Bioluminescent Low-Affinity Ca2+ Indicator for ER with Multicolor Calcium Imaging in Single Living Cells,ACS Chemical Biology

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Bioluminescent Low-Affinity Ca2+ Indicator for ER with Multicolor Calcium Imaging in Single Living Cells
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-03-01 00:00:00 , DOI: 10.1021/acschembio.7b01014
Md Nadim Hossain ₁ , Kazushi Suzuki ₁ , Megumi Iwano ₂ , Tomoki Matsuda _{1,

2} , Takeharu Nagai _{1,

2}

Affiliation

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 μM) 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 μM), 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.

中文翻译：

生物发光的低亲和力Ca ²⁺指示剂，用于在单个活细胞中进行多色钙成像的ER。

肌/内质网（SR / ER）是最重要的细胞间Ca ²⁺储存区（亚毫摩尔浓度），在控制细胞内Ca ²⁺含量中起着至关重要的作用。为了研究细胞中SR / ER Ca ^2+的动力学，已使用具有低Ca ²⁺亲和力的基于荧光蛋白的遗传编码钙指示剂（GECI）。近来，已经报道了具有高亮度的基于生物发光蛋白的GECI，以抵消荧光成像的局限性，例如光毒性。但是，它们的Ca ²⁺亲和力很高，并且在细胞质，细胞核或线粒体中成像受限。在这项研究中，我们开发了一种新颖的青色，低亲和力（K _d= 110μM）强度生物发光GECI，它能够监控HeLa细胞的ER和C2C12衍生的肌管的SR中的Ca ²⁺动态。为了促进细胞器中Ca ²⁺的广泛浓度范围，我们还开发了一种中等亲和力（K _d = 18μM），橙色和生物发光GECI，它能够监控HeLa细胞线粒体中Ca ^2+的动态。有了这些指标，再加上现有的高亲和力，绿色生物发光GECI，我们就成功地同时在三个不同的细胞器（核，线粒体和ER）中进行了多色生物发光Ca ²⁺成像。彩色的，活的，生物发光的Ca ²⁺此处显示的成像可用于稳定地揭示细胞器之间的ER Ca ²⁺稳态和合作性Ca ²⁺调节。这将导致对Ca ²⁺相关的生理功能和病理生理机制的进一步了解。

更新日期：2018-03-01

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