Emmanouilidou E, Teschemacher A G, Pouli A E, Nicholls L I, Seward E P, Rutter G A
Department of Biochemistry School of Medical Sciences University of Bristol Bristol, BS8 1TD, UK.
Curr Biol. 1999 Aug 26;9(16):915-8. doi: 10.1016/s0960-9822(99)80398-4.
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+ 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+ 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.
调节性胞吐作用涉及分泌囊泡与质膜的Ca(2+)触发融合，这是通过激活囊泡膜Ca(2+)结合蛋白实现的[1]。这些蛋白的Ca(2+)结合位点可能位于囊泡表面30纳米范围内，在这个区域内，传统荧光显微镜无法分辨Ca2+浓度的变化。一种名为黄色“钙黄绿素”（Ycam2）的Ca2+荧光指示剂——由绿色荧光蛋白（GFP）的蓝色发射突变体、钙调蛋白、钙调蛋白结合肽M13和增强型黄色发射GFP融合而成，可靶向特定细胞内位置，已被报道[2]。在此，我们构建了一种phogrin（一种定位于分泌颗粒膜的蛋白）与Ycam2（phogrin - Ycam2）的融合蛋白，通过Ycam2中相连的蓝色和黄色荧光蛋白（分别为CFP和YFP）之间荧光共振能量转移（FRET）的变化，来监测分泌囊泡表面Ca2+浓度（[Ca2+]gd）的变化。在神经内分泌PC12细胞和MIN6胰腺β细胞中，整个细胞内胞质[Ca2+]和Ca2+的表观静息值相似。在MIN6细胞中，Ca2+内流激活后，位于质膜约1微米范围内的少数囊泡，其Ca2+的增加显著大于深层囊泡，且大于表观胞质[Ca2+]的变化。利用重组靶向钙黄绿素对全局和局部[Ca2+]变化进行成像的能力，应能扩展这些新型Ca2+探针的用途。
