1. Volume 68, Issue 3, Pages 626-640.e5 (November 2017)
Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle 
Asako Sakaue-Sawano, Masahiro Yo, Naoki Komatsu, Toru Hiratsuka, Takako Kogure, Tetsushi Hoshida, Naoki Goshima, Michiyuki Matsuda, Hiroyuki Miyoshi, Atsushi Miyawaki 
Molecular Cell 
Volume 68, Issue 3, Pages e5 (November 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 68, 626-640.e5DOI: (10.1016/j.molcel.2017.10.001)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Expanded Repertoire of Fucci Probes with Different Ubiquitylation Domains of Human Cdt1
(A) Ubiquitylation regulation of human Cdt1 (hCdt1) by SCFSkp2- and CUL4Ddb1-mediated pathways. Truncated (mutated) constructs used for generating Fucci probes are illustrated below. The constructs are divided into four groups based on the intactness of the PIP box (red box) and the Cy motif (blue box). Gray box: RRL (68–70) are replaced with AAA in the Cy motif.
(B) Fucci(SA) consists of an SCFSkp2-sensitive hCdt1-based probe and an APCCdh1-sensitive hGem-based probe. Fucci(SA) corresponds to the original Fucci.
(C) Fucci(CA) consists of a CUL4Ddb1-sensitive hCdt1-based probe and an APCCdh1-sensitive hGem-based probe. This paper features Fucci(CA).
(D) Fucci(SCA) consists of an SCFSkp2/CUL4Ddb1-sensitive hCdt1-based probe and an APCCdh1-sensitive hGem-based probe. The ubiquitylation domains of Fucci(SCA) can be used as cell-cycle tags.
(B–D) Assuming that the hCdt1- and hGem-based domains are fused to red- and green-emitting FPs, their domain structures (top) and cell-cycle phasing capabilities (bottom) are shown. A theoretical temporal profile of the fluorescence intensity (F.I.) is shown below each domain structure. SCF, SCFSkp2; CUL4, CUL4Ddb1; APC, APCCdh1. 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.
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4. Figure 2
Characterization of Three Fucci Probes for Cell-Cycle Progression in HeLa Cells
(A–L) mCherry and mVenus were used as red and green FPs, respectively, to generate Fucci(SA)2 (A, D, G, J), Fucci(CA)2 (B, E, H, K), and Fucci(SCA)2 (C, F, I, L).
(A–C) Fucci fluorescence and DIC images were merged. These cells were all in an exponentially growing phase. Images were taken every 12.5 min, and each experiment spanned 96 hr. Temporal profiles of fluorescence intensities (F.I.) of mCherry and mVenus are indicated by red and green circles, respectively. M, mitosis. Scale bar, 10 μm. All data were derived from Movie S1.
(D–F) Interphase separation by Fucci reporters.
(G–I) Durations of cell-cycle phases were quantitatively measured for HeLa cells expressing Fucci reporters. The first and second cell cycles of ten cells were analyzed. Data are shown as means ± SD (n = 20).
(E and H) M∗: The duration of M phase was determined with Fucci(CA)2 by the time between NEB and NER. See Figure 3.
(J–L) Flow cytometry analysis. Cells showing red (mCherry[+]mVenus[−]), yellow (mCherry[+]mVenus[+]), and green (mCherry[−]mVenus[+]) fluorescence were gated for quantification of their DNA contents by staining with Hoechst C values denote DNA content as a multiple of the normal haploid genome.
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5. Figure 3
Single-Cell Tracking and Flow Cytometry Analysis of HeLa Cells Expressing Fucci(CA)
(A–C) Cell-cycle tracking of a HeLa cell stably expressing Fucci(CA)2. Sequential image numbers (Frame #) are indicated in (B) and (C).
(A) Images were acquired every 21.9 min. Upon mitosis, two daughter cells are labeled with gray and white arrowheads, and tracking of the latter cell is shown. Scale bar, 10 μm.
(B) Temporal profiles of the fluorescence intensities (F.I.) of mCherry (red circles) and mVenus (green circles). The precise boundary between S and G2 was determined at Frame #42.
(C) Difference in mVenus (mCherry) fluorescence distribution between Frames #2 and #3 indicates the occurrence of NEB. Likewise, a comparison of Frames #5 and #6 reveals the occurrence of NER. Spatial profiles of fluorescence intensity are shown below. Detectable NEB and NER are indicated by downward and upward pointing triangles, respectively, in (B). Scale bar, 10 μm.
(D) Snapshot imaging of a cell island. All four cell-cycle phases were clearly resolved. Scale bar, 10 μm.
(E and F) Flow cytometry analysis of DNA content and synthesis of HeLa cells stably expressing Fucci(CA)2.
(E) Cells showing red (mCherry[+]mVenus[−]), green (mCherry[−]mVenus[+]), and yellow (mCherry[+]mVenus[+]) fluorescence were analyzed for DNA content (DAPI) and synthesis (EdU).
(F) Cells with EdU(+) signals were analyzed on the basis of Fucci(CA)2 signals (mVenus versus mCherry).
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6. Figure 4
Visualization of Cell-Cycle Progression of Rapidly Dividing Mouse Embryonic Stem Cells by Fucci(CA)
(A) mESC/Fucci(CA)2.1: mESCs stably expressing Fucci(CA)2.1.
(B) Timetable of cell plating and time-lapse imaging. The medium was always supplemented with LIF. At 00:00, imaging was initiated after a medium exchange.
(C) Volume-rendered (VR) fluorescence images of Fucci(CA)2.1-expressing mESCs at the indicated times (hour:min). The imaged colony was composed of four cells at 00:00 and grown to a large mass at 36:00 as indicated by a DIC image. Fucci(CA)2.1 labels G1, S, and G2 phases with red, cyan, and white, respectively. Scale bars, 20 μm. All scale bars in the perspective VR images show the distance in the foreground plane.
(D) Temporal profiles of the fluorescence intensities (F.I.) of mCherry (red circles) and AmCyan (cyan circles) spanning one cell-cycle phase of the pointed cell (arrowhead in C). Detectable NEB and NER are indicated by downward- and upward-pointing triangles, respectively.
(E) Durations of cell-cycle phases (G1, S, G2, and M∗) and doubling time (DT∗) were measured for time-lapse-imaged mESCs. The first, second, and/or third cell cycles of two colonies were analyzed. M∗ and DT∗ durations were determined by using NEB and/or NER. Data are shown as means ± SD.
(F) 4D imaging of the colony (shown in C) with a high temporal resolution from 01:00 to 02:50. NEB, cytokinesis, NER, and extinction of red fluorescence were identified on the highlighted cell (C and D). Scale bars, 10 μm. All scale bars in the perspective VR images show the distance in the foreground plane.
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7. Figure 5 UV Sensitivity of S Phase Cells Revealed by Fucci(CA)
(A and B) Cell-cycle alterations in Fucci(CA)2-expressing HeLa cells after ultraviolet (UV) irradiation (24 J/m2).
(A) Top: Timetable of cell preparation, UV irradiation, and time-lapse imaging. Images were taken every 10.6 min. Middle: Eight representative cells exposed to UV irradiation at different cell-cycle phases. Horizontally compressed images are aligned. Scale bar (vertical), 10 μm. Fucci(CA)2 fluorescence and DIC images were merged. X indicates cell death. Bottom: Full images before and after the UV irradiation are shown. Scale bar, 10 μm.
(B) Histogram (fraction %) of occurrence of cell death (black bar) 48 hr after UV irradiation. A total of 180 cells were time-lapse imaged, of which 87, 46, 29, and 18 cells were exposed to UV at G1, S, G2, and M phases, respectively. As reference, 20 cells without UV irradiation were time-lapse imaged.
(C and D) Phosphorylation of H2A.X (γH2A.X) was examined by immunocytochemistry in Fucci(CA)2-expressing cells 15 min after UV irradiation (24 J/m2).
(C) Representative cells at G1, S, G2, and M phases. γH2A.X immunosignals (α-γH2A.X) and Fucci signals without (left) and with (right) UV irradiation are shown. γH2A.X immunosignals are shown with high and low thresholds. Overlay: Fucci and DIC images were merged. Scale bar, 10 μm.
(D) Fluorescence intensities (F.I.) of γH2A.X immunosignals were quantified for UV and cell-cycle dependencies. The number of analyzed cells is indicated in parentheses. Alexa Fluor 633 fluorescence intensity is shown as means ± SD. Statistical significance (∗p < 0.05, ∗∗p < 0.005) was examined by Bonferroni method.
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8. Figure 6 Cell-Cycle Phasing Capabilities of Fucci Technology
Cell-cycle regulations involving E3 ligase activities of CUL4Ddb1, SCFSkp2, and APCCdh1. Molecules whose intracellular concentrations or enzymatic activities change in a cell-cycle-dependent manner are shown in color. PCNADNA: DNA-bound PCNA.
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