Single-fluorophore biosensors based on conformation-sensitive GFP variants

Abstract

The β-strands of GFP form a rigid barrel that protects the chromophore from external influence. Herein, we identified specific mutations in β-strand 7 that render the chromophore sensitive to interactions of GFP with another protein domain. In the process of converting the FRET-based protein kinase A (PKA) sensor AKAR2 into a single-wavelength PKA sensor containing a GFP and a quencher, we discovered that the quencher was not required and that the sensor response relied on changes in GFP intrinsic fluorescence. The identified mutations in β-strand 7 render GFP fluorescence intensity and lifetime sensitive to conformational changes of the PKA-sensing domain. In addition, sensors engineered from the GCaMP2 calcium indicator to incorporate a conformation-sensitive GFP (csGFP) exhibited calcium-dependent fluorescence changes. We further demonstrate that single GFP sensors report PKA dynamics in dendritic spines of neurons from brain slices on 2-photon imaging with a high signal-to-baseline ratio and minimal photobleaching. The susceptibility of GFP variants to dynamic interactions with other protein domains provides a new approach to generate single wavelength biosensors for high-resolution imaging.

Keywords: chromophore; fluorescence lifetime imaging; genetically encoded indicators; green fluorescent protein; high-resolution microscopy; intracellular second messengers.

Figure 1.

Constructs derived from the FRET sensor AKAR2. A) Mechanisms of fluorescence changes reported for AKAR2 and anticipated for the newly generated GAKdY sensor following folding of the substrate peptide (SP) into the Forkhead-associated domain (FHA) on phosphorylation. B) Domain structure of most constructs used in this study, named after their constituent parts (see Abbreviations). Color boxes, gray boxes, and white boxes represent active fluorophores, fluorophores converted into quenchers, and inactivated fluorophores, respectively. Mutations at positions 65–67 (chromophore) and dark-YFP mutations are indicated. Inactivation of the sensing domain has been achieved via mutagenesis in SP (crossed box).

Figure 2.

Characterization of PKA sensors in BHK cells. A) Grayscale fluorescence image shows the distribution of the GAkdY sensor in the cytoplasm and nucleus of BHK cells. Colored numbers (left panel) correspond to traces in panel B. Pseudocolor images of the same field before (T1) and during (T2) application of FSK (13 μM) illustrate the large increase in fluorescence of the GAkdY sensor on PKA activation. B) Traces show the time course and amplitude of fluorescence increases measured in the individual cells indicated in panel A. C) Mean responses of selected PKA sensors. Unless indicated (ns, nonsignificant), means are significantly different. D) Representative examples of emission spectra determined in individual cells illustrate the fluorescence increase of indicated sensors on PKA activation. No variation was observed with the inactive GAkmutdY construct. Each spectrum is the mean of 2 consecutive spectra acquired on the same cell in the presence or absence of FSK. Data were obtained using 2-photon (A–C) or 1-photon confocal microscopy (D).

Figure 3.

Characterization of single-fluorophore sensors using FLIM. Grayscale fluorescence images show BHK cells expressing GAk (A) and GAkdYmut (B) sensors. Pseudocolor images of the same fields before (T1) and during (T2) application of FSK (13 μM) illustrate the decrease in fluorescence lifetime τ_φ on PKA activation. Traces show the variations of τ_φ measured in individual cells indicated in the grayscale images; colors correspond to numbers in top left panels. Bar graphs show variations of the proportion of the short-lifetime (τ₁) and long-lifetime (τ₂) components on FSK application.

Figure 4.

Two-photon excitation spectra. Superimposed excitation spectra obtained in BHK cells expressing constructs that incorporate either 2 active chromophores (GAkdY), an active and an inactivated chromophore (GAkdYmut and GmutAkdY), or a single active chromophore (GAk and dY). The dY spectrum was obtained from a construct expressing only the dark-YFP domain of GAkdY. Note that the GAkdY spectrum exhibited 2 discrete peaks at 920 and 960 nm. The latter was due to fluorescence emission by its dark-YFP moiety since both the GmutAkdY sensor, which contains the chromophore-inactivating glycine triplet (positions 65–67) in its GFP moiety, and dark-YFP alone showed a single fluorescence peak at 960 nm. Conversely, GAkdYmut and GAk exhibited almost identical spectra with a single peak at 920 nm.

Figure 5.

csGFP reports activation of a calcium-sensing domain. A) Domain structure of constructs derived from the GCaMP2 calcium sensor and incorporating a CFP-derived csGFP, as well as variable numbers of a nonapeptide spacer. B, C) Fluorescence images show the distribution of the csGCaMP0.1 sensor in the cytoplasm and nucleus of HEK cells (grayscale), and fluorescence increase of this sensor (pseudocolor) elicited by application of ionomycin (4 μM; B) or carbachol (300 μM; C). Traces illustrate csGCaMP0.1 responses measured in indicated cells (colored numbers in top left panels) and the absence of detectable response of the csGCaMP0.1 I146N mutant on application of these drugs (insets). D) Emission spectra of csGCaMP0.1 and csGCaMP0.1 I146N in the presence or absence of calcium (1 mM), measured in a cell-free assay following bacterial expression of the constructs.

Figure 6.

Single-fluorophore sensors as PKA reporters in brain slice. Grayscale fluorescence images show cytoplasmic and nuclear expression of indicated sensors following viral transfer in neocortical pyramidal neurons. A) Traces illustrate the reversibility of sensors responses to PKA activation by CRF (250 nM) and FSK (13 μM) on washout of the drugs and the virtual absence of photobleaching in our recording conditions. B, C) Pseudocolor images show GAkdYmut (B) and GAk (C) fluorescence intensity in control (T1), CRF (T2), and FSK (T3) conditions. Color traces show the variations of fluorescence intensity measured in the somatic cytosol of individual cells indicated in the corresponding grayscale image. Grayscale traces in panel B were obtained from cell 1 and illustrate the different time course and amplitude of cytosolic and nuclear responses of the sensors. Bar graph in panel C shows mean responses of PKA sensors measured in the somatic cytosol. Unless indicated (ns, nonsignificant), mean responses significantly differed between probes.

Figure 7.

Imaging PKA activity in neuronal processes. Grayscale fluorescence image shows a segment of the apical dendrite and associated spines of a pyramidal neuron expressing the GAkdYmut sensor. Traces show the variations of GAkdYmut fluorescence intensity elicited by CRF (250 nM) and FSK (13 μM) in RPIs including the dendritic shaft and 3 spines (indicated by arrows on the grayscale image). Pseudocolor images of the same field at indicated time points illustrate GAkdYmut responses on drug application.