Fucci: Street Lights on the Road to Mitosis
Robert H. Newman1 and Jin Zhang1,2,*
1Department of Pharmacology and Molecular Sciences
2The Solomon H. Snyder Department of Neuroscience and Department of Oncology
The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA
*Correspondence: jzhang32@jhmi.edu
DOI 10.1016/j.chembiol.2008.02.003
Tracking cell-cycle progression in live cells within their endogenous environment has been an outstanding
challenge. In a recent issue of Cell, Sakaue-Sawano et al. describe Fucci, a technique designed to track
cell-cycle progression with high spatiotemporal resolution in a multicellular context.
The cell division cycle, which describes
the fundamental process by which cells
reproduce, directly impacts the operation
and development of all living organisms
(Nurse, 2000; Nurse et al., 1998). As a consequence,
each step in the process of cell
division—from replication of chromosomal
DNA during S phase to segregation
of sister chromatids during mitosis—must
be coordinated in a manner that ensures
the faithful transmission of hereditary information
from one generation of cells to
the next. Indeed, the timely execution of
each stage of the cell cycle is intimately
linked to key developmental processes
such as differentiation and organogenesis.
On the other hand, failure to precisely
regulate cell-cycle progression leads to
various diseases such as cancer.
To ensure that events such as S phase
and mitosis proceed both in an orderly
fashion and with high fidelity, cells have
developed a series of checkpoints that
act as quality control centers at each
stage of the cell cycle. These checkpoints,
which govern the transitions between
G1/S and G2/M, are designed to
monitor cellular parameters such as genomic
integrity and cell size throughout
the division cycle. If a cell fails to meet
minimal requirements at any point during
the process, regulatory factors prevent
the onset of the next phase until the task
at hand has been completed.
Until recently, it has been difficult to
precisely track cell-cycle progression in
a live, multicellular context. This is because
most of the techniques currently
used to monitor the cell cycle—such as
BrdU incorporation or immunostaining of
cell-cycle markers—require cell fixation
prior to analysis. As a consequence, these
methods do not permit the dynamic behaviors
of cycling cells to be visualized
in real time. Recently, several techniques
have been developed to track cell-cycle
progression in live cells (Leonhardt et al.,
2000; Essers et al., 2005; Easwaran
et al., 2005). Unfortunately, each of these
methods relies upon relatively small
changes in the subcellular distribution
and/or patterning of fluorescently-tagged
proteins. While these approaches may be
useful for monitoring phase transitions
in cultured cells, they may not translate
well to a multicellular context such as
live tissues where a high degree of contrast
is required to discriminate between
cells at various stages of the cell cycle.
Ideally, imaging techniques used to visualize
cell-cycle progression would provide
a means of easily distinguishing between
cells engaged in different stages of the
cell cycle with minimal perturbation to
the system under study, allowing the
dynamic behavior of cycling cells to be
monitored in real-time.
In a report published in a recent issue
of Cell, Sakaue-Sawano and colleagues
describe an exciting technique designed
to track cell-cycle progression with high
spatiotemporal resolution in a multicellular
context (Sakaue-Sawano et al.,
2008). This method, termed fluorescent
ubiquitination-based cell-cycle indicator
(Fucci), exploits cell-cycle-dependent
proteolysis of the ubiquitination oscillators,
Cdt1 and Geminin, to specifically
mark the G1/S transition in living cells.
By fusing the red- and green-emitting
fluorescent proteins mKO2 and Azami
Green (mAG) to portions of Cdt1 and
Geminin, respectively, the authors are
able to achieve striking contrast between
various stages of the division cycle. Specifically,
the nuclei of cells in G1 phase
(and G0) appear red, while those of cells
in S/G2/M appear green (Figure 1). During
the transition from G1 to S phase, cell nuclei
turn yellow, clearly marking cells that
have initiated DNA replication.
The dramatic color changes exhibited
by Fucci are based upon the reciprocal
activities of the ubiquitin E3 ligase complexes
APCCdh1
and SCFSkp2
(Vodermaier,
2004). For instance, while APCCdh1
functions primarily during G1 phase,
SCFSkp2
is most active during S, G2, and
early M phases (Figure 1). Consequently,
the APCCdh1
and SCFSkp2
substrates
Geminin and Cdt1 are specifically degraded
during G1 and S/G2/M, respectively
(Nishitani et al., 2004). After testing
several truncated forms of each protein,
the authors identified two protein fragments,
Cdt1(30/120) and Geminin(1/
110), that exhibit the same oscillatory
behavior as the wild-type species. Importantly,
neither of these constructs perturbed
cell-cycle progression. Therefore,
the fusion proteins mAG-Geminin(1/110)
and mKO2-Cdt1(30/120) employed by
Fucci function as benign cell-cycle
markers that effectively label the nuclei
of cells in S/G2/M green and those in G1
red. Since both chimeras are stabilized
to varying degrees as the activities of
SCFSkp2
and APCCdh1
converge during
the G1/S transition, yellow nuclei are
observed in these cells. The high contrast
afforded by Fucci allows the cell cycle to
be correlated with many important physiological
processes. For instance, to examine
cell-cycle progression of tumor
cells during metastasis, the authors injected
Fucci-expressing HeLa cells into
live animals and tracked their distribution
over time. Although most of the cells in
the blood vessels existed in G1, many
underwent a G1/S phase transition as
they migrated into neighboring tissues
during extravasation.
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Aside from its high contrast, Fucci also
exhibits several characteristics that make
it particularly well suited for studying cellcycle
progression in a multicellular context.
For instance, because it relies upon
ubiquitin-mediated proteolysis rather
than transcriptional regulation, expression
of Fucci can be driven by constitutive
promoters. Not only does this property
reduce the variability in protein expression
levels often observed at different
developmental stages, it also facilitates
the generation of transgenic animals.
Thus, Fucci is a very attractive tool for
cell-cycle analysis during development.
To this end, the authors used transgenic
mice expressing mKO2-Cdt1(30/120) and
mAG-hGem(1/110) under the control of
the CAG promoter to visualize, in neural
tissues, the distribution of proliferating
neuronal progenitor cells and postmitotic
cells marked with bright red nuclei, a consequence
of mKO2-Cdt1(30/120) accumulation
after cell-cycle exit. Importantly,
because time-lapse imaging of live tissue
sections can be achieved over relatively
short time intervals using Fucci, the
migration patterns of individual neuronal
progenitors could be correlated with their
cell-cycle progression. Together, these
experiments demonstrate Fucci’s great
promise for studying the coordination of
the cell cycle and development.
Fucci also has the potential to significantly
enhance our understanding of the
molecular mechanisms governing cellcycle
progression. For instance, because
the fluorescent chimaeras utilized by
Fucci are distributed exclusively in the
nucleus, this method is amenable to coimaging
studies using fluorescent biosensors
designed to track signaling dynamics
in live cells (Zhang et al., 2002). This can
be accomplished either by excluding the
reporter from the nucleus or by utilizing
fluorescent protein variants with emission
spectra distinct from those of the Fucci
probes. The ability to simultaneously image
cell-cycle progression and signaltransduction
pathways inside living cells
will be vital to unraveling the spatiotemporal
dynamics underlying cell-cycle checkpoint
control mechanisms (Lukas et al.,
2004). Importantly, since Fucci provides
a means of easily distinguishing between
cells engaged in different stages of the
cell cycle, synchronization procedures
that may otherwise perturb the cellular
system are not necessary using this approach.
This property can also facilitate
high-throughput analysis of cell-cycle
progression in RNAi- or small-moleculebased
screens. In this way, Fucci promises
to play an integral part in the identification
of new cell-cycle regulators as well
as chemical biology tools for studying the
cell cycle.
Fucci, as a powerful tool to visualize
cell-cycle progression in living cells, lays
a foundation for studying the cell cycle in
a variety of cellular contexts. In particular,
its ability to mark the G1/S transition with
high contrast in transgenic animals opens
new and exciting avenues of research.
Moreover, the anticipated development
of complementary cell-cycle probes designed
to mark phase transitions other
than G1/S will further expand the applications
of this innovative technology.
Together, these reporter systems promise
to provide unprecedented insights into
the regulation and coordination of cellcycle
progression in many physiological
processes.
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Figure 1. Schematic Representation of the Fucci Technology
Fucci relies upon cell-cycle-dependent proteolysis of the ubiquitination oscillators Cdt1 and Geminin to
specifically mark the G1/S transition in living cells. During G1 phase, the nuclei of Fucci-expressing cells
appear red due to both the stabilization of mKO2-Cdt1(30/120) and ubiquitin-mediated proteolysis of
mAG-hGem(1/110). As cells transition from G1 to S phase, each chimera is stabilized to varying degrees,
resulting in nuclei with a yellowish hue. Once the cells have transitioned to S phase, mAG-hGem(1/110) is
stabilized and mKO2-Cdt1(30/120) is degraded, causing the nuclei of these cells to appear green. Green
fluorescence is maintained throughout S, G2, and M phases until the fluorescent signal is lost for a brief
period between M and G1 due to the simultaneous destruction of both probes.
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