Abstract：
The endoplasmic reticulum (ER) is the main reservoir of Ca2+ of the cell. Accurate and quantitative measuring of Ca2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca2+ indicators have been developed, including a family of fluorescent Ca2+ indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca2+-binding protein aequorin. GAP Ca2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca2+ concentrations; they are insensible to variations in the Mg2+ concentrations or pH variations (in the 6.5-8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca2+ affinity version of GAP, GAP3 (KD ≅ 489 µM), has been engineered to conform with the estimated [Ca2+] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca2+. The ratiometric measurements provide a quantitative method to assess accurate [Ca2+]ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca2+ indicators to simultaneously monitor ER and cytosolic Ca2+. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescence Basic Protocol 2: Monitoring ER Ca2+ Basic Protocol 3: Monitoring ER- and cytosolic-Ca2+ Support Protocol: Generation of a stable cell line expressing erGAP3.
摘要：
内质网(ER)是细胞Ca2+的主要储库。准确和定量地测量内质网内腔内的Ca2+动力学一直是具有挑战性的。在过去的十年中，已经开发了一些基因编码的Ca2指标，包括一系列荧光Ca2+指示剂，称为GFP-水母蛋白(GAP)。它们基于两种水母蛋白的融合，绿色荧光蛋白（GFP）和Ca2+结合蛋白aequorin。GAPCa2+指标表现出几个特征的组合：它们是激励比率指标，随着在405和470nm激发的荧光的相互变化，这对于成像实验是有利的;它们表现出Hill系数为1，这有助于将荧光信号校准为Ca2+浓度;它们对Mg2+浓度的变化或pH值的变化（在6.5-8.5范围内）不敏感;和，由于缺乏哺乳动物同源物，这些蛋白质在转基因动物中具有良好的表达。低Ca2+亲和力版本的GAP，GAP3(KD489µM),已被设计为符合ER中估计的[Ca2+]。靶向ER内腔的GAP3(erGAP3)可用于腔内Ca2+成像。比率测量提供了一种定量方法来评估准确的[Ca2+]ER，动态和静止。此外,erGAP3可以与合成的胞浆Ca2+指示剂结合，同时监测ER和胞浆Ca2+。这里,我们提供详细的方法来评估erGAP3表达和进行Ca2+成像，要么局限于ER腔，或同时在ER和细胞质中。©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案1:通过免疫荧光检测ER中的erGAP3基本方案2:监测ER-Ca2+基本方案3:监测ER-和胞质-Ca2+支持方案:生成表达erGAP3的稳定细胞系。
