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"Fluorescent Timer": Protein That Changes Color with Time

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Science  24 Nov 2000:
Vol. 290, Issue 5496, pp. 1585-1588
DOI: 10.1126/science.290.5496.1585

Abstract

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock–dependent promoter in Caenorhabditis elegansand from the Otx-2 promoter in developing Xenopusembryos. Thus, E5 is a “fluorescent timer” that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

Green fluorescent protein (GFP) from the luminescent jellyfish Aequorea victoria is an important tool in molecular and cellular biology as a transcriptional reporter, fusion tag, or biosensor (1). The recent discovery of GFP-like fluorescent proteins from nonbioluminescentAnthozoa species (2), in particular the red fluorescent protein drFP583, has opened new horizons for multicolor labeling and fluorescence resonance energy transfer applications.

An earlier report (2) suggested that the red fluorophore of drFP583 requires an additional autocatalytic modification of a GFP-like fluorophore. We thus generated mutants of drFP583 using error-prone polymerase chain reaction (PCR) (3) and screened for mutants exhibiting a green intermediate fluorescence (4).

Mutations resulted in proteins with varying fluorescent properties, such as faster maturation, double emission (green and red), or exclusive green fluorescence. Of particular interest was the E5 mutant, which changes its fluorescence over time. This mutant changed from initial bright green fluorescence to yellow, orange, and finally red over time (Fig. 1, A and B). Yellow and orange fluorescence indicate that the protein species with green and red fluorophores are both present (Fig. 1B, color insert). The existence of a green-emitting intermediate suggests that E5 maturation involves the modification of a GFP-like fluorophore to give the red fluorophore. Changing the temperature had the same effect on the rates of decay of green fluorescence and growth of red fluorescence, which suggests that these processes reflect the same chemical reaction (Fig. 1B). In addition, the overall reaction speed was independent of the initial concentration of E5 protein in the range from 10 μg/ml to 1 mg/ml (as in Fig. 1B). It was also insensitive to variations in ionic strength in the range from 10 mM to 1 M NaCl, to the presence of 150 mM EDTA, or to changes in pH between 7.0 and 8.0. pH values below 4.5 or above 12 resulted in the disappearance of the red-shifted absorption and the appearance of 386-nm or 448-nm absorption peaks for acid and alkali, respectively (this was also observed for drFP583) (Fig. 1D). This is strikingly similar to the absorption spectra of GFP— 383 nm and 446 nm for acid and alkali, respectively (5)—in which the pH-dependent shift between 383 and 446 nm is due to ionization of the fluorophore phenolic group (6). The maturation from green to red fluorescence slows down considerably in deoxygenated buffer, suggesting that the fluorophore modification requires molecular oxygen. The properties of E5 provide insight into the nature of the red fluorophore; for instance, we detected similar fluorescence dynamics in drFP583, although barely detectable changes in green fluorescence make it unsuitable for practical application (Fig. 1C).

Figure 1

In vitro analysis of the E5 mutant. All spectra are normalized; 0 hours refers to the first measurement performed on the freshly purified protein. (A and C) Emission spectra of E5 (A) and drFP583 (C), excited at 280 nm during the course of fluorescence development. (B) Time course of green (500 nm) and red (580 nm) fluorescence development in E5 at 37° and 50°C; the color bar represents the overlay color of green and red fluorescence at each time point at 37°C. (D) Absorption spectra of acid- (NaOAc, pH 4.5) or alkali- (NaOH, pH 12) denatured E5; PBS denotes E5 absorption in PBS.

As compared to drFP583, E5 has two substitutions: Val105→ Ala105 (V105A) and Ser197 → Thr197 (S197T). The impact of each substitution on the fluorescent properties of E5 was assessed in single mutants. Mutation V105A resulted in a twofold increase in fluorescence quantum yield as compared to drFP583 but no spectral shifts, whereas the S197T mutant essentially recapitulated the fluorescent timer phenotype. We modeled the structure of drFP583 on the basis of GFP crystal structure (7) (details of modeling are available atwww.sciencemag.org/cgi/content/full/290/5496/1585/DC1and the atomic coordinates are available athttp://cmm.info.nih.gov/kajava) and found that Ser197 in drFP583 is analogous to Thr203 in GFP. Thr203 is in direct contact with the fluorophore (7, 8), and replacements at this position invariably alter the fluorescent properties of GFP (9–12).

E5 has the potential to function as a fluorescent clock, giving temporal and spatial information on target promoter activity. Green fluorescent areas would indicate recent activation, yellow-to-orange regions would signify continuous promoter activity, and red fluorescent cells and tissues would denote areas in which promoter activity has ceased after an extended “on” period. In vitro, the rate of color conversion (the red:green ratio) is independent of the protein concentration, suggesting that it will not depend on the expression level within a cell. Given the superior in vivo stability of drFP583 as compared to GFP (2), protein degradation of either the green or red form is unlikely to be a problem. This approach would make it possible to discriminate changes in gene expression from the effects of morphogenetic displacement of expressing and nonexpressing cells. None of the existing techniques (such as in situ hybridization, immunostaining, or tracing of any known reporter) can achieve that. We analyzed the expression of E5 in three heterologous systems: mammalian cells, C. elegans, and Xenopus.

We characterized E5 in a HEK 293 mammalian cell line engineered with Tet-On or Tet-Off expression systems (13). For 293 Tet-On cells transfected with E5, a distinct green fluorescence was visible and was detected by flow cytometry between 6 and 9 hours after induction, whereas cells with both green and red fluorescence appeared after 9 hours after induction (Fig. 2A). Similar to bacterial expression, in mammalian cells, red fluorescence developed faster in the E5 mutant than in wild-type drFP583 protein (Fig. 2A). When 293 Tet-Off cells were used, the majority of cells initially demonstrated strong green and red fluorescence at the beginning (a fully induced promoter) but lost the strong green fluorescence upon transcription shutdown. These changes could be readily observed under a fluorescence microscope (Fig. 2B). The persistence of strong green fluorescence in some cells was due to the inevitable heterogeneity of transient transfection, including the abnormal accumulation of mRNA in some cells and promoter leakiness.

Figure 2

Transient expression of drFP583 and the E5 mutant in Tet-On/Off systems. (A) FACS analysis of 293 Tet-On cells. Transcription was induced at 0 hours. Triangles, fluorescence in the FL1 (FITC) channel; circles, fluorescence in the FL2 (PE) channel. (B) Fluorescence images of 293 Tet-Off cells transfected with the E5 mutant; transcription was shut down at 0 hours.

To demonstrate the utility of the fluorescent timer as a tool for studying promoter activity in specific organisms, the E5 mutant was placed under the control of the C. elegans heat shock promoter hsp 16-41. This promoter exhibits minimal expression in unstressed animals, robust induction of transcription after heat shock, and rapid inactivation upon subsequent recovery to ambient temperature (14). An hsp-E5transgene was microinjected into worms, and several independent lines carrying the transgene as an extrachromosomal array were established (15). No fluorescence was observed in [hsp-E5(+)] worms maintained at ambient temperatures (16). However, after a standard heat shock regime (1 hour at 33°C), green fluorescence was observed in embryos as early as 2 hours into the recovery period (Fig. 3). Red fluorescence was detected in [hsp-E5(+)] embryos at 5 hours after heat shock (Fig. 3) and increased in intensity over time, so that at 50 hours after heat shock, the red:green signal ratio was close to 9:1. Similar kinetics of the fluorescent timer were observed in [hsp-E5(+)] worms at larval and adult stages. The prolonged periods of green fluorescence observed in these experiments are due to stabilization of the E5 mRNA, caused by the presence of a 3′ untranslated region (UTR) derived from the unc-54 gene (17). In our experiments, the color hue of transgenic embryos at different time points after heat shock could be readily distinguished by eye (Fig. 3A, overlay). Moreover, within experimental error, the red:green fluorescence ratio changed linearly with time (at least within the first 14 hours), thus providing a unique measurement of time elapsed since the heat shock. Remarkably, despite considerable heterogeneity in the absolute fluorescence intensities of individual embryos at any given time point, the red:green fluorescence ratios among embryos at the same time point were similar, as is expected for an autocatalytic reaction causing the color transition. In addition, the fluorescent ratio was uniform throughout the embryo (Fig. 3, overlay), despite differences in monitored cell types; indicating that, at least under our experimental conditions, the process of E5 maturation is independent of the cellular environment.

Figure 3

Heat shock–regulated expression of the E5 mutant in C. elegans. Representative images of [hsp-E5(+)] embryos are shown: the bright field (DIC), FITC filter, PE filter, and the overlay, after 2, 5, 10, and 50 hours after the heat shock.

We also used the E5 mutant to trace the activity of theOtx-2 promoter. The homeobox gene Otx-2 is involved in the patterning of anterior structures, which are common to all bilaterian animals (18). In Xenopus, at the midgastrula stage, the major domain of Otx-2 expression is in the head neuroectoderm. As development proceeds, the expression is almost completely suppressed in parts of this domain, namely, in the presumptive rostral area, telencephalon, and ventral diencephalon (19). Thus, in the tadpole's brain, Otx-2expression revealed by in situ hybridization is strong in the mesencephalon and dorsal diencephalon but is much weaker in the telencephalon and ventral diencephalon (Fig. 4C). We assembled a plasmid containing the E5 gene under the control of the Xenopus Otx-2 promoter and microinjected this into both dorsal blastomers of the X. laevis embryo at the eight-cell stage (20). The representative mosaic fluorescent image composed from clones of cells, which acquired the plasmid during blastomere cleavage (21), reflects the in situ hybridization data accurately (Fig. 4, A to C). The telencephalon and rostral region of the tadpole are marked orange, indicating that the Otx-2 promoter was once active there but is now mostly silent, giving the accumulated protein time to mature. Simultaneously, the mesencephalon and ventral diencephalon are green, indicating that Otx-2 promoter activity is driving expression of E5 in these regions. In a control experiment, the expression of E5 was driven by the promoter of another homeobox gene,Xanf-1. The expression of Xanf-1 also occurs in neuroectoderm, but, unlike Otx-2, does not have distinct spatiotemporal domains and ceases before the tadpole stage (21, 22). Correspondingly, the signal from theXanf-1/E5 construct appeared uniformly orange in the tadpole brain (Fig. 4D).

Figure 4

Expression of E5 (timer) in a developingXenopus embryo; fully matured E5 appears orange because of the FITC filter set. (A) Dorsal view of the tadpole expressing E5 under the control of the Otx-2 promoter; only some cells express the E5 protein because of the mosaic distribution of plasmids within the embryo. (B) Brain region of the tadpole shown in (A). Telencephalic (Tel) and di- and mesencephalic (Di and Mes) borders are designated by a dotted line (C) Dorsal view of the whole-mount in situ hybridization of the tadpole brain with an Otx-2 probe. (D) Dorsal view of the brain region of the tadpole expressing E5 under the control of theXanf-1 promoter.

“Fluorescent timer” provides an easy and reliable way to analyze the “history” of gene expression and gives the ability to monitor two equally important processes: activation and down-regulation of gene expression. The ability to evaluate promoter activity over a wide time range by analyzing a single developmental stage raises the possibility of large-scale screening for new time-dependent promoters, many of which are associated with development control genes.

  • * To whom correspondence should be addressed. E-mail: Alexey.Terskikh{at}Stanford.edu

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