Fluorescence Lifetime Imaging of pH along the Secretory Pathway

Abstract

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.

Figure 1

FLIM of recombinant RpHLuorin2. (a) Representative confocal images of 10 μM recombinant RpHLuorin2 in calibration buffers with defined pH. The intensity image (left column) was convoluted with the fluorescence lifetime value per pixel and pseudo-colored (right column). (b) Representative fluorescence lifetime histograms of recombinant RpHLuorin2 in pH 4.87 solution (red-dashed line) or pH 7.5 solution (pink-dashed line). Fits with monoexponential decay functions (pH 4.87, solid red line; pH 7.5, solid pink line) convoluted with the instrumental response function (gray-dotted line). Graphs are normalized to the maximum photon counts. (c) Average lifetime histograms from the images of panel (a). 30 regions of interest (i.e., ∼10 × 10 μm of imaged area) were selected per pH buffer and the average lifetime τ was measured. (d) pH dependence of recombinant RpHLuorin2 in defined pH calibration buffers from the images of panel (a).

Figure 2

Calibration of RpHLuorin2 by FLIM in HeLa cells expressing GPI-RpHLuorin2. (a) Representative confocal micrographs of HeLa cells expressing GPI-RpHLuorin2 in defined calibration buffers. The intensity image (left column) was convoluted with the fluorescent lifetime value per pixel and pseudo-colored (right column). Scale bars, 10 μm. (b) Average lifetime histograms from the images of panel (a). N = 86 (pH 4.87), 108 (pH 5.31), 90 (pH 5.67), 115 (pH 6.17), 122 (pH 6.68), 113 (pH 7.03), and 120 (pH 7.5) cells from three independent experiments. (c) pH dependence of HeLa cells expressing GPI-RpHLuorin2 in defined pH calibration buffers from the images of panel (a).

Figure 3

Steady-state pH measurements of secretory pathway markers. (a) Schematic overview of all RpHLuorin2 constructs used in this study. The signal sequence of LAMP1 is removed following cotranslational ER insertion and is not shown in the diagram. ER-RpHLuorin2 contains the N-terminal signal sequence of the ER-resident protein calreticulin and a C-terminal ER retention signal KDEL. MW: molecular weight; RUSH: retention using selective hooks; and SBP: streptavidin-binding protein. (b) Representative confocal micrographs of HeLa cells expressing the mentioned RpHLuorin2 fusion constructs. The intensity image (left column) was convoluted with the fluorescent lifetime value per pixel and pseudo-colored (middle column). The intensity image was also convoluted with the calculated pH per pixel and pseudo-colored (right column). FLIM: fluorescence lifetime imaging microscopy. Scale bars, 10 μm. (c) Quantification of average pH values from panel (b). N = 88 (ER), 188 (MGAT2), 193 (GalT), and 134 (LAMP1) cells from three to five independent experiments.

Figure 4

Incomplete blockage of Golgi acidification by Bafilomycin A1. (a) Representative confocal micrographs of HeLa cells expressing MGAT2-RpHLuorin2 incubated for 1 h in the absence (solvent control DMSO) or presence of Bafilomycin A1 (200 nM BafA1). To generate the FLIM images (middle column), the intensity images (left column) were convoluted with the fluorescent lifetime value per pixel and pseudo-colored. To generate the pH images, the lifetimes were converted to the calculated pH per pixel and also convoluted with the fluorescence intensities (right column). Scale bars, 10 μm. (b) Quantification of average pH values from panel (a). N = 72 (DMSO) and 77 (BafA1) cells from four independent experiments. The dashed lines indicate the average pH of the ER from Figure 3c. (c,d) Same as panels (a,b), but now for GalT-RpHLuorin2. N = 68 (DMSO) and 50 (BafA1A) cells from four independent experiments.

Figure 5

Dynamic pH measurements along the secretory pathway. (a) Representative confocal micrographs of HeLa cells expressing MGAT2-RpHLuorin2 in the absence (Ctrl, green) or presence of BFA (orange). The intensity image (left column) was convoluted with the fluorescent lifetime value per pixel and pseudo-colored (middle column). The intensity image was also convoluted with the calculated pH per pixel and pseudo-colored (right column). FLIM, fluorescence lifetime imaging microscopy. Scale bars, 10 μm. (b) Quantification of average pH values from panel (a). N = 110 (DMSO) and 165 (BFA) cells from 2–3 independent experiments. (c) Representative confocal micrographs of HeLa cells expressing RUSH TNFα-RpHLuorin2 in the absence of biotin (0 min) or 20, 40, and 60 min after biotin addition. The intensity image (left column) was convoluted with the fluorescent lifetime value per pixel and pseudo-colored (middle column). The intensity image was also convoluted with the calculated pH per pixel and pseudo-colored (right column). FLIM, fluorescence lifetime imaging microscopy. Scale bars, 10 μm. See also Supporting Information Movie S1. (d) Quantification of average pH values of the cell shown in panel (c) and Supporting Information Movie S1. Plotted is the average apparent lifetime for all pixels of the projected imaged area of the cell. (e) Average pH measured of all cells expressing RUSH TNFα-RpHLuorin2. N = 29 from two independent experiments.