{"title":"Real-Time FRET-Based Measurements of Ca2+/PKA Dynamics in Plasma Membrane Microdomains of Primary Hippocampal Neurons","authors":"Marta Sobolczyk-Prawda, Tomasz Boczek","doi":"10.1002/cpz1.1048","DOIUrl":null,"url":null,"abstract":"<p>Both Ca<sup>2+</sup> and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca<sup>2+</sup>/PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca<sup>2+</sup> and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.1048","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Both Ca2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca2+/PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.
基于 FRET 的原发性海马神经元质膜微域 Ca2+/PKA 动态实时测量
Ca2+ 和蛋白激酶 A(PKA)都是多方面、无处不在的信号分子,对于调节错综复杂的信号通路网络至关重要。然而,它们在特化膜区域内的动态特性仍不十分明确。通过使用基因编码的荧光指示剂特异性地靶向于不同的质膜微域,我们建立了一种方案,允许在离散的神经元微域中以高空间和时间分辨率观察 Ca2+/PKA 动态。该方法采用了带有灵敏相机和专用 CFP/YFP/mCherry 滤光片组的荧光显微镜,可同时检测供体-受体发射和红色荧光信号。在这份详细的分步指南中,我们概述了实验过程,包括大鼠原代神经元的分离和转染靶向脂质筏或质膜非移植区的生物传感器。我们提供了记录所需的必要设备和成像设置信息,同时强调了实时测量的关键参数和故障排除指南。最后,我们举例说明了在特定细胞区中观察到的 Ca2+ 和 PKA 变化。这项技术的应用可能对研究第二信使之间的交叉对话及其在各种病理条件下的变化具有重要意义。© 2024 Wiley Periodicals LLC.
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