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

Live cell imaging has revealed that calcium ions (Ca2+) pass in and out of many cellular organelles. However, technical hurdles have limited measurements of Ca2+ in acidic organelles, such as endosomes. Although evidence hints that endosomes play a role in Ca2+ signaling, direct measurements within endosomal lumina represent one of the final frontiers in organelle imaging. To measure Ca2+ in a TiVAMP-positive endosome sub-population, the pH-resistant ratiometric Ca2+ biosensor GEM-GECO1 and the ratiometric pH biosensor mKeima were used. A positive correlation between acidic endosomal pH and higher Ca2+ was observed within these Rab5a- and Rab7-positive compartments. Ca2+ concentration in most endosomes was estimated to be below 2 mu M, lower than Ca2+ levels in several other intracellular stores, indicating that endosomes may take up Ca2+ during physiological stimulation. Indeed, endosomes accumulated Ca2+ during glucose-stimulation, a condition where endosomal pH did not change. Our biosensors permitted the first measurements revealing a role for endosomes in cellular Ca2+ homeostasis during physiological stimulation. (C) 2015 The Authors. Published by Elsevier Ltd.