pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

Abstract

Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation.

Figure 1.

Theoretical optimization of pH sensor properties. (A) Theoretical calculation of optimal pK_a for the largest fluorescence change from 5.5 to 7.5, with a n_H of 1.0. Black solid line, relative fluorescence at pH 7.5 (F_7.5/F_max); black dashed line, relative fluorescence at pH 5.5 (F_5.5/F_max); red line, fluorescence fold change (F_7.5/F_5.5); blue line, relative fluorescence intensity change ((F_7.5-F_5.5)/F_max); magenta line, intensity scaled fold change, which is the multiplication product of fold change and intensity change. (B) Effect of n_H on the intensity scaled fold change at different pK_a values. The pH-sensitive FPs covered in this work are mapped according to pK_a and n_H values. From top to bottom (n_H descending), right to left (pK_a descending): SEP; pHuji; pHoran4 (orange, same as mOrange and mOrange variants), pHoran3, A-17 (blue, same as mApple and mApple variants), pHoran2, and pHoran1; A-47, mNectarine (white), and mOrange; mCherry-TYG (black), A-9, and mApple; and pHTomato.

Figure 2.

Spectra and pH titration curves of pH-sensitive FPs. (A) Excitation and emission spectra of pH-sensitive FPs SEP, pHTomato, pHoran4, and pHuji at pH 5.5 (dashed line) and 7.5 (solid line). (B) pH titration curves of the indicated FP. (C) Fluorescence of HeLa cells expressing pDisplay proteins fused to the indicated FP as a function of pH. Error bars represent SEM.

Figure 3.

Detection of exocytosis events with TfR-pHuji. (A) Detection of exocytosis events with TfR-SEP. (a) Example of an exocytosis event recorded at 10 Hz. (b) Quantification of the fluorescence of the event shown in panel a. A background image (average of five frames before the event) was subtracted before quantification. Circles correspond to images in panel a. (c) Mean of 56 events quantified as in panel b in three cells. Dotted line is an extrapolation of the linear fit of the nine data points before event detection to account for photobleaching. (B) Fluorescence of a representative cell (out of four recorded cells) expressing TfR-pHuji illuminated at the time marked by the yellow arrowhead and imaged continuously at 10 Hz. (C) Time-lapse recording of the same cell as in B (100-ms illumination, 0.25 Hz) similar to the ones used for imaging CCV formation. (D) Same as in A for exocytosis events detected in TfR-pHuji–expressing cells. (c) Mean fluorescence values for 40 events in three cells. Error bars represent SEM.

Figure 4.

Detection of endocytic vesicles containing TfR-red FPs. (A) Images of NIH-3T3 cells transfected with TfR fused to SEP, pHTomato, pHoran4, or pHuji at pH 7.4. (B) Details corresponding to the boxed areas in A, at pH 7.4 (left) and 2 s later at pH 5.0 (middle, same contrast; right, increased contrast as indicated). Note the complete quenching of some clusters (blue arrowheads), whereas others are still visible (yellow arrowheads) at pH 5.0. (C) Examples of events detected in cells transfected with the four FPs. (D) Mean frequency of scission events detected with the different markers. The number of cells tested is indicated. (E) Proportion of terminal scission events with the different markers. (F) Mean fluorescence of nonterminal (dark green, 984 events) and terminal (light green, 802 events) scission events at pH 7.4 (top) and 5.0 (bottom) aligned to their time of detection in 14 cells transfected with TfR-SEP. The black lines indicate 95% confidence intervals for significant enrichment. (G) Same as F for eight cells transfected with TfR-pHuji (dark red, 598 nonterminal events; light red, 447 terminal events). Error bars represent SEM.

Figure 5.

TfR-pHuji colocalizes with CCPs and dynamin is recruited at the time of scission detected with TfR-pHuji. (A) Portion of a 3T3 cell cotransfected with TfR-pHuji and clc-GFP. The clusters of TfR-pHuji colocalize with CCPs (merged). (B) Fluorescence in the green and red channels of segmented clusters in a representative cell. Yellow dots, clusters detected in both channels (274 clusters); green dots, clusters detected in the GFP channel only (no matching structure in the red channel, 39 clusters); red dots, clusters detected in the pHuji channel only (no matching structure in the green channel, 34 clusters). Black dashed line shows linear regression of all clc-GFP clusters (R = 0.75). Green and red lines show the lower detection limits of GFP and pHuji clusters, respectively. (C, a) Proportion for n = 5 cells of clusters detected in both channels (yellow) or with either TfR-pHuji (red) or clc-GFP (green) as shown in B. (b) clc clusters enriched in TfR (yellow) or not (green) according to the top-right quadrant defined by the GFP and pHuji lower detection limits determined as in B and vice-versa (yellow and red). (D) Examples of nonterminal (a) and terminal (b) scission events in a cell coexpressing TfR-pHuji and clc-GFP. (c) Example of a scission event in a cell coexpressing TfR-pHuji and dyn1-GFP. Note the maximal recruitment of dyn1-GFP at −4 s. (E) Mean fluorescence of scission events aligned to their time of detection (left, 320 events in five cells cotransfected with TfR-pHuji and clc-GFP; right, 283 events in five cells cotransfected with TfR-pHuji and dyn1-GFP). Error bars represent SEM.

Figure 6.

Co-detection of TfR-SEP and TfR-pHuji within the same endocytic vesicle. (A) Portion of a 3T3 cell cotransfected with TfR-SEP and TfR-pHuji. (B) Fluorescence in the green and red channels of segmented clusters at pH 7.4 in a representative cell. Yellow dots, clusters detected in both channels (422 clusters); green dots, clusters detected in the SEP channel only (no matching structure in the red channel, 38 clusters); red dots, clusters detected in the pHuji channel only (no matching structure in the green channel, 89 clusters). Black dashed line shows linear regression of all TfR-SEP clusters (R = 0.85). Green and red lines show the lower detection limits of SEP and pHuji clusters, respectively. (C, a) Proportion for n = 5 cells of clusters detected in both channels (yellow) or with either TfR-pHuji (red) or TfR-SEP (green) as shown in B. (b) TfR-SEP clusters enriched in TfR-pHuji (yellow) or not (green) according to the top-right quadrant defined by the SEP and pHuji lower detection limits determined as in B and vice-versa (yellow and red). (D) Example of a scission event co-detected with TfR-SEP and TfR-pHuji. (E) Same analysis as in B performed on the fluorescence of CCVs at their time of detection at pH 5.0. Yellow dots, 303 CCVs; green dots, 570 CCVs; red dots, 119 CCVs. Black dashed line shows linear regression of all TfR-SEP–detected scission events (R = 0.59). Green and red lines show the lower detection limits of SEP and pHuji CCVs, respectively. (F) Same analysis as in C performed on detected CCVs at pH 5.0. SEP and pHuji lower detection limits were determined as in E. (G) Mean TfR-SEP and TfR-pHuji fluorescence at pH 7.4 (top) and 5.0 (bottom) of scission events detected in the green channel (3,721 events in five cells) aligned to the time of CCV detection. The black lines indicate 95% confidence intervals for significant enrichment. (H) Same as G with 2,177 scission events detected in the red channel. Error bars represent SEM.

Figure 7.

SEP-β2AR relocalizes and internalizes at TfR-pHuji clusters upon stimulation. (A) Portion of a HeLa cell cotransfected with SEP-β2AR and TfR-pHuji before (top) and during (bottom) application of the β2AR agonist isoproterenol (20 µM). (B) Fluorescence in the green and red channels of segmented clusters at pH 7.4 in a representative cell before (a) and during (b) application of isoproterenol. Yellow dots, clusters detected in both channels (238 [a] and 204 clusters [b]); green dots, clusters detected in the SEP channel only (77 [a] and 75 clusters [b]); red dots, clusters detected in the pHuji channel only (127 [a] and 96 clusters [b]). Black dashed lines show linear regressions of all TfR-pHuji clusters (R = 0.41 [a] and R = 0.87 [b]). Green and red lines show the lower limits of SEP and pHuji cluster detection, respectively. (C) Proportion for n = 7 cells of TfR-pHuji clusters enriched in SEP-β2AR (yellow) or not (red) according to the top-right quadrant defined by the SEP and pHuji lower detection limits determined as in B and vice-versa (yellow and green), before (a) and after (b) application of isoproterenol. (D, a) Frequency of events represented as cumulative number in seven cells of events detected with TfR-pHuji (left) and SEP-β2AR (right) during the course of the experiment, normalized at the end (17 min). Dotted lines are regression lines of the data before agonist application, extrapolated to the entire recording. (b) The frequency of scission events detected with SEP-β2AR (green) is significantly higher during isoproterenol application as compared with baseline (*, P < 0.01), whereas the frequency of TfR-pHuji–detected scission events (red) remains unchanged. Error bars represent SEM.

Figure 8.

TfR-pHuji and SEP-β2AR are differentially sorted at the level of CCV formation. (A and B) Examples of a scission event visible with both TfR-pHuji and SEP-β2AR (A) or with TfR-pHuji only during isoproterenol application (B). Note the enrichment of β2AR at pH 7.4 but not in the CCV. (C) Fluorescence at the time of detection in the green and red channels of CCVs detected in a representative cell during isoproterenol application. Yellow dots, CCVs detected in both channels (11 CCVs); green dots, CCVs detected in the SEP channel only (14 CCVs); red dots, clusters detected in the pHuji channel only (76 CCVs). Black dashed line shows linear regression of all TfR-pHuji clusters (R = 0.33). Green and red lines show the lower limits of SEP and pHuji CCV detection, respectively. (D, a) Proportion for n = 7 cells of vesicles detected in both channels (yellow) or with either TfR-pHuji (red) or SEP-β2AR (green). (b) TfR-pHuji CCVs enriched in SEP-β2AR (yellow) or not (red) according to the top-right quadrant defined by the SEP and pHuji lower detection limits determined as in B and vice-versa (yellow and green). (E) Mean TfR-pHuji and SEP-β2AR fluorescence at pH 7.4 (top) and 5.0 (bottom) of scission events detected in the red channel (570 events in seven cells) aligned to the time of CCV detection. The black lines indicate 95% confidence intervals for significant enrichment. (F) Same as E with 324 scission events detected in the green channel. (G) Working model in which TfR (red lollipops) is constitutively clustered at all clathrin-coated structures (brown lines) via AP-2 binding (orange squares) and internalized in vesicles via the action of dynamin (blue rings) regardless of isoproterenol application. On the opposite, SEP-β2AR (green lollipops) in basal conditions is homogenously distributed at the cell surface (black line) and occasionally internalized (a), whereas during application of isoproterenol it is actively targeted to CCPs via recruitment of cytosolic β-arrestin (green squares; b). This relocalization of ligand-bound receptors to all CCPs leads to potential surface-localized intracellular signaling platforms (yellow bolts), whereas ligand-induced internalization of β2AR only occurs in a subpopulation of CCVs. Error bars represent SEM.