title: Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.
abstract: Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-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 beta 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 approximately 1 microm 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.
authors.affiliation: Department of Biochemistry School of Medical Sciences University of Bristol Bristol, BS8 1TD, UK.
authors.forename: E
authors.initials: E
authors.lastname: Emmanouilidou
authors.forename: A G
authors.initials: AG
authors.lastname: Teschemacher
authors.forename: A E
authors.initials: AE
authors.lastname: Pouli
authors.forename: L I
authors.initials: LI
authors.lastname: Nicholls
authors.forename: E P
authors.initials: EP
authors.lastname: Seward
authors.forename: G A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D016716:PC12 Cells; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D002118:Calcium