Institute of Biological Information Processing, IBI-1, Research Center Jülich, 52428 Jülich, Germany.
Institute of Biological Information Processing, IBI-3, Research Center Jülich, 52428 Jülich, Germany.
Int J Mol Sci. 2022 Jun 13;23(12):6593. doi: 10.3390/ijms23126593.
Calcium (Ca) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca concentration ([Ca]) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca] are ideally suited to modulate Ca-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca, the introduction and the ongoing development of genetically encoded Ca indicators (GECI) opened a new era to study Ca-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from cells to study their biophysical properties in solution. All versions were examined to monitor Ca signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca signals at the (sub)-cellular level with pronounced temporal resolution.
钙离子在生理学和细胞信号转导中起着关键作用。细胞内钙离子浓度([Ca])比细胞外浓度低三个数量级，导致跨膜浓度梯度陡峭。因此，[Ca]的空间和时间动态非常适合调节 Ca 介导的细胞对外界信号的反应。已经开发了多种高度复杂的方法来深入了解细胞内 Ca 动力学。除了电生理测量和应用合成染料外，这些染料在与 Ca 相互作用时会改变其荧光特性，还引入并不断开发遗传编码的 Ca 指示剂（GECI），开创了研究活细胞和生物体中 Ca 驱动过程的新时代。在这里，我们专注于一种成熟的 GECI，即 GCaMP3.0。我们系统地用序列基序修饰该蛋白，使传感器定位于核、线粒体基质、线粒体外膜和质膜。个别变体和 GCaMP3.0 的细胞质版本从细胞中过表达和纯化，以研究它们在溶液中的生物物理特性。所有版本都进行了检查，以监测稳定转染细胞系和用重组腺相关病毒(rAAV)转导的原代皮质神经元中的 Ca 信号。在这项比较研究中，我们提供了证据，证明这是一种可靠的方法，可以可靠地在亚细胞水平上以显著的时间分辨率追踪 Ca 信号。
