Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations

Abstract

Live cell imaging has revealed that calcium ions (Ca(2+)) pass in and out of many cellular organelles. However, technical hurdles have limited measurements of Ca(2+) in acidic organelles, such as endosomes. Although evidence hints that endosomes play a role in Ca(2+) signaling, direct measurements within endosomal lumina represent one of the final frontiers in organelle imaging. To measure Ca(2+) in a TiVAMP-positive endosome sub-population, the pH-resistant ratiometric Ca(2+) biosensor GEM-GECO1 and the ratiometric pH biosensor mKeima were used. A positive correlation between acidic endosomal pH and higher Ca(2+) was observed within these Rab5a- and Rab7-positive compartments. Ca(2+) concentration in most endosomes was estimated to be below 2μM, lower than Ca(2+) levels in several other intracellular stores, indicating that endosomes may take up Ca(2+) during physiological stimulation. Indeed, endosomes accumulated Ca(2+) during glucose-stimulation, a condition where endosomal pH did not change. Our biosensors permitted the first measurements revealing a role for endosomes in cellular Ca(2+) homeostasis during physiological stimulation.

Structure and function of ER membrane contact sites with other organelles

“…Specifically, early endosomes marked by RAB5 GTPase have a luminal Ca 2+ concentration of around 0.5 μM, and late endosomes marked by RAB7 GTPase have a luminal Ca 2+ concentration of around 2.5 μM. Late endosomes and lysosomes contain Ca 2+ levels that are close to ER Ca 2+ levels 91,92 (FIG. 4a).…”

Environmental Effects on Reactive Oxygen Species Detection—Learning from the Phagosome

“…There are only a few FP-based pH biosensors available at the edges of the visible spectrum: the cyan ECFP (60) and the red pHRed engineered from mKeima (69). The combined detection of pH with pHRed and calcium concentration with the ratiometric and slightly pH sensitive GEM-GECO1 (pK a = 6.1) in the endosomal lumen of the same cells has been recently described (1). Besides the pH measurement itself, pHRed allowed the calibration and correction from pH variations of the signal measured by the GEM-GECO1 (1).…”

Organellar channels and transporters

“…Cl − influx regulates lysosomal acidification by providing counter ions for H + pumping [37, 38]. [Ca 2+ ] lumen is ~ 0.5 mM for lysosomes [28], higher than the low micromolar ranges for early and late endosomes [39]. Ca 2+ efflux from lysosomes is important for signal transduction [28].…”