Mechanism of a genetically encoded dark-to-bright reporter for caspase activity

Abstract

Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.

FIGURE 1.

CA-GFP becomes fluorescent only upon cleavage by active caspase. A, CA-GFP fusion protein consists of green fluorescent protein (GFP, green), the four amino acid caspase-7 cleavage site Asp-Glu-Val-Asp (DEVD, blue) and the quenching peptide (QP, gray). B, model of uncleaved CA-GFP (non-fluorescent). C, model of cleaved CA-GFP (fluorescent). D–F, cleavage and fluorescence of bacterially co-expressed CA-GFP with active (WT) or inactive (C186A) caspase-7. Full-length caspase-7 undergoes auto-zymogen processing to generate the active, two-chain form of caspase-7. Mutation of the catalytic cysteine in the C186A mutant yields the inactive single-chain procaspase-7 zymogen. D, fluorescence of CA-GFP in lysates (n = 3 (S.E.)). E, immunoblot probed with anti-caspase-7 large subunit antibody. F, immunoblot probed with anti-GFP antibody.

FIGURE 2.

CA-GFP fluorescence is robust enough for fluorescence microscopy and flow cytometry. BL21 (DE3) E. coli cells expressing (A) CA-GFP alone or (B) CA-GFP with inactive caspase-7 C186A or (C) CA-GFP with active wild-type caspase-7. DIC images (left). Fluorescent microscopic images (center) of live cells. Intact cell flow cytometric analysis of GFP fluorescence (right) listing the fraction of cells with GFP fluorescence. Scale bars represent 10 μm.

FIGURE 3.

CA-GFP is fluorescently activated in mammalian cells undergoing apoptosis. A, fluorescent response of CA-GFP-transfected NIH 3T3 cells at indicated times after induction of apoptosis with staurosporine (STS). Scale bar represents 50 μm. Br; Brightfield microscopy. G, GFP fluorescence. B, CA-GFP cleavage after induction of apoptosis is observed as a function of time by immunoblotting with an anti-GFP antibody. C, appearance of cleaved caspase-3 was observed after induction of apoptosis by immunoblotting with an anti-caspase-3 antibody. Tubulin was probed as a loading control. D, fraction of transfected cells that are fluorescent (white bars) and fraction of cleaved CA-GFP (black bars) in cells induced to undergo apoptosis. *, significance level; p < 0.05 relative to zero time point.

FIGURE 4.

The response of CA-GFP can be measured in single cells undergoing apoptosis. A, time-lapse confocal images of NIH 3T3 cells co-expressing CA-GFP and mLumin were recorded at the indicated times following treatment with STS to induce apoptosis. DIC; differential interference contrast images showing changes in cell morphology. G, green channel monitoring CA-GFP fluorescence. R, red channel, monitoring mLumin (control) fluorescence. Scale bar represents 25 μm. B and C, ratio of green (GFP)/red (mLumin) fluorescence for (B) untreated control or (C) staurosporine-treated cells. Ratios for six (B) or four (C) independent cells were measured. The G/R ratio of cells 1 and 2 shown in panel (A) are indicated as 1 or 2 in plot (C).

FIGURE 5.

CA-GFP can be activated in vitro. A, cleavage of CA-GFP by wild-type caspase-7 from 0 min to 18 h. (overnight). B, gain of fluorescence during in vitro cleavage of CA-GFP by caspase-7 (black line) with error bars (gray). C, CA-GFP cleavage precedes appearance of fluorescence. Percentage of total fluorescence (white) and cleaved CA-GFP (black) at each time point (n = 3 (S.D.)).

FIGURE 6.

CA-GFP silencing relies on tetramerization and prevention of chromophore maturation. A, absorbance spectra of GFP and CA-GFP suggest that CA-GFP chromophore is non-functional. B, mass spectra of GFP and CA-GFP tryptic fragments indicate that the CA-GFP chromophore has not undergone the maturation reaction. C, circular dichroism spectra of GFP and CA-GFP indicate that both proteins are folded into a predominantly β-sheet structure. D, size exclusion chromatogram for GFP, CA-GFP, and cleaved CA-GFP. The observed molecular weights demonstrate that GFP and cleaved CA-GFP are monomers, while uncleaved CA-GFP is predominantly tetramer. The molecular weights for the standards are marked as diamonds on the x-axis. E, expected and observed molecular weights by size exclusion chromatography for GFP, CA-GFP, and cleaved CA-GFP.

FIGURE 7.

CA-GFP is activated by cleavage and maturation of the chromophore. This scheme depicts the model of how fluorescence of tetrameric CA-GFP is activated. Dark CA-GFP contains an immature chromophore. Cleavage by active caspase releases GFP from the tetramer. Free GFP is then able to undergo the required conformational changes allowing chromophore maturation by the reaction scheme shown and production of fluorescent, bright GFP.