Figure 1.
Converting an eGFP based Ciona FP voltage sensor to one like ArcLight.
A) Comparison of the mean response amplitudes of Ciona voltage sensor domain-eGFP-S249 carrying different combinations of amino acids that are the same as those in super ecliptic pHluorin A227D. A probe with poor plasma membrane localization is indicated with the Greek letter Δ. Means with the same letter do not differ significantly (Tukey-Kramer HSD test). eGFP (n = 10), eGFP-A227D (n = 5), eGFP-A227D-S147D (n = 4), eGFP-A227D-S147D-N149Q (n = 4), eGFP-A227D-S202F-Q204T (n = 5), eGFP-A227D-S147D-S202F-Q204T (n = 4), ArcLight-S249 (n = 10). All the measurements were made in HEK293 cells (See Methods). Insert is the 3D structure of eGFP (PDB file 1EMG). The non-identical amino acids of eGFP and super ecliptic pHluorin A227D that lie on the facing surface of eGFP are colored and numbered. B) Mutations to eGFP and their combinations in making those probes tested in panel A are given in corresponding columns in the table. Values are means ± SEM.
Figure 2.
pH sensitivities of free fluorescent proteins carrying different combinations of amino acids.
A) The fitted pH sensitivity of super ecliptic pHluorin with the indicated mutations that make it more like eGFP. B) The fitted pH sensitivity of eGFP with the indicated amino acids that make the eGFP more like super ecliptic pHluorin A227D.
Figure 3.
Replacing the super ecliptic pHluorin A227D in ArcLight with the pH sensitive fluorescent proteins YFP or ratiometric pHluorin.
A) Averaged optical traces of two probes, YFP (n = 6) or YFP-A227D (n = 5) inserted at S249 of the Ciona voltage sensitive domain, in response to 100 mV depolarizations of 300 ms from the holding potential of −70 mV. The traces are processed by low pass FFT filtering of 50 Hz. B) The mean response amplitude of the two probes in panel A. (*** P<0.001) C) Averaged optical traces of two probes, ratiometric pHluorin (n = 6) or ratiometric pHluorin-A227D (n = 3) inserted at S249 of Ciona voltage sensitive domain, in response to 100 mV depolarizations of 300 ms from the holding potential of −70 mV. D) The mean response amplitude of the two probes shown in panel C. No significant difference was detected between the two groups. Values are means ± SEM.
Figure 4.
ArcLight does not simply respond to environmental pH changes.
The fluorescence signals change as expected when the composition of S4 is altered. A) Plots of ΔF/F vs membrane voltage of three probes: ArcLight-S249-Q217, ArcLight-S249-R217 and ArcLight-A242-R217. The smooth curves are sigmoid fits. B) The normalized sigmoid curves of the three probes. Values are means ± SEM.
Figure 5.
Using two-photon polarization microscopy to study the orientation and movement of the FP moiety in ArcLight.
A) Linear dichroism of CiVSD-EP, ArcLight-S249 and ArcLight-Q239. Excess of fluorescence elicited by light polarized horizontally and vertically is shown by red and green color, corresponding to a dichroic ratio indicated by the color scale bar. B) ΔF/F as a function of membrane voltage. C) Changes in dichroic ratio (Δrmax/rmax) as a function of membrane voltage. D) Correlation of ΔF/F with Δrmax/rmax. E) Dynamics of fluorescence change during a 100 mV depolarization and repolariztion observed with two-photon polarization microscopy. F) Dynamics of changes in dichroic ratio (Δrmax/rmax) during a 100 mV depolarization and repolariztion observed with two-photon polarization microscopy. G) Correlation of ΔF/F with Δrmax/rmax measured during the depolarization and repolarization of a 100 mV step. Values are means ± SEM.
Figure 6.
The relationship of ArcLight response amplitude to linker length.
A) Top: Schematic topology of the fusion protein in the plasma membrane; Bottom: The linker length modified derivatives of ArcLight. The sequence of the Ciona voltage sensitive domain and linker are framed in orange, the linker sequence is indicated with red; the three amino acids sequence “GDP” was introduced during plasmid construction and is shaded with blue; the sequence of the super ecliptic pHluorin A227D is shaded with green. The range of linker modification is from A231 to S249. B) The mean response amplitude of the linker length modified derivatives of ArcLight. Probes with poor plasma membrane localization are indicated with the Greek letter Δ. Groups that have significant difference with ArcLight S249 were indicated with the asterisks (*** P<0.001). n = A231: 6; R232: 7; S236: 8; H237: 5; Q238: 6; Q239: 7; M240: 8; K241: 7; A242: 7; S243: 7; S244: 8; R245: 5; R246: 7; T247: 8; I248: 8; S249: 10. C) The mean tau-on-fast component (Tao-on-1)and its amplitude contribution (A-on-1) in the double exponential fitting of the fluorescence response during depolarization. No significant difference was detected between these groups. n = A231: 1; R232: 7; S236: 7; H237: 5; Q238: 6; Q239: 6; M240: 8; K241: 7; A242: 6; S243: 7; S244: 8; R245: 4; R246: 7; T247: 8; I248: 8; S249: 9. D) The tau-off-fast component (Tau-off-1) and its amplitude contribution (A-off-1) during repolarization. Groups that have significant difference with ArcLight S249 were indicated with the asterisks (* P<0.05; *** P<0.001). n = A231: 4; R232: 6; S236: 7; H237: 4; Q238: 5; Q239: 6; M240: 8; K241: 7; A242: 6; S243: 7; S244: 5; R245: 4; R246: 6; T247: 8; I248: 7; S249: 10. E) The mean response amplitude of ArcLight-Q239 (n = 7), ArcLight-A242 (n = 7) and ArcLight-A242-GDPdel (n = 4). No significant difference was detected between these groups. F) The mean fast (Tau-on-1) and slow components (Tau-on-2) for the double exponential fitting of the fluorescence response during depolarization. ArcLight-Q239 (n = 6), ArcLight-A242 (n = 6) and ArcLight-A242- GDPdel (n = 4). Groups that have significant difference with ArcLight A242 were indicated with the asterisks (*** P<0.001). G) The mean fast (Tau-off-1) and slow components (Tau-off-2) for the double exponential fitting of the fluorescence response during repolarization. ArcLight-Q239 (n = 6), ArcLight-A242 (n = 6) and ArcLight-A242- GDPdel (n = 4). Groups that have significant difference with ArcLight A242 were indicated with the asterisks (*** P<0.001). Values are means ± SEM.
Table 1.
Dynamic properties of the linker modified ArcLight derivatives.
Figure 7.
The effect of deletion of non-essential residues at the N- or C-terminus of super ecliptic pHluorin A227D on the response properties of ArcLight.
A) The schematic structure of super ecliptic pHluorin A227D and truncations. The N-terminal segment deleted in ArcLight-Q239-NΔ5 and the C-terminal segment deleted in ArcLight-Q239-CΔ9 are indicated with red. B) Top: sequence of ArcLight-Q239-NΔ5 and ArcLight-Q239-CΔ9. Bottom: The mean response amplitude of ArcLight-Q239 (n = 7), ArcLight-Q239-NΔ5 (n = 6) and ArcLight-Q239-CΔ9 (n = 8). Groups that have significant difference with ArcLight Q239 were indicated with the asterisks (*** P<0.001). C) The mean fast component (Tau-on-1) and its amplitude contribution (A-on-1) in the double exponential fitting of the fluorescence response during depolarization. ArcLight-Q239 (n = 6), ArcLight-Q239- NΔ5 (n = 6) and ArcLight-Q239- CΔ9 (n = 8). Groups that have significant difference with ArcLight Q239 were indicated with the asterisks (* P<0.05). D) The mean fast component (Tau-off-1) and its amplitude contribution (A-off-1) in the double exponential fitting of the fluorescence response during repolarization. ArcLight-Q239 (n = 6), ArcLight-Q239- NΔ5 (n = 6) and ArcLight-Q239- CΔ9 (n = 8). Groups that have significant difference with ArcLight Q239 were indicated with the asterisks (* P<0.05). Values are means ± SEM.
Figure 8.
The effect of farnesylation at the C-terminus of super ecliptic pHluorin A227D on the response properties of ArcLight.
A) Top: Schematic topology of ArcLight-S249 with a farnesylation site. Bottom: Sequence of ArcLight-S249 with a farnesylation site. The farnesylation site added to the C-terminus of the fluorescent protein is shaded with pink. B) Left: Non- filtered, averaged optical responses of ArcLight-S249 (red, n = 10 cells; single trial for each cell) and ArcLight-S249 with a farnesylation site (blue, n = 6 cells; single trial for each cell) to +100 mV depolarizations of 300 ms from a holding potential of −70 mV. In this and subsequent figures, the timing of the depolarization is indicated with the black line. Right: The mean response amplitude of ArcLight-S249 (n = 10) and ArcLight-S249 with a farnesylation site (n = 6). (* P<0.05) C) The mean fast (Tau-on-1) and slow components (Tau-on-2) of the “on” dynamics of ArcLight-S249 (n = 9) and ArcLight-S249 with a farnesylation site (n = 6) during depolarization fitted with a double exponential equation. (* P<0.05) D) The mean fast (Tau-off-1) and slow components (Tau-off-2) of the “off” dynamics of ArcLight-S249 (n = 10) and ArcLight-S249 with a farnesylation site (n = 3) during repolarization fitted with a double exponential equation. (* P<0.05). Values are means ± SEM.
Figure 9.
The effect of using tandem super ecliptic pHluorin A227D on the response properties of ArcLight.
A) Schematic topology of ArcLight-S249 with tandem fluorescent proteins (tandem ArcLight-S249). The linker sequence between the two fluorescent proteins is shaded with pink. Non-essential amino acids “MDELYK” at the C-terminus of the front fluorescent protein and “MSKGEE” at the N-terminus of the back fluorescent protein were deleted in the probe. B) Left: Non-filtered, averaged optical responses of ArcLight-S249 (red, n = 10 cells; single trial for each cell) and tandem ArcLight-S249 (blue, n = 4 cells; single trial for each cell) to 100 mV depolarizations of 300 ms from the holding potential of −70 mV. Right: The mean response amplitude of ArcLight-S249 (n = 10) and tandem ArcLight-S249 (n = 4). (*** P<0.001) C) The dynamics of tandem ArcLight-S249 (n = 4) during depolarization is best fit with a single exponential equation, the mean tau of tandem ArcLight-S249 is compared with the fast (Tau-on-1) and slow components (Tau-on-2) of ArcLight-S249 (n = 9). (** P<0.005) D) The mean tau of tandem ArcLight-S249 (n = 4) during repolarization compared with the fast (Tau-off-1) and slow components (Tau-off-2) of ArcLight-S249 (n = 10). (*** P<0.005). Values are means ± SEM.