In vitro and in vivo calibration of low affinity genetic Ca2+ indicators

IF 4.3 2区 生物学 Q2 CELL BIOLOGY Cell calcium Pub Date : 2023-11-08 DOI:10.1016/j.ceca.2023.102819
Alba Delrio-lorenzo , Jonathan Rojo-ruiz, Patricia Torres-vidal, Maria Teresa Alonso, Javier García-sancho
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Abstract

Calcium is a universal intracellular messenger and proper Ca2+concentrations ([Ca2+]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca2+ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca2+ levels result in malfunction and disease. Selective intraorganellar Ca2+measurements are best achieved by using targeted genetically encoded Ca2+ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca2+] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca2+]ER/SR based on imaging of fluorescence after heating the tissue at 50–52 °C, since this value coincides with that obtained in the absence of Ca2+ (Rmin). Knowledge of the dynamic range (Rmax/Rmin) and the Ca2+-affinity (KD) of the indicator permits calculation of [Ca2+] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca2+ affinity green and red GECIs.

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体外和体内低亲和力遗传Ca2+指标的标定
钙是一种普遍的细胞内信使,适当的Ca2+浓度([Ca2+])在细胞质细胞器的细胞质溶胶和管腔中是细胞功能所必需的。Ca2+稳态是通过在质膜和内膜上的微妙泵/泄漏平衡来实现的,不适当的Ca2+水平会导致功能障碍和疾病。选择性胞内Ca2+测量最好通过使用靶向遗传编码的Ca2+指标(GECIs)来实现,但要将腔内荧光信号校准为准确的[Ca2+]是具有挑战性的,特别是在体内,由于难以在各种组织或条件下标准化和校准荧光信号。我们在这里报告了一种方法,在50-52°C加热组织后,根据荧光成像将靶向肌浆内网(ER/SR)的GAP (GFP-Aequorin Protein)的比率信号校准为[Ca2+]ER/SR,因为该值与缺乏Ca2+ (Rmin)时获得的值一致。该指标的动态范围(Rmax/Rmin)和Ca2+亲和力(KD)的知识允许通过应用一个简单的算法计算[Ca2+]。我们已经在体外用几种细胞类型(HeLa、HEK 293T和小鼠星形胶质细胞)以及果蝇体内验证了这一过程。此外,该方法适用于其他低Ca2+亲和力的绿色和红色geci。
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来源期刊
Cell calcium
Cell calcium 生物-细胞生物学
CiteScore
8.70
自引率
5.00%
发文量
115
审稿时长
35 days
期刊介绍: Cell Calcium covers the field of calcium metabolism and signalling in living systems, from aspects including inorganic chemistry, physiology, molecular biology and pathology. Topic themes include: Roles of calcium in regulating cellular events such as apoptosis, necrosis and organelle remodelling Influence of calcium regulation in affecting health and disease outcomes
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