Measuring pH in insulin secretory granules using phasor fluorescence lifetime imaging of a genetically encoded sensor

It is widely accepted that the pH of insulin granules is acidic, and that its active regulation during granule maturation plays a role in the process of insulin secretion by β-cells. Yet, a calibrated measurement of the absolute granule pH with organelle specificity is still lacking. To tackle this issue, we used the genetically encoded E ¹ GFP pH reporter inserted into the C-peptide of proinsulin and expressed in Insulinoma 1E cells. Following verification of correct targeting of the E ¹ GFP reporter in the insulin granules, phasor-based Fluorescence Lifetime Imaging Microscopy (FLIM) was applied to obtain a calibrated and probe-concentration-independent measurement of insulin-granule pH. Our results confirmed the acidic nature of insulin granules under maintenance cell-culturing conditions, with an average luminal pH of ~ 5.8, and showed that acidity is actively maintained, as evidenced by its near-neutralization upon treatment with the vacuolar H ⁺ -ATPase inhibitor Concanamycin. Additionally, by exploiting the intrinsic spatial resolution of FLIM, we highlighted that granules which are proximal to the plasma membrane are slightly more acidic (~ 0.1 pH units) than those which are distal, a difference preserved even during the early phase of glucose-induced insulin secretion. This study lays the foundations for future investigations of granule pH in physiology and disease.