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Genetically Encoded Tools for Optical Dissection of
the Mammalian Cell Cycle
Graphical Abstract
Asako Sakaue-Sawano, Masahiro Yo,
Naoki Komatsu, ..., Michiyuki Matsuda,
Hiroyuki Miyoshi, Atsushi Miyawaki
In Brief
Sakaue-Sawano et al. developed
Fucci(CA), a genetically encoded optical
sensor that monitors cell-cycle
interphase. It employs an S phasespecific CUL4Ddb1-mediated
ubiquitylation to sharply distinguish
major cell-cycle transitions and phases,
particularly G1, S, and G2. In the
repertoire of Fucci probes, Fucci(CA) is
ideal as an all-purpose, high-resolution
interphase sensor.
Fucci(CA) is a genetically encoded optical sensor for live cellcycle monitoring
The mechanism of Fucci(CA) requires S phase-specific
CUL4Ddb1 ubiquitylation
Fucci(CA) demarcates interphase with boundaries between
G1, S, and G2
Fucci(CA) visualized short G1 phases during rapid divisions
of embryonic stem cells
Sakaue-Sawano et al., 2017, Molecular Cell 68, 1–15
November 2, 2017 ª 2017 Elsevier Inc.
Please cite this article in press as: Sakaue-Sawano et al., Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle, Molecular Cell
Molecular Cell
Genetically Encoded Tools for Optical
Dissection of the Mammalian Cell Cycle
Asako Sakaue-Sawano,1 Masahiro Yo,1 Naoki Komatsu,1 Toru Hiratsuka,2,3 Takako Kogure,1 Tetsushi Hoshida,4
Naoki Goshima,5 Michiyuki Matsuda,2 Hiroyuki Miyoshi,6 and Atsushi Miyawaki1,4,7,*
for Cell Function Dynamics, BSI, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan
of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
3King’s College London Centre for Stem Cells and Regenerative Medicine, 28th Floor, Tower Wing, Guy’s Campus, Great Maze Pond, London
4Biotechnological Optics Research Team, Center for Advanced Photonics, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan
5Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Koutou
135-0064, Japan
6Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
7Lead Contact
Eukaryotic cells spend most of their life in interphase
of the cell cycle. Understanding the rich diversity of
metabolic and genomic regulation that occurs in
interphase requires the demarcation of precise
phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable realtime monitoring of interphase and cell-cycle biology.
We re-engineered the Cdt1-based sensor from
the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in
combination with SCFSkp2-mediated ubiquitylation.
In cultured cells, Fucci(CA) produced a sharp triple
color-distinct separation of G1, S, and G2, while
Fucci(SCA) permitted a two-color readout of G1
and S/G2. Fucci(CA) applications included tracking
the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UVirradiation damage in S phase. These results show
that Fucci(CA) is an essential tool for quantitative
studies of interphase cell-cycle regulation.
The ‘‘licensing’’ machinery of DNA replication origins ensures
faithful reproduction only once during the S phase of each cell
cycle (Arias and Walter, 2007; Blow and Laskey, 1988; Diffley,
1996; Masai et al., 2010; Nurse, 2000). To prevent over-replication in higher eukaryotes, the licensing factor Cdt1 (chromatin
licensing and DNA replication factor 1, Cdc10-dependent transcript 1) is inhibited by ubiquitin-mediated proteolysis and Geminin binding (Kim and Kipreos, 2007; Lee et al., 2004; Nishitani
et al., 2001; Wohlschlegel et al., 2000). Human Cdt1 (hCdt1) consists of 546 amino acids, and after entry into S phase, the middle
domain participates in binding to Geminin, whereas the N-terminal ubiquitylation domain (approximately 100 amino acids) is targeted for proteolysis by two E3 ubiquitin ligases. First, CUL4Ddb1
(Cullin 4, damage-specific DNA-binding protein 1) E3 ligase
recognizes six highly conserved amino acids in the PCNA interaction protein motif (PIP box) at the N terminus (approximately
10 amino acids) of hCdt1. This CUL4Ddb1-mediated proteolysis
occurs only in S phase or after DNA damage, because this ubiquitin ligase promotes the ubiquitylation of substrates only on the
DNA-bound fraction of PCNA (Havens and Walter, 2009; Ishii
et al., 2010; Nishitani et al., 2006; Roukos et al., 2011). Second,
SCFSkp2 E3 ligase targets the remainder of the ubiquitylation
domain, which is phosphorylated on Ser31 and/or Thr29, during
the S and G2 phases. These phosphorylations are catalyzed by
cyclin A-dependent kinases and require the cyclin-binding motif
(Cy motif) of hCdt1, Arg68-Arg69-Leu70 (Liu et al., 2004; Nishitani et al., 2006; Takeda et al., 2005).
The original Fucci (fluorescent, ubiquitination-based cell cycle
indicator) technology harnessed the cell cycle-dependent proteolysis of Cdt1 and Geminin (Sakaue-Sawano et al., 2008). Geminin, the inhibitor of Cdt1, is degraded under the control of
APCCdh1 (anaphase-promoting complex, Hct1/srw1/fizzyrelated CDC20 homologue 1) E3 ligase. Over the course of the
cell cycle, SCFSkp2 and APCCdh1 E3 ligase activities oscillate
reciprocally, and the protein levels of their direct substrates
oscillate accordingly. To label S/G2/M phase nuclei green, the
original Fucci probe had mAG (monomeric Azami-Green fluorescent protein) fused to the APCCdh1-mediated ubiquitylation
domain (1–110) of human Geminin (hGem) (Fucci-S/G2/M =
mAG-hGem[1/110]); this chimeric protein is the direct substrate
of APCCdh1 E3 ligase. On the other hand, to label G1 phase nuclei
red, the probe had mKO2 (monomeric Kusabira-Orange 2 fluorescent protein) fused to residues 30–120 of hCdt1 (Fucci-G1 =
mKO2-hCdt1[30/120]) (Figure 1A, [PIP()Cy(+)]). Because
hCdt1(30/120) contains Ser31 and the Cy motif, it can serve as
a direct substrate for SCFSkp2 E3 ligase. In contrast, due to the
truncation of 29 N-terminal amino acids, hCdt1(30/120) is not
degraded by the CUL4Ddb1-mediated pathway and is accordingly indifferent to DNA damage. Although a variety of color
Molecular Cell 68, 1–15, November 2, 2017 ª 2017 Elsevier Inc. 1
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Table 1. Terminology for Different Versions of Fucci
Fucci Type
hCdt1-Based Probe
hGem-Based Probe
Fucci (Sakaue-Sawano et al., 2008)
Fucci2 (Sakaue-Sawano et al., 2011)
(Nishimura et al., 2013)
FR and NIR FUCCI (Rodriguez et al.,
Reported Name (Reference)
Fucci4 (Bajar et al., 2016)
NIR cell cycle reporter (Shcherbakova
et al., 2016)
variants of Fucci, including Fucci2 containing mCherry and
mVenus (Sakaue-Sawano et al., 2011), were developed after
the initial publication, most of them carry hCdt1(30/120) and
can therefore be called Fucci(SA) (Figure 1B and Table 1),
because they monitor the balance between SCFSkp2 and
APCCdh1 E3 ligase activities.
Although Fucci(SA) highlights the G1-to-S phase transition
with yellow fluorescence, none of the existing probe designs provide a fluorescent readout for distinct interphase boundaries.
Also, Fucci(SA) has a fluorescence gap in very early G1 phase.
This gap makes it possible in principle to distinguish early G1
from middle/late G1 phases. For example, Pauklin and Vallier
(2013) used the original Fucci to discover that human embryonic
stem cells (hESCs) in early G1 phase could only initiate differentiation into endoderm, whereas hESCs in late G1 phase were
limited to neuroectoderm differentiation. However, the fluorescence gap generates three problems. First, it makes it difficult
to continuously track cell position in all phases of the cell cycle.
The gap may affect the efficiency of single-cell lineage tracking
due to substantial changes in the position and number of
observed cells at the start of G1 phase. Second, it is impossible
to distinguish non-fluorescent early G1 cells from non-transfected cells in samples with heterogeneity of Fucci transgene
expression. Third, most embryonic stem cells with high pluripotency, such as mouse embryonic stem cells (mESCs), transit in
G1 phase for a very short time, and G1 in such samples can
easily be missed.
We engineered the hCdt1-based probe to make it sensitive to
CUL4Ddb1 instead of, or in addition to, SCFSkp2. By combining
the resultant probe with hGem(1/110)-containing probes sensitive to APCCdh1, we developed Fucci(CA) (Figure 1C) and
Fucci(SCA) (Figure 1D) probes (Table 1) to increase the versatility
of Fucci technology for new biological studies of cell-cycle interphase regulation. Figures 1B, 1C, and 1D depict the domain
structures and cell-cycle color profiles of Fucci(SA), Fucci(CA),
and Fucci(SCA), which contain hCdt1(30/120), hCdt1(1/100)
Cy(), and hCdt1(1/100) fusions, respectively. hCdt1(30/120) is
a specific substrate of SCFSkp2 because it lacks the PIP box
but retains Ser31 and the Cy motif (PIP[]Cy[+]) (Figure 1B).
Since the first publication on Fucci, hCdt1(30/120) fused to
red-emitting fluorescent proteins (FPs) has been widely used
as a Fucci-G1 probe. In this study, hCdt1(1/100)Cy() was newly
constructed as a specific substrate of CUL4Ddb1; it retains the
PIP box but loses the function of the Cy motif (PIP[+]Cy[]) (Nishitani et al., 2006) (Figure 1C). Lastly, hCdt1(1/100) is targeted by
both SCFSkp2 and CUL4Ddb1 because it retains both the PIP box
and the Cy motif (PIP[+]Cy[+]) (Nishitani et al., 2006) (Figure 1D).
Although we previously observed delayed division of cells that
expressed mKO2-hCdt1(1/100) (Sakaue-Sawano et al., 2008),
the negative effect was due to an unintentional excess of probe
expression; we confirmed the normal proliferation of cells stably
expressing mCherry-hCdt1(1/100). Each of these hCdt1-based
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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probes can be combined with the hGem(1/110) fusion to create
a complete set of Fucci probes with distinct properties:
Fucci(SA), Fucci(CA), or Fucci(SCA). Figure S1 illustrates how
acquired images were processed for quantitative measurements
in this study.
Re-engineering Fucci for High-Resolution Dissection of
Interphase Boundaries
Figure 2 illustrates the performance of the three Fucci versions
containing mCherry and mVenus. We generated HeLa cells
that stably express Fucci(SA)2, Fucci(CA)2, and Fucci(SCA)2.
The suffix ‘‘2’’ indicates that mCherry and mVenus are used for
fluorescence labeling. We examined the temporal profiles of
the fluorescence intensities of mCherry and mVenus by singlecell tracking analysis under a light microscope (Figures 2A–2F,
Movie S1). We noted two major differences in cell-cycle phasing
between Fucci(SA)2 (Figures 2A and 2D) and Fucci(CA)2 (Figures
2B and 2E). First, whereas the mCherry-hCdt1(1/100)Cy()
signal decreased abruptly to zero upon entering S phase (emergence of mVenus-hGem[1/110]), the mCherry-hCdt1(30/120)
signal decreased slowly with a significant delay, yielding a yellow
fluorescence during the G1/S transition. Second, because
CUL4Ddb1-mediated proteolysis was active only during S phase,
the mCherry-hCdt1(1/100)Cy() signal emerged before mitosis.
As a result, G2-phase nuclei were labeled with yellow fluorescence, and after mitosis the nuclei were labeled with strong
red fluorescence throughout G1 phase. On the other hand,
both red and green signals of Fucci(SCA)2 decreased rapidly
and emerged gradually, and their oscillatory changes were
completely reciprocal, resulting in the absence of yellow signal
throughout the cell cycle (Figures 2C and 2F). Although
Fucci(CA)2 and Fucci(SCA)2 showed a small gap in fluorescence
at the G1/S transition, this was not problematic, because the
cell-cycle transition involves no drastic change in either the position or number of observed cells. We also created an algorithm
that tracked Fucci(SA)2-expressing HeLa cells through the gap
in early G1, and our automatic single-cell tracking system with
the algorithm made it possible to quantitatively measure the temporal profiles of more than 100 trackings for each fluorescence
component of Fucci reporters (Figures S1 and S2). The speed
of hCdt1(30/120) degradation by SCFSkp2 was characterized
with a time constant (t) of 28.6 min (Figure S2D, SCFOn). In
contrast, hCdt1(1/100)Cy() and hCdt1(1/100) were degraded
by CUL4Ddb1 more rapidly, with ts of 3.4 min (Figure S2E,
CUL4On) and 3.5 min (Figure S2F, SCFOn/CUL4On), respectively.
On the other hand, hGem(1/110) showed further faster kinetics of
degradation, with a t of 2.1 min (Figure S2G, APCOn). The rate
constants (ks) of both degradation and accumulation are summarized (Figure S2H). These statistical data demonstrate reproducibility of the temporal profiles shown in Figures 2 and S2,
allowing us to quantitate the differential speeds of degron degradation by SCFSkp2, CUL4Ddb1, and APCCdh1. Our quantitative
measurements revealed that only Fucci(CA)2 can accurately
measure the durations of all three cell-cycle interphases and
M phase (Figures 2G–2I).
Next, we investigated the cell-cycle monitoring behaviors of
Fucci(SA)2, Fucci(CA)2, and Fucci(SCA)2 by population analysis
(Figures 2J–2L). After staining with Hoechst 33342 for 30 min, the
cells were harvested and analyzed alive by flow cytometry.
Notably, the cells labeled with yellow fluorescence by
Fucci(SA)2 and Fucci(CA)2 had DNA content of 2–4 C and 4 C,
respectively. In summary, these three Fucci versions have
different cell-cycle phasing capabilities. Fucci(SA)2 effectively
highlights the transition process from G1 phase to S phase;
Fucci(CA)2 fully labels the G1 phase with sharp boundaries
and distinguishes S and G2 phases; and Fucci(SCA)2 clearly
separates the cell cycle into G1 and S-G2 phases.
Analysis of Cell-Cycle-Resolving Capabilities of
Fucci(CA) in Cultured Cells
In this study, we focused on Fucci(CA) and intensively investigated its performance using HeLa cells stably expressing
Fucci(CA)2. A typical series of differential interference contrast
(DIC) and Fucci(CA)2 fluorescence images is shown in Figure 3A.
Fucci(CA)2 labeled nuclei in G1, S, and G2 phases with red
(mCherry), green (mVenus), and yellow (mCherry+mVenus) fluorescence, respectively (Figure 3B). The remaining cell-cycle
phase, M, involves a drastic change in morphology, and is easily
identifiable by common bright-field microscopy techniques,
such as phase contrast (PC) and DIC. DIC images were used
in combination when cells and nuclei were manually tracked.
When DIC is merged with Fucci(CA)2 fluorescence, mitosis can
be monitored as the splitting of one round yellow object into
two red cells (Figure 3A, #4/#5, #50/#51). In addition, the
use of relatively high NA (numerical aperture) objectives empowers observation of Fucci(CA)2 fluorescence during M phase,
the duration of which is defined by the time between breakdown
and reformation of the nuclear envelope (NEB and NER) (Araujo
et al., 2016). The G2/M transition can be marked by the spread of
fluorescence into the cytoplasm due to NEB (Figure 3C, #2/#3);
the M/G1 transition can be marked by the sequestration of red
fluorescence into the nucleus due to NER (Figure 3C, #5/#6).
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 mm. 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 33342. C values denote DNA content as a multiple of the normal haploid genome.
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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 mm.
(legend continued on next page)
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Altogether, when an image-based approach is taken, Fucci(CA)2
can discriminate M from other phases and is thus capable of
resolving all four cell-cycle phases (Figures 2E and 2H). Notably,
the pan-cell-cycle resolving capability of Fucci(CA)2 was proved
in snapshot imaging (Figure 3D). G2 cells can be discriminated
from S cells by the presence of red fluorescence (Figure 3B),
and S/G2 resolution is clear at single time points.
To characterize S phase discrimination of Fucci(CA)2, we
further examined DNA synthesis as well as DNA content of
the stable transformant HeLa cells (Figures 3E and 3F). After incubation with 5-ethynyl-20 -deoxyuridine (EdU) for 15 min, the
cells were fixed and subjected to our original Click-iT reaction
for labeling with Alexa Fluor 647 (Sakaue-Sawano et al., 2013)
and finally stained with 4’,6-diamidino-2-phenylindole (DAPI).
We carried out multi-color flow cytometric analysis using
four laser lines (Table S1). Then we determined the correlation among the levels of hCdt1(1/100)Cy() (mCherry),
hGem(1/110) (mVenus), DNA synthesis (Alexa Fluor 647), and
DNA content (DAPI). The cells were analyzed on the basis of
Fucci(CA)2 signals, and three subpopulations were gated:
red, mCherry(+)/mVenus(); green, mCherry()/mVenus(+);
and yellow, mCherry(+)/mVenus(+) (Figure 3E, left). The DNA
contents of these subpopulations quantified by DAPI staining
were 2 C, 2–4 C, and 4 C, respectively (Figure 3E, middle),
consistent with the results obtained from Hoechst 33342stained live cells (Figure 2K). Then the short pulse labeling
with EdU showed that the green cells in the mCherry()/
mVenus(+) fraction indeed underwent DNA replication (Figure 3E, right). This observation was verified by analysis with
the gating order reversed (Figure 3F).
Remarkably, G2 cells can be distinguished from S cells qualitatively by the presence of the mCherry-hCdt1(1/100)Cy()
signal. Very recently, Bajar et al. (2016) used a degron of
SLBP (SLBP[18–126]) in combination with a Fucci(SA) probe
to distinguish S and G2 phases in their version, termed Fucci4.
It is noted, however, that the SLBP degron is degraded
gradually, in contrast to the rapid and full degradation of
hCdt1(1/100)Cy() and hGem(1/110). We attempted stable
expression of mTurquoise-SLBP(18–126) in conjunction with
Fucci(CA)2 in HeLa cells. Time-lapse imaging and quantitative
measurements confirmed the slow decline of mTurquoise fluorescence during G2 phase (Figures S3A and S3B), suggesting
that multiple images (measurements) at different time points
are required to retrospectively detect the S/G2 transition.
Then we used the HeLa transformant cells for flow cytometry
analysis and pharmacological snapshot imaging. We observed
mTurquoise-SLBP(18–126) signals in all interphase cell-cycle
phases (Figure S3C) and both S- and G2-arrested cells (Fig-
ure S3D), indicating that the mTurquoise readout cannot be
used for separating S and G2 phases in single-time-point
Tracking the G1 Phase in Rapidly Dividing mESCs
mESCs self-renew in a naive state of pluripotency and proliferate
very rapidly (Pauklin et al., 2011; Stead et al., 2002; White and
Dalton, 2005). Thus, mESCs exhibit unusual cell-cycle regulation
characterized by a short G1 phase. Furthermore, mESCs divide
and move in a 3D culture space (colonies on feeder cell layers).
To test if Fucci(CA) can be used to follow cell-cycle progression
under these complex, dynamic conditions, we performed 4D imaging experiments using mESCs (Oikawa et al., 2013) expressing a color variant of Fucci(CA) (Figure 4). To enable tracking of
differentiation state as well as proliferation of mESCs and utilize
differentiation markers with yellow-emitting FPs, we generated
Fucci(CA)2.1 containing AmCyan fluorescent protein (AmCyan)
instead of mVenus (Nishimura et al., 2013) (Figure 4A).
Fucci(CA)2.1-expressing mESCs on feeder cell layers proliferated reasonably quickly in the presence of leukemia inhibitory
factor (LIF), and we imaged cells in 4D in a colony with 10-min intervals for 36 hr (Figure 4B, Movie S2). During the imaging time
period, all imaged cells cycled approximately three times with
a similar doubling time. Fucci(CA)2.1 labeled mESC nuclei in
G1, S, and G2 phases with red (mCherry), cyan (AmCyan), and
white (mCherry+AmCyan) fluorescence, respectively (Figure 4C).
After tracking individual nuclei manually, we made quantitative
measurement of their cell-cycle progression. For example, the
first cell cycle of a given cell (arrowhead in Figure 4C) is highlighted in Figure 4D. It was shown that Fucci(CA)2.1 detects a
short G1 phase reliably and distinguishes S and G2 phases.
Other typical examples from the experiment are shown (Figure S4A). We quantified durations of all cell-cycle phases for
mESCs that grew in two colonies (Figure 4E). They were found
to proliferate with a doubling time of 596 ± 79 min (n = 24). Importantly, since Fucci(CA)2.1 demarcates the G1 phase sharply, we
were able to determine its duration precisely (62 ± 14 min, n = 43)
with a relatively short sampling interval (10 min). A full series of
images during the G1 phase (Figure 4F) indicates the significance of increasing both the spatial and temporal resolution of
this 4D imaging procedure.
We also generated Fucci(SA)2.1 and compared the G1-detecting capability between Fucci(CA)2.1 and Fucci(SA)2.1 (Figure S4B). In one experiment, mESCs expressing Fucci(CA)2.1
and Fucci(SA)2.1 in colonies were imaged for 63.5 hr after the
final addition of LIF. They proliferated at the same rate, with a
doubling time of approximately 16 hr. In mESCs/Fucci(SA)2.1,
mCherry fluorescence indicating G1 phase could not be
(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 mm.
(D) Snapshot imaging of a cell island. All four cell-cycle phases were clearly resolved. Scale bar, 10 mm.
(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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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 mm. 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 mm. All scale bars in the perspective VR images show the distance in the foreground plane.
detected (Figure S4B, top). Accordingly, it was difficult to track
the lineages of single cells. At time points later than 50 hr, however, the proliferation rate decreased due to LIF depletion, and
mCherry fluorescence gradually became detectable. By
contrast, Fucci(CA)2.1 always labeled mESCs in the short G1
phase with mCherry fluorescence, enabling efficient single-cell
lineage tracking (Figure S4B, bottom).
8 Molecular Cell 68, 1–15, November 2, 2017
Sensitivity of Fucci(CA) Signals to UV Light
Because the CUL4Ddb1-mediated pathway is activated not only
in S phase but also after DNA damage, we examined to what
extent the red fluorescence of Fucci(CA)2 would be affected by
UV irradiation that causes DNA damage. In conventional studies,
UV irradiation involves aspiration of culture medium, after which
the cultured cells are directly exposed to 20–50 J/m2 UV light
Please cite this article in press as: Sakaue-Sawano et al., Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle, Molecular Cell
(254 nm) from a UV crosslinker. In this study, however, with the
goal of understanding the effects of UV in a healthy state, we
UV-irradiated cultured cells in normal culture medium (Figure S5A). To determine how much UV light was transmitted
through the medium (2 mL in a glass-bottom dish with a diameter
of 34 mm), we used the surface-enhanced third-harmonic generation (THG) process (Tsang, 1995). Through an objective lens, a
focused beam of an ultrashort pulse laser (762 nm) was administered on the upper surface of a polycrystalline ceramics-based
coverslip that had been coated with a fluoropolymer. At the ceramics/polymer interface, THG-light (254 nm) was generated,
and its intensity was measured over the medium (Figure S5B).
Our measurement revealed approximately 20% transmission
of 254-nm UV light through normal culture medium. Accordingly,
to obtain an effective energy of 24 J/m2, for example, we set the
UV crosslinker energy level to 120 J/m2. Hereafter we use effective energy levels to indicate UV exposure doses.
To survey UV effects, Fucci(CA)2-expressing HeLa cells were
exposed to UV (24 or 80 J/m2) and time-lapse imaged for a few
days (Figure S5C, Movie S3). Previous biochemical studies (Hu
et al., 2004; Nishitani et al., 2006) showed that the majority of
endogenous Cdt1 was degraded promptly after UV irradiation,
and that the degradation lasted for at least 2 hr. Therefore, to
examine the effects of UV on Fucci(CA)2 signals, rather than
cell viability, flow cytometry analysis was performed 2 hr
post-UV irradiation (Figure S5D). No appreciable cell death
was observed with 24 or 80 J/m2 UV irradiation in the early stage
(2 hr). Interestingly, the degradation of mCherry-hCdt1(1/100)
Cy() was more prominent in G1 than G2 phases (Figure S5D).
In G1 cells, substantial degradation of the mCherry-containing
probe was detected at 24 J/m2 UV, and the degradation degree
was greater at 80 J/m2 UV. In G2 cells, however, less degradation was observed with both 24 and 80 J/m2 UV. The time-lapse
imaging (Figure S5C, Movie S3) revealed different cell responses
to 24 and 80 J/m2 UV in the late stage (later than 12 hr). At
24 J/m2 UV, cell proliferation was clearly slowed, but mCherry
fluorescence gradually recovered, whereas at 80 J/m2 UV, the
majority of cells died.
Differential Sensitivity to UV between S and G2 Phases
Because 24 J/m2 UV does not cause severe cell death and is in
the range employed in common UV irradiation experiments, we
used this energy level to study UV-induced cell-cycle alterations.
Since our protocol had no medium removal/exchange, we were
able to perform UV irradiation during time-lapse imaging over
short time intervals. Thus, we could precisely determine the
cell-cycle phase at which each cell was exposed to the UV light
(Figure 5A). We then followed individual cells in growing phase by
their Fucci(CA)2 fluorescence signals for 2 days (Figure 5A). We
also increased the number of cells UV-irradiated/time-lapse
imaged (Figure S6). While the red fluorescence from mCherry
was decreased substantially and transiently 1–2 hr after UV irradiation during G1 phase, the fluorescence was less sensitive to
UV irradiation during G2 phase, consistent with the flow cytometry data (Figure S5D).
Prolongation of G1, S, or G2 phase was observed for all of the
tracked cells, but cell-cycle arrest occurred mostly in S phase
(Figure S6B). Remarkably, cell death was most prominent for
cells that experienced UV irradiation at S phase; fewer than
10% of the cells survived 2 days after UV irradiation (Figures
5B and S6B). Vulnerability of cells to UV irradiation differed
significantly between S and G2 phases.
Next, we studied how cellular responses to UV irradiation vary
with the cell cycle. Previous studies showed that UV irradiation
induces redistribution of phosphorylation of histone H2A.X on
Ser139 (gH2A.X), which ranges from foci to high-level uniform
pan-nuclear staining (de Feraudy et al., 2010; Marti et al.,
2006), depending on the cell-cycle phase. To examine early formation of gH2A.X, we fixed cells in all four cell-cycle phases
15 min after UV irradiation (24 J/m2) and assessed the integrated
intensity of immunofluorescence signals (Figures 5C, 5D,
and S7). In these endpoint experiments, G1, S, G2, and M phases
were clearly identified (Figure S7B), as was evidenced by snapshot imaging (Figure 3D). Quantitative analysis with a high dynamic range revealed that UV irradiation caused the greatest
gH2A.X formation at S phase; the staining appeared to be pannuclear with a low threshold, but was composed of a large
number of foci of different sizes and intensities (high threshold).
A significant but moderate increase in gH2A.X formation upon
UV irradiation was also observed in G2 cells. By contrast, for
both endogenous and UV irradiation-induced gH2A.X formation,
the lowest levels of staining were observed in G1 cells.
New Fucci-Based Imaging Tools for Resolving CellCycle Interphase
The original version of Fucci, Fucci(SA), highlights the cell-cycle
transition from G1 to S phase with high color contrast, like a
traffic signal; red and green mean ‘‘stop’’ and ‘‘go,’’ respectively,
for the transition (Sakaue-Sawano et al., 2008). Fucci probes
have since diversified in color (Table 1); for example, replacement of green fluorescence with cyan allows Fucci compatibility
with GFP imaging (Nishimura et al., 2013). Very recently, far-red
or near-infrared FPs have been substituted in order to generate
Fucci variants for intravital deep imaging (Rodriguez et al.,
2016; Shcherbakova et al., 2016). However, visualizing cell-cycle
transitions other than G1/S remains important (Zielke and Edgar,
2015). Therefore, we re-engineered a new version, Fucci(CA), to
distinguish clear interphase boundaries between G1, S, and G2
phases. Fucci(CA) monitors the balance between CUL4Ddb1 and
APCCdh1 E3 ligase activities. Considering its simple principle of
operation and reproducibility compared to alternate probes,
Fucci(CA) sets a new standard for cell-cycle monitoring methods
(Table S2). In this study, we used Fucci(CA) to fully highlight the
short G1 phase of rapidly proliferating mESCs and, in general, to
continuously track cell position in all phases of the cell cycle. We
also used Fucci(CA) to examine S phase sensitivity to UV irradiation. It will be interesting to use this Fucci variant to explore
G2-specific intracellular signaling (Krenning et al., 2014; Naetar
et al., 2014).
Cell-Cycle Phasing Capabilities of Fucci Probes
In principle, genetically encoded probes for cell-cycle progression exploit either translocation or degradation of proteins that
occurs in a cell-cycle-dependent manner. Translocation-based
Molecular Cell 68, 1–15, November 2, 2017 9
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(legend on next page)
10 Molecular Cell 68, 1–15, November 2, 2017
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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.
This terminology might mislead readers
into believing that Fucci contains the
entire protein of Cdt1 or Geminin.
probes require high (subcellular)-resolution observation and are
therefore not amenable to flow cytometry analysis or low-spatialresolution intravital imaging. In contrast, Fucci technology harnesses the cell-cycle-dependent degradation of human Cdt1
and Geminin (Figure 6). We rigorously screened truncated
mutants of these two proteins to obtain the best performance,
and our original Fucci probes contained two ubiquitylation domains: hCdt1(30/120) and hGem(1/110). Because the Fucci
probes monitor the balance between SCFSkp2 and APCCdh1 E3
ligase activities, they are called Fucci(SA), and we extensively
evaluated performance by both single-cell tracking analysis
(time-lapse imaging) and population single-time-point analysis
(flow cytometry) (Sakaue-Sawano et al., 2008, 2011, 2013).
Importantly, our evaluation always included BrdU/EdU incorporation analysis that directly monitors DNA replication after short
pulse (15 min) labeling. In addition, we showed that sufficient
levels of expression of Fucci(SA) probes do not perturb the
cell-cycle regulation of recipient cells; their inertness was
demonstrated most directly by successful generation and maintenance of several transgenic mouse lines that constitutively express the probes, including tandem Fucci (tFucci) (Table S3)
(Mort et al., 2014; Sakaue-Sawano et al., 2008; Yo et al., 2015;
Zielke and Edgar, 2015).
Unfortunately, an increasing number of papers disregard
the ubiquitylation domain information (hCdt1[30/120] and
hGem[1/110]) of the Fucci(SA) probes. Those papers describe
mKO2-Cdt1/mAG-Geminin and mCherry-Cdt1/mVenus-Geminin as the two components of Fucci and Fucci2, respectively.
Resolving All Four Cell-Cycle
Fucci(CA) probes can effectively resolve
G1, S, and G2 phases in time-lapse imaging (Figures 2B, 2E, and 3A–3C), snapshot
imaging (Figure 3D), and flow cytometry (Figures 2K, 3E, and 3F).
M phase can be identified by common bright-field microscopy
techniques. Nevertheless, all four cell-cycle phases can be
resolved merely by fluorescence readouts (Table S2). To develop
cell-cycle-dependent biosensors, Hahn et al. (2009) used proliferating cell nuclear antigen (PCNA) and the C terminus of human
DNA helicase B (HDHB) as an FP fusion. Recently, Araujo et al.
(2016) studied duration control of each cell-cycle phase in single
cells expressing hCdt1(30/120)-YFP and PCNA-mCherry. The
length of M was defined as the time between NEB and NER
that could be detected by fluorescence redistribution of mCherry
and YFP, respectively. The length of G1 was monitored by the
appearance and disappearance of the YFP fluorescence; the
length of S was defined as the time between the appearance
and disappearance of nuclear speckles (mCherry); and the
length of G2 was defined as the time between disappearance
of the nuclear speckles and NEB. However, these two approaches require high spatial resolution observation and cannot
be assessed by flow cytometry. In addition, although the PCNA
foci are well documented, their reliability as an S phase marker
remains controversial (Johnson et al., 2016).
The beginning and end of M phase are detected by Fucci(CA).
On the one hand, NEB can be identified by redistribution of the
hCdt1- and hGem-based probes (Figure 1C), and this applies
to both Fucci(SA) and Fucci(SCA) (Figures 1B and 1D). On the
other hand, NER can be identified by redistribution of the
hCdt1-based probe, which is specific to Fucci(CA) (Figure 1C).
With a reasonably high NA objective, therefore, resolving all
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 mm. 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 mm.
(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 (gH2A.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. gH2A.X immunosignals (a-gH2A.X) and Fucci signals without (left) and with (right) UV irradiation are shown.
gH2A.X immunosignals are shown with high and low thresholds. Overlay: Fucci and DIC images were merged. Scale bar, 10 mm.
(D) Fluorescence intensities (F.I.) of gH2A.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.
Molecular Cell 68, 1–15, November 2, 2017 11
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four cell-cycle phases is possible in principle in Fucci(CA)-expressing cells. However, in this study our focus was on developing a tool for reliably resolving the rich metabolic and genomic
regulation of interphase G1, S, and G2 phases. In fact, the study
by Araujo et al. (2016) found that the durations of G1, S, and G2
phases are quite variable, whereas the duration of M phase is
short and constant. Considering that Fucci(CA) employs only
two FPs for distinguishing interphase boundaries between G1,
S, and G2 phases, it is expected that this probe can be easily
combined with a CyclinB1-based probe (Santos et al., 2012) of
a different color to resolve all four cell-cycle phases more clearly.
Bajar et al. (2016) reported the development of Fucci4, which
could resolve all four cell-cycle phases (Tables 1 and S2). First,
the original Fucci (Fucci[SA]) was modified slightly by substituting Clover for mAG. The modified Fucci(SA) was then combined
with mTurquoise2-SLBP(18–126) and H1.0-mMaroon1. In
addition to the G1/S transition reported by Fucci(SA), the S/G2
transition could be followed by the two intensiometric reporters,
Clover-hGem(1/110) and mTurquoise2-SLBP(18–126). Because
the cyan fluorescence from mTurquoise2 decreases gradually
during G2 phase, however, the S/G2 transition can be identified
only retrospectively by time-lapse imaging. Accordingly, Fucci4
cannot clearly discriminate S and G2 phases in single-time-point
observations (Figure S3 and Table S2), such as flow cytometry
and snapshot imaging, techniques that are essential for highcontent analysis, high-throughput screening, and in vivo highresolution cell-cycle profiling such as in xenograft tumors (Chittajallu et al., 2015). Furthermore, although the G2/M and M/G1
transitions can be followed in Fucci4 based on the fluorescence
of mMaroon1 on chromatin, M phase imaging requires high
spatial resolution to discern chromatin condensation, alignment,
and separation, and therefore cannot be combined with flow
cytometry analysis.
Application of Fucci(CA) to Pluripotent Stem Cell Biology
The cell cycles in pluripotent primitive cells of embryonic origin,
such as mESCs, often progress very rapidly. Previous studies
based on ensemble endpoint measurements, such as flow cytometry, determined the cell-cycle length of mESCs to be
approximately 11 hr (Stead et al., 2002), and according to a review by Pauklin et al. (2011) it is 10–14 hr. On the other hand, a
more recent study by Nakai-Futatsugi and Niwa (2016) using
single-cell time-lapse imaging with Fucci(SA) found significant
variations of mESC cell-cycle length, ranging from 10 to
30 hr. When placed inside a tiny stage-top incubator for fluorescence imaging, however, it may be natural that continuously
imaged mESCs exhibit different behaviors. In this study, nevertheless, we successfully minimized observation-derived effects
in time-lapse imaging to let mESCs proliferate homogenously
quickly, with a cell-cycle length of 9.9 ± 1.3 hr (n = 24) (Figure 4E). Remarkably, we used Fucci(CA) to quantify their G1
phase duration to be 1.0 ± 0.2 hr (n = 43) (Figure 4E). Because
the dead time of Fucci(SA) for detecting G1 is 2 hr, this kinetic
limitation of the conventional Fucci probes is a serious limitation for studying the cell-cycle dynamics of fast-dividing
mESCs with full pluripotency; in consequence, the use of
Fucci(CA) with shorter sampling intervals is the only solution
to this rapid biological timing issue.
12 Molecular Cell 68, 1–15, November 2, 2017
mESCs are derived from the inner cell mass of late pre-implantation embryos. Pluripotent cells of late pre-implantation and
early post-implantation embryos exhibit rapid division rates,
with a cell-cycle length of approximately 10 hr (Stead et al.,
2002). Moreover, pluripotent cells in the embryonic epiblast are
thought to divide much faster, spending most of the time in S
phase. Thus, unlike current cell-cycle sensors, the Fucci(CA)
technology will be useful for exploring transient G1 phase regulation during embryogenesis. Because a unique and sometimes
variable dynamics of cell-cycle regulation is associated with pluripotency, Fucci(CA) reporters should be used in combination
with a variety of differentiation markers. Currently existing
marker constructs contain either GFP or YFP and can be combined with Fucci(CA)2.1.
Application of Fucci(CA) to Detect S Phase Sensitivity to
UV Irradiation
Cell responses to UV irradiation vary, and this may reflect differential regulation in DNA repair, cell-cycle checkpoints, and cell
death. Using population and time-lapse imaging analyses of
HeLa cells expressing Fucci(CA)2, we examined the diversity
of the cell-cycle alterations induced by UV irradiation (24 J/m2).
UV-induced, CUL4Ddb1-mediated degradation of mCherryhCdt1(1/100)Cy() was obvious during G1 phase, but only faint
during G2 phase. Although intact (endogenous) Cdt1 is mostly
absent in G2 phase, due to its degradation by SCFSkp2-mediated
pathway, it is possible that hCdt1(1/100)Cy() during G2 phase
is tolerant of the DNA damage-induced degradation. Despite
the effects of UV on Fucci(CA)2 signals, however, cell-cycle
progression was practically trackable (Figures 5A and S6). In
particular, cell-cycle observation during S and G2 phases was
not perturbed by UV irradiation. Accordingly, we were able to
discriminate between S and G2 phases to find that UV irradiation
caused the greatest gH2A.X formation at S phase (Figures 5C
and 5D) and that UV exposure at S phase caused most
(90%) cells to die (Figures 5A, 5B, and S6).
Fucci Variants for Cell-Cycle Tags
Activation-induced deaminase (AID) deaminates cytosines in
DNA to initiate immunoglobulin gene diversification and reprogram CpG methylation in early development. AID undergoes
nuclear degradation more slowly in G1 than in S/G2/M phase
(Le and Maizels, 2015). To examine the role of cell-cycle regulation, Fucci(SA) probes were used for fusion to AID. AID-mCherryhCdt1(30/120) was found to be more effective in accelerating
somatic hypermutation and class-switch recombination than
AID-mCherry or AID-mCherry-hGem(1/110) (Le and Maizels,
2015). However, the overlapping presence of hGem(1/110) and
hCdt1(30/120) at the G1/S transition should diminish the contrast
between the G1- and S/G2/M-specific tags. Accordingly, we
believe that the fully reciprocal cell-cycle-sensing domains of
Fucci(SCA) (hCdt1[1/100] and hGem[1/110]; Figures 2C, 2F, 2I,
and 2L) could be used as comparable cell-cycle tags.
CRISPR/Cas9 induces DNA double-strand breaks (DSBs),
which are repaired by homology-directed repair (HDR) or nonhomologous end-joining (NHEJ). HDR activity is virtually absent
during G1 phase, increases sharply in S phase, and begins to
decrease by G2/M, whereas NHEJ is active throughout the cell
Please cite this article in press as: Sakaue-Sawano et al., Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle, Molecular Cell
cycle. It was speculated that the absence of Cas9 in G1 would
reduce DNA DSBs that would invariably be repaired by errorprone NHEJ. Fusion of Cas9 to the N-terminal region of human
Geminin (Cas9-hGem[1/110]) increases the rate of HDR up to
1.87-fold relative to wild-type Cas9 (Gutschner et al., 2016).
Another study verified that Cas9-hGem(1/110) minimizes
the formation of undesirable indels induced by NHEJ compared
to wild-type Cas9 or Cas9-hCdt1(30/120) (Howden et al., 2016).
In this case, hCdt1(30/120) could be replaced with hCdt1(1/100)
for a clearer comparison.
Cell-cycle tags, such as Fucci(SCA) probes or their cell-cyclesensing domains, are expected to be useful for studies of DNA
repair/recombination and RNA biogenesis for the purpose of
genome engineering.
First, although Fucci(CA) probes can effectively dissect phases
within mammalian interphase, the S/G2 boundary is less
distinct than G1/S boundary. While the G1/S transition is characterized by a rapid and complete decline of hCdt1(1/100)Cy()
signal as well as a gradual emergence of hGem(1/110) signal,
the S/G2 transition is defined by the emergence of an
hCdt1(1/100)Cy() signal. Although S/G2 separation can be
made qualitatively at any time, G2 cells at earlier phases are
harder to identify than at later phases. Second, because
Fucci(CA) probes use the CUL4Ddb1-mediated pathway, the intensity of hCdt1(1/100)Cy() signal may be reduced by stimulations that cause DNA damage, such as strong UV irradiation.
However, this influence is transient and observed principally
during the G1 phase and, accordingly, does not affect Fucci(CA)
cell-cycle phasing capabilities. Finally, the original Fucci,
Fucci(SA), cannot be fully replaced with Fucci(CA); they should
be used complementarily and/or combined with different
colors. As the repertoire of Fucci probes is expanded, users
should be aware of their operational principles, including these
limitations, to choose the best combinations.
Detailed methods are provided in the online version of this paper
and include the following:
B Gene Construction
B Cell Culture
B Lentivirus Production
B Flow Cytometry
B Long-term Time-lapse Imaging
B Automated Cell Tracking
B Numerical Analysis
B Confocal Imaging
B Manual Cell Tracking
B EdU Incorporation
B UV Irradiation
B Surface-Enhanced Third-Harmonic Generation
B Immunocytochemistry
Supplemental Information includes seven figures, three tables, and three
movies and can be found with this article online at
A.S.-S. and A.M. conceived and designed the study. A.S.-S. performed all the
experiments, analyzed the data, and designed the manuscript. M.Y. and H.M.
prepared Fucci-expressing mESCs and imaged them. N.K., T. Hiratsuka, and
M.M. created a software platform for quantitative Fucci analysis. T. Hoshida
performed THG experiments. T.K. performed gene construction and immunocytochemistry. N.G. performed cDNA cloning. A.M. designed and wrote the
manuscript and supervised the project.
The authors thank K. Ohtawa and RIKEN BSI-Olympus Collaboration Center
(BOCC) for technical assistance, Drs. K. Midorikawa and H. Kawano for valuable advice on THG imaging, Drs. M. Hirose and A. Ogura for providing mESCs
(BRC6), and Dr. C. Yokoyama for manuscript editing. This work was supported
in part by grants from the Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research on Innovative Areas:
Resonance Bio (15H05948) and Living In Space (16H01653), the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS)
(17dm0207001h0004) and AMED-CREST (15664816) from Japan Agency for
Medical Research and Development, AMED, and the Uehara Memorial Foundation (F1506004).
Received: March 31, 2017
Revised: August 16, 2017
Accepted: September 29, 2017
Published: October 26, 2017
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Anti-phospho-Histone H2A.X (Ser139) Antibody
Merck Millipore
clone JBW301; RRID: AB_310795
Alexa Fluor 633-conjugated goat anti-mouse IgG
Molecular Probes
Neurobasal medium
N2 supplement
B27 supplement
Chemicals, Peptides, and Recombinant Proteins
recombinant human LIF
Cat. E1383
Hoechst 33342
EdU 5-ethynyl-20 -deoxyuridine
Life Technologies
Life Technologies
Ceramic Y2O3
Kong et al., 2015; CoorsTek
Transparent Y2O3 ceramics
Teflon AF1600
Lowry et al., 1992; DuPont
Teflon AF 1600
Sakaue-Sawano et al., 2011
DDBJ/EMBL/GenBank database (AB512478)
Sakaue-Sawano et al., 2011
This paper
This paper
This paper
This paper
This paper
This paper
This paper
This paper
Original, unprocessed data
This paper; Mendeley Data
HeLa cells, a subclone of HeLa.S3
Sakaue-Sawano et al., 2008,
2011; this paper
RIKEN, Mikoshiba Lab
mESC (BRC6), mouse embryonic stem cell line
Oikawa et al., 2013; this
RIKEN, BRC BioResource Engineering Division
HeLa/Fucci(CA)2 (clone #3)
This paper
(BioResource Center Cell Bank) (http://cell.brc.
Deposited Data
Experimental Models: Cell Lines
(Continued on next page)
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CyAAA-F: 50 -cag gcc agg cca ccg gcc cgc gcg
gcc gcg cgg ctg tcg gtg gac gag gtt tcc
This paper
CyAAA-R: 50 -gga aac ctc gtc cac cga cag ccg cgc
ggc cgc gcg ggc cgg tgg cct ggc ctg
This paper
pCSII-EF-MCS vector
Miyoshi et al., 1997
pCAG-HIVgp packaging plasmid
Miyoshi et al., 1997
pCMV-VSV-G-RSV-Rev VSV-G-/Rev-expressing
Miyoshi et al., 1997
FlowJo software
Tree Star
Molecular Devices
Windows Live Movie Maker
ImageJ, FIJI
FIJI plugin, Trackmate
Schindelin et al., 2012
Excel software
Microsoft Corporation
MATLAB software
Imaris software
Recombinant DNA
Software and Algorithms
Homepage for Fucci-related materials
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Atsushi
Miyawaki (
Gene Construction
cDNA encoding mCherry-hCdt1(30/120) [DDBJ/EMBL/GenBank data-base AB512478] and mVenus-hGem(1/110) [AB512479] were
used for Fucci(SA)2 (Sakaue-Sawano et al., 2011). DNA fragments encoding hCdt1(1/100) and hCdt1(1/100)Cy(-) were amplified using primers containing 50 -XhoI and 30 -XbaI sites, and digested products were substituted for the hCdt(30/120) gene in mCherryhCdt1(30/120) in pcDNA3 vector. Likewise, a DNA fragment encoding SLBP(18–126) (Goshima et al., 2008) was amplified, digested,
and substituted for the hGem(1/110) gene in mTurquoise-hGem(1/110) in pcDNA3 vector. The entire construct was transferred to
pCSII-EF vector using EcoRI and XbaI sites (Sakaue-Sawano et al., 2008). For introduction of CyAAA mutation, the following complementary oligonucleotide primers containing NotI site were used. CyAAA-F: 50 -cag gcc agg cca ccg gcc cgc gcg gcc gcg cgg ctg
tcg gtg gac gag gtt tcc; CyAAA-R: 50 -gga aac ctc gtc cac cga cag ccg cgc ggc cgc gcg ggc cgg tgg cct ggc ctg. The sequence of
mCherry-hCdt1(1/100)Cy(-) and mCherry-hCdt1(1/100) have been deposited in the DDBJ/EMBL/GenBank data-base [LC171208]
and [LC192885]. A tFucci probe was constructed by concatenating the hCdt1-based probe, P2A sequence (Fang et al., 2005;
Kim et al., 2011), and the hGem-based probe using In-Fusion cloning system. The accession numbers in the DDBJ/EMBL/GenBank
data-bases are [LC192886] for tFucci(SA)2, [LC192887] for tFucci(SA)2.1, [LC192888] for tFucci(SA)2.2, [LC212776] for tFucci(CA)2,
[LC212777] for tFucci(CA)2.1, and [LC212778] for tFucci(CA)2.2.
Cell Culture
HeLa cells (a subclone of HeLa.S3) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) and penicillin/streptomycin. HeLa.S3 has been characterized to proliferate relatively fast with a doubling time of 15–18 hr
(Sakaue-Sawano et al., 2008) (see Figures 2G-2I). A mouse embryonic stem cell line (mESC) (BRC6) (Oikawa et al., 2013) was obtained
from BioResource Engineering Division, BioResource Center, RIKEN. This cell line was well validated to form teratomas
and express endogenous pluripotent markers. The mESCs were plated onto mitomycin-C-treated MEF cells at a concentration of
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4 3 104 cells/cm2 on the 0.1% gelatin-coated dish. The culture medium of mESCs was generated by including 48% DMEM/F12
(Invitrogen), 48% Neurobasal medium (Invitrogen), 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 10 ng/ml recombinant human LIF (WAKO), 2 mM L-Glutamine (Invitrogen), 0.1 mM NEAA (Invitrogen), 0.1 mM 2-Mercaptoethanol (Sigma), 5 mg/mL
BSA (Sigma), 1 mM PD0325901 (WAKO), and 3 mM CHIR99021 (WAKO). Passage of mESCs was performed using 0.25% TrypsinEDTA (Invitrogen). Etoposide (Cat. E1383) and Bleomycin (ab142977) were purchased from Sigma and abcam, respectively.
Lentivirus Production
Replication-defective, self-inactivating lentiviral vectors were used (Miyoshi et al., 1997). The pCSII-EF-MCS vector encoding a Fucci
probe was co-transfected with the packaging plasmid (pCAG-HIVgp) and the VSV-G-/Rev-expressing plasmid (pCMV-VSV-G-RSVRev) into 293T cells. High-titer viral solutions were prepared and used for transduction into HeLa cells (MOI = 1 – 10) or mESCs
(MOI = 50 – 100). Stable transformants were established by 2-step FACS with both green and red channels and by diluting cells
for single-cell cloning.
Flow Cytometry
Hoechst 33342 solution (56 mL 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) (Table S1).
mVenus was excited by a 488-nm laser line (laser diode) and its emission was collected through 530/30BP; mCherry was excited by a
561-nm laser line and its emission was collected through 610/20 BP; mTurquoise was excited by a 445-nm laser line and its emission
was collected through 525/50 BP. Hoechst 33342 was excited by a UV Laser at 355 nm, and its emission was collected through
450/50 BP. Alexa Fluor 647 was excited by a 640 nm laser line and its emission was collected through 670/30 BP. The data were
analyzed using FlowJo software (Tree Star).
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 3 /340 N.A. = 0.90), a halogen lamp, a red LED (620 nm), a CCD camera (Olympus, DP30), differential interference contrast (DIC) optical components, and interference filters. The halogen lamp was used with a 500AF25 excitation filter, a
525DRLP dichroic mirror, and a 545AF35 emission filter for observing the mVenus fluorescence, and a 565WB20 excitation filter,
a 595DRLP dichroic mirror, and a 635DF55 emission filter for observing the mCherry fluorescence, and a 440DF20 excitation filter,
a 455DRLP dichroic mirror, and a 480DF30 emission filter for observing the mTurquoise fluorescence (Table S1). 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 software (Molecular Devices), respectively. Movies were assembled using Windows Live Movie Maker. LCV100 accommodates up to eight 35-mm glass-bottom dishes, which can be sequentially imaged. Thus, comparison imaging experiments were
performed in parallel (Figure 2, S3, S5, S6 and Movie S1, S3).
Automated Cell Tracking
Image processing was performed using functions implemented in ImageJ (1.51n); acquired images were low-pass filtered and
smooth backgrounds were removed. Fucci-mCherry and Fucci-mVenus images were merged for tracking cell nuclei. Tracking
was performed by a script-based operation of the FIJI plugin, Trackmate ( (Schindelin et al., 2012).
‘‘Detector’’ parameters were optimized for effective detection of nuclei with Fucci fluorescence as follows:
settings.detectorFactory = LogDetectorFactory(),
DetectorKeys.KEY_RADIUS = 6.7 (for image binning 4) / 8.0 (for image binning 2),
DetectorKeys.KEY_THRESHOLD = 0.25,
For cells showing the ‘‘fluorescence gap’’ where the two color Fucci signals switch from one to the other, ‘‘Tracker’’ parameters
were optimized to maximize the spatio-temporal correlation between the two tracks.
TrackerFactory = SparseLAPTrackerFactory(),
Mean fluorescence intensities were calculated on identified nuclear regions of Fucci-mCherry and Fucci-mVenus images. The dataset composed of XY location, time, and mean intensities was exported to Excel software (Microsoft Corporation, Redmond, WA) or
MATLAB software (MathWorks, Natick, MA) for numerical analysis.
Numerical Analysis
Exported datasets were aligned according to time and track number, and cells successfully tracked for >20 hr were analyzed. Representative time courses are shown in Figure S1. To estimate degradation and accumulation rates of Fucci degrons (Figure S2D-H) and
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SLBP degron (Figure S3B), logistic growth equations (see below) were fitted to the data points (Santos et al., 2012). Briefly, time
courses were excised in time windows around local maxima. The Findpeak function of MATLAB was used to identify time positions
of intensity peaks. Then excised time courses were normalized and aligned to the respective peaks.
ðintensityÞ = min +
1 + eðkðtto ÞÞ
min and amp are minimum and amplitude of fluorescent intensities, respectively. k is the rate of degradation (k < 0)/accumulation
(k > 0) of Fucci reporters with a unit of 1/h. to is midpoint of the logistic curve with a unit of hour. k and to were obtained as free parameters. Levenberg-Marquardt method implemented in nlinfit function (MATLAB) was used for curve fitting. The time constant of
Fucci signal t [min] were calculated by the equation: t = 60*ln(2)/abs(k), where abs(x) is absolute number of x.
Confocal Imaging
mESCs expressing Fucci(SA)2.1 or Fucci(CA)2.1 in a naive state were cultured onto mitomycin-C-treated MEF cells on the 0.1%
gelatin-coated multi-well plate or glass-bottom dish. Cells were subjected to long-term, time-lapse imaging using an Olympus
FV3000 or FV1000 confocal microscope. On the one hand, FV3000 was equipped with a UPLSAPO 30 3 S objective lens (N.A. =
1.05, W.D. = 0.8 mm), a 445-nm diode laser and a 460–500 emission filter for observing the AmCyan fluorescence, and a 594-nm
diode laser and 610–710 emission filter for observing the mCherry fluorescence (Figure 4, Movie S2) (Table S1). Confocal images
were taken with a z-step of 1.23 mm. This system was used to perform time-lapse imaging with a 10 min interval. On the other
hand, FV1000 was equipped with a UPLSAPO 20 3 objective lens (N.A. = 0.75, W.D. = 0.6 mm) (Figure S4) (Table S1, FV1000-a).
For observing the AmCyan fluorescence, a 440-nm diode laser, a DM440–458/515/559–561 multichroic mirror, and a 460–500 emission filter were used. For observing the mCherry fluorescence, a 559-nm diode laser, the DM440–458/515/559–561 multichroic
mirror, and a 570–670 emission filter were used. Confocal images were taken every 5 mm along the z-axis. Image analysis was performed using Imaris software (Bitplane, Concord, MA) to create volume rendered images.
Manual Cell Tracking
Image processing was performed manually using the ‘‘Journal’’ functions implemented in MetaMorph (Molecular Devices). First,
Fucci-mCherry and Fucci-mVenus or Fucci-AmCyan images 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 and therefore for filling up the fluorescent gap in early G1 phase. Time sequence data of the XY positions of tracked cells
are saved in ‘‘TrackRef’’ files. The mean fluorescence intensities of tracked nuclei were calculated using the ‘‘Region measurements’’
EdU Incorporation
After incubation with 5-ethynyl-20 -deoxyuridine (100 mM, EdU) (Life Technologies) for 15 min, cells were subjected to a Click-iT reaction for labeling with Alexa Fluor 647. The reaction was performed according to our original procedure (Sakaue-Sawano et al.,
2013). Cells were fixed with 4% paraformaldehyde (PFA) for 10 min on ice, and then were treated with azide-conjugated Alexa Fluor
647 by the Click-iT reaction (Life Technologies) with a slight modification. As 4 mM copper ion for the conjugation between EdU and
Alexa dyes in the Click-iT reaction tended to quench fluorescent proteins, we attempted to lower the copper ion concentration. We
found that 1.3 mM copper ion was sufficient for the reaction while effectively avoiding the quenching reaction. Finally, the cell samples were stained with 3 mM DAPI and analyzed using a FACSAria II (BD Biosciences) (Table S1).
UV Irradiation
UV irradiation was carried out using a FUNA UV Cross-linker (FS-1500, Funakoshi, Tokyo). To obtain an effective energy of 24 and
80 J/m2 on the coverslip surface, we set the UV-cross-linker energy level to 120 and 400 J/m2, respectively. To perform UV-irradiation
during time-lapse imaging with a short time interval, the turret with multiple dishes was transiently placed below the UV Cross-linker.
Surface-Enhanced Third-Harmonic Generation
A 0.17-mm thick plate was generated from transparent polycrystalline ceramics (Ceramic Y2O3, CoorsTek) (Kong et al., 2015). Next,
the surface of the plate was spin-coated with Fluoropolymer (Teflon AF1600, DuPont) (Lowry et al., 1992), which was then baked at
170 C. The coating/baking step was repeated five times. Through an objective lens, the surface of the plate was irradiated with a
focused beam from an ultrashort pulse laser (Pu, 762 nm). At the ceramics/polymer interface THG (P3u, 254 nm) was achieved,
and its intensity was measured over the medium.
Cells were fixed with 4% PFA for 10 min at 4 degree and then permeabilized in 0.4% Triton X-100/phosphate-buffered saline for
30 min at room temperature. The antibodies used were: Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 (Merck Millipore) and Alexa Fluor 633-conjugated goat anti-mouse IgG (Molecular Probes). The cell samples were stained with 3 mM DAPI. Image acquisition was performed using an FV1000 (Olympus) confocal microscope system equipped with a UPLSAPO 60 3 objective
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lens (N.A. = 1.35, W.D. = 0.15 mm) (Table S1, FV1000-b). For observing DAPI fluorescence, a 405-nm diode laser and a 415-455
emission filter were used. For observing mVenus fluorescence, a 515-nm diode laser and a 525–555 emission filter were used.
For observing mCherry fluorescence, a 559-nm diode laser and a 575–620 emission filter were used. For observing Alexa Fluor
633 fluorescence, a 635-nm diode laser and a 655–755 emission filter were used. For all the channels, a DM405–440/515/
559/635 multichroic mirror was used. Confocal images were taken every 0.8 mm along the z-axis to create z stacks (8 slices). Image
analysis was performed using Metamorph software (Universal Imaging, Media, PA) and snapshot images were prepared by z-projection mode.
A Bonferroni method (Figure 5D) was used. Differences were considered to be significant at *p < 0.05 or **p < 0.005. Data are reported
as mean ± SD.
Stable cell lines; HeLa/Fucci(CA)2 (clone #3) cells will be distributed from the RIKEN BioResource Center Cell Bank (http://cell.brc. The information about Fucci-related materials are available in our website (
All the original, unprocessed data have been deposited to Mendeley Data and are available at
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