Bioluminescent Low-Affinity Ca2+ Indicator for ER with Multicolor Calcium Imaging in Single Living Cells
Bioluminescent Low-Affinity Ca2+ Indicator for ER with Multicolor Calcium Imaging in Single Living Cells
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DOI：
10.1021/acschembio.7b01014
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发表时间：
2018-07-01
影响因子：
4
通讯作者：
Nagai, Takeharu
中科院分区：
文献类型：
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作者：
Hossain, Md Nadim;Suzuki, Kazushi;Nagai, Takeharu
肌浆/内质网 (SR/ER) 是最重要的细胞间 Ca2+ 储存（亚毫摩尔浓度），在控制细胞内 Ca2+ 水平方面发挥着至关重要的作用。为了研究细胞中 SR/ER Ca2+ 动力学，已使用具有低 Ca2+ 亲和力的基于荧光蛋白的基因编码钙指示剂 (GECI)。最近，据报道，基于生物发光蛋白的高亮度 GECI 可以克服荧光成像的限制，例如光毒性。然而，它们的 Ca2+ 亲和力很高，因此在细胞质、细胞核或线粒体中的成像受到限制。在本研究中，我们开发了一种新型青色、低亲和力（K-d = 110 μM）强度生物发光 GECI，它能够监测 HeLa 细胞 ER 和 C2C12 衍生肌管 SR 中的 Ca2+ 动态。为了促进细胞器中广泛的 Ca2+ 浓度范围，我们还开发了中等亲和力 (K-d = 18 μM)、橙色和生物发光 GECI，从而能够监测 HeLa 细胞线粒体中的 Ca2+ 动态。借助这些指标，结合现有的高亲和力、绿色生物发光 GECI，我们成功地同时对三种不同的细胞器（细胞核、线粒体和内质网）进行多色生物发光 Ca2+ 成像。这里展示的多色、活体、生物发光 Ca2+ 成像可用于稳定地揭示细胞器之间的 ER Ca2+ 稳态和协同 Ca2+ 调节。这将有助于进一步了解Ca2+相关的生理功能和病理生理机制。
The sarco/endoplasmic reticulum (SR/ER) is the foremost intercellular Ca2+ store (at submillimolar concentrations), playing a crucial role in controlling intracellular Ca2+ levels. For the investigation of SR/ER Ca2+ dynamics in cells, fluorescent protein-based genetically encoded calcium indicators (GECIs) with low Ca2+ affinity have been used. Recently, bioluminescent protein-based GECIs with high brightness have been reported to counter the constraints of fluorescence imaging, such as phototoxicity. However, their Ca2+ affinity is high and limited for imaging in the cytosol, nucleus, or mitochondria. In this study, we developed a novel cyan color, low-affinity (K-d = 110 mu M) intensiometric bioluminescent GECI, which enables monitoring of the Ca2+ dynamics in the ER of HeLa cells and the SR of C2C12-derived myotubes. To facilitate the broad concentration range of Ca2+ in cellular organelles, we additionally developed an intermediate affinity (K-d = 18 mu M), orange color, and bioluminescent GECI, which enables monitoring of Ca2+ dynamics in the mitochondria of HeLa cells. With these indicators, in conjunction with an existing high-affinity, green, bioluminescent GECI, we succeeded in multicolor bioluminescent Ca2+ imaging in three distinct organelles (nuclei, mitochondria, and ER) simultaneously. The multicolor, live, bioluminescent Ca2+ imaging demonstrated here can be used to stably reveal the ER Ca2+ homeostasis and cooperative Ca2+ regulation among organelles. This will lead to the further understanding of Ca2+-related physiological functions and pathophysiological mechanisms.