Optogenetic toolkit for precise control of organellar calcium signaling

内质网 (ER) 或肌肉细胞中的肌浆网，充当最大的细胞内网 Ca2 存储并在塑造 Ca2 信号的时空动态以维持 细胞内 Ca2 稳态。相反，钙也密切参与调节 ER 功能， 包括脂质合成、蛋白质合成、折叠、修饰和易位。因此， ER 采用一系列调节器来控制膜两侧的 Ca2 浓度， 通过膜接触位点介导 Ca2 与周围细胞器的转移。 ER Ca2 解除管制 稳态不仅导致 ER 应激和未折叠蛋白反应 (UPR)，而且还参与 心血管疾病、神经系统疾病、代谢综合征等疾病。 药理学调节和基因操作通常用于研究 ER Ca2 处理 机械，但这些基本上不可逆的方法往往缺乏空间精度和特异性。 光遗传学技术提供了一种调节 ER Ca2 稳态的新方法 优越的时空分辨率和高可逆性。因此，团队建议创建一个 创新的光遗传学工具包，称为基因编码内质网钙执行器（GEECA）， 实现内质网 Ca2 稳态、内质网细胞器 Ca2 通讯和细胞器的光学询问 Ca2 调节多个生物系统中的活性。在具体目标 1 中，团队将设计一组 基于内质网局部钙通道和/或其光开关致动器进行光调谐的 GEECA ER Ca2 信号具有不同的动力学和动态特性。在具体目标 2 中，团队力求开发 一个光遗传学平台来控制内质网与其之间的细胞器间束缚和Ca2+转移 周围的细胞器。该项目的成功实施将为 实现内质网 Ca2 稳态和细胞器间 Ca2 信号传导的无创和精确调节 具有高时空精度，从而对细胞生理学进行远程控制，包括 ER 应激、能量代谢、自噬和 mTOR 信号传导。从翻译的角度来看，分子 该项目产生的工具将为人类疾病提供新的介入方法 与异常的 ER Ca2 信号传导有关。

Endoplasmic reticulum (ER), or sarcoplasmic reticulum in muscle cells, acts as the largest intracellular Ca2+ store and plays a pivotal role in shaping the spatiotemporal dynamics of Ca2+ sigals to maintain intracellular Ca2+ homeostasis. Reciprocally, calcium is also intimately involved in regulating ER functions, including lipid synthesis, protein synthesis, folding, modifications and translocation. Consequently, the ER employs a series of regulators to control Ca2+ concentration on both sides of the membrane and mediate Ca2+ transfer with the surrounding organelles via membrane contact sites. Deregulated ER Ca2+ homeostasis not only results in ER stress and unfolded protein response (UPR), but also is involved in cardiovascular diseases, neurological diseases, metabolic syndromes and other diseases. Pharmacological modulation and genetic manipulations are often applied to study the ER Ca2+ handling machinery, but these largely irreversible approaches often lack spatial precision and specificity. Optogenetics technology provides a novel approach for modulating ER Ca2+ homeostasis with superior spatiotemporal resolution and high reversibility. Hence, the team proposes to create an innovative optogenetic toolkit, named as Genetically Encoded ER Calcium Actuators (GEECAs), that enable optical interrogation of ER Ca2+ homeostasis, ER-organelle Ca2+ communications and organellar Ca2+-modulated activities in multiple biological systems. In Specific Aim 1, the team will design a set of GEECAs based on ER-localized calcium channels and/or their photoswitchable actuators to photo-tune ER Ca2+ signals with varying kinetic and dynamic properties. In Specific Aim 2, the team seeks to develop an optogenetic platform to control inter-organellar tethering and Ca2+ transfer between ER and its surrounding organelles. The successful execution of this project will provide novel opportunities to achieve noninvasive and precise modulation of ER Ca2+ homeostasis and inter-organellar Ca2+ signaling with high spatiotemporal precision, thereby exerting remote control over cellular physiology, including ER stress, energy metabolism, autophagy and mTOR signaling. From a translational perspective, molecular tools generated from this project will provide novel interventional approaches for human diseases associated with aberrant ER Ca2+ signaling.