Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon

Abstract

AbstractRegulated exocytosis involves the Ca2+-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca2+-binding proteins [1]. The Ca2+-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow ‘cameleon’ (Ycam2) – comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP – which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin–Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic β cells, apparent resting values of cytosolic [Ca2+] and [Ca2+]gd were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within ∼1 μm of the plasma membrane underwent increases in [Ca2+]gd that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes