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

Regulated exocytosis involves the Ca2+-triggered fusion of secretory vesicl es with the plasma membrane, by activation of vesicle membrane Ca2+-binding proteins [1], The Ca2+-binding sites of these proteins are likely to lie w ithin 30 nm of the vesicle surface, a domain in which changes in Ca2+ conce ntration cannot be resolved by conventional fluorescence microscopy. A fluo rescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (G FP), 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-Y cam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory ve sicle surface ([Ca2+](gd)) through alterations in fluorescence resonance en ergy transfer (FRET) between the linked cyan and yellow fluorescent protein s (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN 6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [C a2+](gd) were similar throughout the cell. In MIN6 cells, following the act ivation of Ca2+ influx, the minority of vesicles that were within similar t o 1 mu m of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and great er than the apparent cytosolic [Ca2+] change. The ability to image both glo bal and compartmentalised [Ca2+] changes with recombinant targeted cameleon s should extend the usefulness of these new Ca2+ probes.