. In contrast, a structural model of mMaroon0.1 predicts a π-π stacking interaction between Trp160 and the bottom of the chromophore.

Supplementary Figure 2 Evolution of Maroon fluorescent proteins.

(a) Evolutionary history leading to Maroon1. Mutations accumulated during each engineering step are on the left. (b) Native PAGE of purified protein (5 μM). tdTomato was used as a dimeric fluorescent protein standard. (c) Fluorescence images of patches of bacteria expressing Maroon mutants grown for 2 d. (d) Relative brightness of Maroon mutants (normalized to CFP expression) in HeLa cells, quantified 2 d post-transfection.

Supplementary Figure 3 Basic characteristics of mMaroon1.

(a) Sequence alignment of mNeptune2 and mMaroon1. Mutated amino acids are in red. (b) Absorbance, excitation and emission spectra of purified Maroon1. Excitation peak occurs at 609 nm and emission peak occurs at 657 nm. (c) Absorbance spectra of RFPs with peak excitation ≥ 605 nm: E2-Crimson, eqFP670, mMaroon1, TagRFP657. (d) pH dependence of fluorescence. Error bars are s.e.m. of triplicate measurements. (e) Photobleaching kinetics of purified protein in oil under arc lamp illumination with 615/30 nm excitation. Curve represents the mean of 6 measurements. Time values were normalized to simulate excitation power resulting in an emission rate of 1000 photons per s per molecule.

Supplementary Figure 4 Performance of mMaroon1 in fusions in mammalian cells.

Widefield fluorescence imaging was performed on HeLa (a,b) and U2OS (c-f) cells expressing fusion proteins designed to label subcellular structures: (a) Lifeact-7aa-mMaroon1 (actin), (b) paxillin-22aa-mMaroon1 (focal adhesions), (c) PDHA-10aa-mMaroon1 (mitochondria), (d) mMaroon1-10aa-lamin B1 (nuclear envelope), (e) calnexin-14aa-Maroon1 (endoplasmic reticulum), (f) mannosidaseII-10aa-mMaroon1 (Golgi complex). Scale bar, 10 μm.

Supplementary Figure 5 Four-color imaging with a CFP, GFP, OFP, and mMaroon1.

(a) Left, calculated fluorescence emission for equimolar concentrations of mKO2 and far-RFPs in upon excitation by a 550/10-nm filter, superimposed with the transmission spectrum of the 575/25-nm emission filter used in this study. Less than 0.1% of mMaroon1 emission occurs below 588 nm. Right, the red-shifted emission spectrum of mMaroon1 should also allow collection of more OFP emission compared to other far-RFPs when a 561-nm laser is used for OFP excitation. (b) HEK293 cells transfected in parallel with mCardinal or mMaroon1 were imaged in cyan, green, orange, and far-RFP channels defined as in Fig. 1a. Images were acquired with exposure times of 400 ms in each channel in 12-bit mode (4096 grayscale levels), then scaled for display to the first 256 counts above background for cyan, green, and orange channels, or the full intensity range for the far-red channel. Scale bar, 10 μm. (c) Mouse NIH3T3 cells expressing mTurquoise2-SLBP, Clover-Geminin(1-110), mKO2-Cdt(30-120), or H1.0-mMaroon1 were each imaged in cyan, green, orange, and far-red channels. Images were acquired with exposure times of 100 ms for cyan, 50 ms for green, 25 ms for orange, and 200 ms for far-red, then scaled for display to the first 256 counts above background in each channel. Scale bar, 10 μm.

Supplementary Figure 6 Four-construct imaging with a CFP, GFP, OFP, and mMaroon1.

Confocal images of U2OS cells simultaneously expressing MannII-10aa-mTurquoise2 (Golgi), calnexin-14aa-Clover (endoplasmic reticulum), PDHA-10aa-mKO2 (mitochondria), and H1.0-10aa-mMaroon1 (nucleus) in four channels defined as follows: cyan, 440-nm excitation, 460- to 500-nm emission; green, 488-nm excitation, 500- to 550-nm emission; orange, 559-nm excitation, 570- to 670-nm emission; far-red, 635-nm excitation, 650- to 750-nm emission. Note while calnexin and PDHA fusions show some overlapping distributions in perinuclear regions, some PDHA signal appears in regions lacking calnexin signals, and vice versa, indicating the overlap is not complete and thus not due to channel bleed-through. Scale bar, 10 μm.

Supplementary Figure 7 mTurquoise2-SLBP(18-126) is cyclically degraded.

(a) Amino acid sequence of mTurquoise2-SLBP(18-126). Linker sequences are in gray. (b) mTurquoise2-SLBP(18-126) shows degradation at G2 and reaccumulation in G1 through two cell divisions. To help follow cells as they divide, each cell is labeled in a hierarchical dot-decimal scheme. Scale bar, 10 μm.

Supplementary Figure 8 mTurquoise2-SLBP(18-126) does not perturb the cell cycle.

(a) Stable expression of mTurquoise2-SLBP(18-126) does not affect cell cycle progression as assessed by DNA content distributions in flow cytometry. (b) Stable expression of mTurquoise2-SLBP(18-126) does not noticeably affect S-phase distribution as assessed by uptake of an EdU label. For each treatment group, three plates were cultured and labelled, then 10,000 cells from each plate were analyzed by flow cytometry. Measurements of EdU-positivity were performed blinded to sample identity. (c) Stable of expression of mTurquoise2-SLBP(18-126) with EYFP-PCNA does not alter cell cycle phase durations in HeLa cells compared to cells expressing EYFP-PCNA alone. p = 0.37 for G1 phase, p = 1.0 for S phase, p = 0.55 for G2 phase, and p = 0.17 for M phase by Welch’s two-tailed t-test, with n = 14 cells per group. Error bars are standard deviation. Measurements and statistics were performed blinded to sample identity.

Supplementary Figure 9 H1.0-mMaroon1 reports chromosome condensation and movements during mitosis.

(a) Amino acid sequence of H1.0-mMaroon1. Linker sequences are in gray. (b) Visualization of in interphase, prophase, metaphase, and analphase with H1.0-mMaroon1 in HeLa and U2OS cells.

Supplementary Figure 10 Quantification of fluorescent signal in time-lapse imaging of Fucci4 in HeLa cells.

Representative trace of fluorescent signal from all four reporters over two cell divisions, tracing one arbitrarily chosen daughter and granddaughter cell. Fluorescence was normalized to maximum value. H1.0-mMaroon1 signal was set at 1.0 during the presence of chromosomal aggregates, manually scored. Analysis was performed via manual scoring. Onset of S phase was defined by the appearance of Geminin, following Sakaue-Sawano et al. (ref. 6). Onset of G2 was defined as the commencement of SLBP degradation, following Koseoglu et al. (ref. 21).

Supplementary Figure 11 Fucci4 functions in human U2OS cells and mouse NIH3T3 cells.

A lentivirus co-expressing mKO2-Cdt1(30-120) and Clover-Geminin(1-110), and another coexpressing mTurquoise2-SLBP(18-126) and H1.0-mMaroon1 were used to transduce U2OS cells (a) or NIH3T3 cells (b). (a) The U2OS cell in the center was expresses all four reporters and proceeds through S, G2, M, and G1 phases. Another cell partially visible in the corner appears to be transduced with only the lentivirus co-expressing mTurquoise2-SLBP(18-126) and H1.0-mMaroon1. (b) The NIH3T3 cell in the center expresses all four reporters and proceeds through S, G2, M, and G1 phases. Other cells partially visible at the edges appear to be transduced with only the lentivirus co-expressing mKO2-Cdt1(30-120) and Clover-Geminin(1-110). Deduced cell cycle phases are indicated to the right. Scale bar, 10 μm.

Supplementary Figure 12 Visualizing progression through four cell cycle phases with mTurquoise2-PCNA.

(a) Cartoon diagram of four cell cycle reporters mTurquoise2-PCNA, Clover-Geminin(1-110), mKO2-Cdt1(30-120), and H1.0-Maroon1. (b,c) Tracking of cell cycle stages in HeLa cells via time-lapse imaging of Fucci4, including a mother cell dividing into two daughter cells (b) and daughter cells dividing into granddaughter cells (c). Scale bar, 10 μm. (d) Quantification of cell populations in G1, S, G2, and M phases after no treatment (n = 316 cells) or double thymidine block (n = 539 cells). Numbers are percentage of cells in each phase. (e) Representative images of Fucci4 components in HeLa cells after synchronization and drug treatment. Scale bar, 10 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Tables 1 and 2, and Supplementary Note. (PDF 2545 kb)

Tracking of cell cycle phases by live-cell fluorescence microscopy using Fucci4 in human HeLa cells.

Cells expressing mKO2-Cdt1(30-120), Clover-Geminin(1-110), mTurquoise2-SLBP(18–126), and H1.0 Maroon1 were imaged through two cell divisions. The inferred cell cycle phase is indicated with a text label in the channel that is the most informative for identifying that phase. Timestamp shows time in hours:minutes. (MP4 3463 kb)

Tracking of cell cycle phases by live-cell fluorescence microscopy using Fucci4 in mouse NIH3T3 cells.

Cells expressing mKO2-Cdt1(30-120), Clover-Geminin(1-110), mTurquoise2-SLBP(18-126), and H1.0-Maroon1 were imaged through one cell division. The inferred cell cycle phase is indicated with a text label in the channel that is the most informative for identifying that phase. Timestamp shows time in hours:minutes. (MP4 10121 kb)

Tracking of cell cycle phases by live-cell fluorescence microscopy using Fucci4 in human HeLa cells.

Cells expressing mKO2-Cdt1(30-120), mTurquoise2-PCNA, Clover-Geminin(1-110), and H1.0-Maroon1 were imaged through two cell divisions. The inferred cell cycle phase is indicated with a text label in the channel that is the most informative for identifying that phase. Timestamp shows time in hours:minutes:seconds. (MP4 6800 kb)

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Bajar, B., Lam, A., Badiee, R. et al. Fluorescent indicators for simultaneous reporting of all four cell cycle phases. Nat Methods 13, 993–996 (2016). https://doi.org/10.1038/nmeth.4045

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