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 (GFP), pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution unsuitable to follow the rapid transit of cargo between organelles. We therefore 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. Then, we analyze the dynamic pH changes within cells treated with Brefeldin A, a COPI coat inhibitor. Finally, we followed the pH changes of newly-synthesized molecules of the inflammatory cytokine tumor necrosis factor (TNF)-α while it was in transit from the endoplasmic reticulum 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.

Progress in pH-Sensitive sensors: essential tools for organelle pH detection, spotlighting mitochondrion and diverse applications

“…The trans/medial-Golgi pH ranges from 6.4 to 6.81, and the pH can be measured using β-1,4-galactosyltransferase (GT) fused with pH-sensitive EYFP (Llopis et al, 1998;Lázaro-Diéguez et al, 2006;Rivinoja et al, 2006). Various markers, including B4GALT1/ST6GAL1 (Qasba et al, 2008;Hassinen et al, 2010;Peanne et al, 2011;Khoder-Agha et al, 2019), and GalT (Linders et al, 2022), as well as TGN38 (Bos et al, 1993), are employed for trans-Golgi measurements, while the cis-Golgi markers GM130 (Tie et al, 2016;Zheng et al, 2022), GMAP-210 (Infante et al, 1999), GP73 (Bachert et al, 2007;Hu et al, 2011), MGAT2/GnTII (Hassinen et al, 2010), and Giantin (Linstedt and Hauri, 1993;Satoh et al, 2019) are utilized for cis/medial-Golgi measurements.…”

“…Lysosomes are acidic organelles, and their pH can be measured using fluorescent proteins sensitive to acidic conditions, such as mTFP1 (FIRE-pHLy) (Chin et al, 2021) and LAMP1 conjugated with RpHluorin (Linders et al, 2022). Additionally, markers such as Dnase2B (Shpak et al, 2008;Ohkouchi et al, 2013), and Cathepsin D (Chai et al, 2019) are used for lysosome localization.…”