Fred Schaufele (freds@metabolic.ucsf.edu)
is assistant professor in the Department of Medicine and Metabolic Research
Unit, University of California, San Francisco, CA
94143-0540.
It has been only seven years since the green fluorescent protein (GFP)
of the jellyfish Aequorea victoria was first shown to retain its fluorescent
properties when expressed in a wide variety of cells and organisms1,
2.
Because neither cell fixation nor disruption is required to detect GFP, it
has catalyzed the rapid development of an astonishingly wide number of applications
as a noninvasive marker in living cells3. It has proved useful
as a simple reporter of gene expression, and GFP derivatives with differing
fluorescence properties have been shown to provide excellent direct visualization
of the spatial relationships and physical interactions of differentially tagged
proteins in living cells.
Now, two recent studies describe a new wrinkle for these versatile intracellular
tracers4,
5. Both reports describe that a distant GFP homolog,
drFP583 (more commonly referred to as DsRed), previously isolated from coral
of the genus Discosoma6, fluoresces green immediately
after synthesis but then matures to fluoresce red. There are minor discrepancies
in the reported amount of the initial green fluorescence from the parental
DsRed4,
5. However, the papers both demonstrate that the rate
of green to red fluorescence maturation of DsRed5, or certain
derivatives of DsRed4,
5, is stable to wide fluctuations in
pH, ionic strength, or protein concentration, and appears to be linear with
time. These properties indicate that the ratio of red to green fluorescence
can be a useful monitor of the time since DsRed was initially expressed. This
DsRed-based "fluorescent timer" will open up new avenues of investigation
in which the timing of a variety of events can be noninvasively determined
in living cells.
If the fluorescent timer is continuously expressed, then its steady-state
red:green fluorescence ratio will be a function of the rate of synthesis of
the initially green fluorescent conformer, the rate of maturation to the red
fluorescent conformer, and the rate by which fluorescence is lost through
protein degradation or photobleaching (Fig. 1). Because
of the very long half-life of DsRed6, the relative resistance
of DsRed to photobleaching5, and the uniform rate of green to
red fluorescence maturation4,
5, Terskikh et al. were
able to specifically analyze perturbations in the synthesis of a DsRed-based
promoter/ reporter as a change in the steady-state ratio of red to green fluorescence4. This will be useful for a number of studies, particularly in transgenic
organisms, in which the kinetics of alteration in reporter expression can
be compared in different cells and tissues after application of a stimulus.
Figure 1. A fluorescent timer for characterizing the sequence of intracellular
events.
Derivatives of the fluorescent protein DsRed fluoresce green immediately
after synthesis and then mature to fluoresce red. The steady-state ratio of
red fluorescence to green fluorescence within a cell will be a function of
the rate of synthesis of the initially green conformer, the rate of green
to red fluorescence maturation (which appears to be a constant), and the rate
of loss of fluorescence via protein degradation. Photobleaching may also contribute
to fluorescence loss, but DsRed is relatively resistant to photobleaching.
Dimerization of the green and red conformers that leads to a fluorescence
resonance energy transfer of green fluorescent energy to red fluorescence
(FRET) will also affect the red/green fluorescence ratio. Cells or stuctures
within a cell that possess different red/green fluorescence ratios therefore
differ in the synthetic and/or degradation rates of the protein and/or in
the oligomeric status of the protein.
The DsRed-based reporter also can be used for ascertaining whether a stimulus
has similar effects on the kinetics of promoter induction or repression in
different cultured cell types. The excitation and emission properties of drFP583
in the green and the red channels minimize the pesky background fluorescence
of cells in the absence of expressed DsRed such that fluorescent timer applications
may readily lend themselves to high-throughput ratiometric screening. Tried
and true technologies already exist for high-throughput screening of gene
reporter activity in cultured cells, so at least initially the DsRed-based
reporter is more likely to be used as a secondary screening tool. Eventually,
the kinetic attributes and versatility of DsRed-based reporters for other
applications discussed below may even permit it to supplant existing primary
screens.
Immediate advances may be found in the application of fluorescence timer
technology to problems that are more difficult to monitor by existing techniques.
The rate of protein degradation also will affect the red:green fluorescence
ratio of DsRed (Fig. 1). Protein turnover is an important
component of physiological action and drug response that, to date, has been
laborious to study. An in-frame fusion of a target protein to the extremely
stable DsRed likely will assume the turnover rate of the target protein. Altered
stability of the target protein following a physiological or pharmacological
stimulus then would be reflected in the altered stability of the fusion protein,
which would be measured as an alteration in the red:green fluorescence ratio.
The fluorescence timer also will be useful for determining the temporal
sequence of intracellular trafficking. Specific cellular substructures can
be correlated by microscopy with the intracellular distributions in the red:green
fluorescence emitted from DsRed-linked proteins. A structure displaying more
green fluorescence character would be one that is occupied by the linked protein
at an earlier time after synthesis than is a structure displaying more red
fluorescence character. This will facilitate the identification of new classes
of compounds that affect a protein's movement and location, which in many
cases is just as important as a protein's amount and isolated activity.
The properties of DsRed and its derivatives are not yet optimal. The propensity
of DsRed to form a tetramer5 is currently one such limiting
property that may cause some linked proteins to acquire properties not present
in the native protein. Baird et al.5 used this oligomerization
to demonstrate that green fluorescence from the immature, green conformer
of DsRed is transferred to an adjacent mature red conformer by fluorescence
resonance energy transfer (FRET). Therefore, FRET will affect the ratio of
red:green fluorescence and complicate measurements of the timer function if
FRET changes between measurements. Even once mutants deficient in oligomerization
are identified, FRET between immature and mature DsRed brought into close
proximity by multimerization of a linked target protein still will need to
be measured in parallel and accounted for.
The development of fluorescent timer technology is still in its infancy
but offers much promise. A drug or stimulus found in an initial screen to
alter the red:green ratio of DsRed may have an effect at any of a diverse
set of levels (Fig. 1), from the synthesis of the reporter
or fusion protein, to protein degradation, to alterations in the multimeric
status of the target protein or DsRed itself (FRET), to even the maturation
of DsRed. This breadth of responses may allow the investigator to rapidly
identify compounds that affect a variety of events that can be readily discerned
in follow-up experiments. The properties and known structures of the many
GFP homologs and derivatives identified to date will certainly aid the identification
of DsRed derivatives with more optimal qualities3,
4,
5,
6.
These advances, together with existing GFP-based technologies, will allow
the noninvasive spatial, temporal, and physical characterization of a wide
variety of biochemical events within the context of the living cell. The ability
to rapidly measure these effects before and after the addition of a stimulus
will make these assays readily amenable to high-throughput screening.
