Waldeck-Weiermair Markus, Bischof Helmut, Blass Sandra, Deak Andras T, Klec Christiane, Graier Thomas, Roller Clara, Rost Rene, Eroglu Emrah, Gottschalk Benjamin, Hofmann Nicole A, Graier Wolfgang F, Malli Roland
Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.
Sensors (Basel). 2015 Jun 4;15(6):13052-68. doi: 10.3390/s150613052.
Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.
钙调蛋白是复杂的基因编码荧光探针，可用于定量细胞内的Ca2+信号。这些探针基于位于末端的荧光蛋白（FP）之间的Förster共振能量转移（FRET），当Ca2+与中央钙调蛋白肌球蛋白轻链激酶M13结构域结合时，它们会一起移动。大多数现有的钙调蛋白由青色和黄色荧光蛋白（CFP和YFP）作为FRET对组成。然而，具有绿色和橙色或红色荧光蛋白（GFP、OFP、RFP）的红移版本具有一些优点，例如光毒性较小，并且与细胞的自发荧光和fura-2（一种著名的化学Ca2+指示剂）的光谱重叠最小。虽然基于GFP/OFP或GFP/RFP的钙调蛋白已成功用于研究细胞质和线粒体Ca2+信号，但用于可视化内质网（ER）Ca2+动态的红移钙调蛋白迄今尚未开发出来。在本研究中，我们生成并测试了几种内质网靶向的红移钙调蛋白。我们的结果表明，基于GFP/OFP的钙调蛋白由于靶向错误及其高Ca2+结合亲和力，不适用于记录内质网Ca2+信号。然而，内质网靶向的基于GFP/RFP的探针适合以可靠的方式检测内质网Ca2+。通过这项研究，我们增加了用于可视化主要细胞内Ca2+储存库中Ca2+动态的钙调蛋白种类。
