Optogenetic toolkit for precise control of organellar calcium signaling

内质网(ER)，或肌肉细胞中的肌浆网，是细胞内最大的 CA2储存并在塑造钙信号维持的时空动力学中起着关键作用 细胞内钙动态平衡。反过来，钙也密切参与调节内质网功能， 包括脂质合成、蛋白质合成、折叠、修饰和易位。因此， ER采用了一系列调节剂来控制膜两侧的钙浓度和 通过膜接触部位介导与周围细胞器的钙转移。去调节内质网钙离子 动态平衡不仅导致内质网应激和未折叠蛋白反应(UPR)，而且还参与 心血管疾病、神经疾病、代谢综合征等疾病。 药物调控和遗传操作常被用来研究内质网钙通道的调控 但这些基本上不可逆转的方法往往缺乏空间精确度和特异性。 光遗传学技术为调控内质网钙稳态提供了一种新途径 优越的时空分辨率和高度的可逆性。因此，该团队提议创建一个 创新的光遗传工具包，名为遗传编码的ER钙执行器(GEECA)， 实现内质网钙稳态、内质网钙通讯和细胞器的光学询问 多种生物系统中钙离子的调节活性。在具体目标1中，团队将设计一套 基于内质网局部钙通道和/或其可光开关致动器的光可调GEECA 内质网钙信号具有不同的动力学和动力学特性。在具体目标2中，团队寻求开发 调控内质网和ITS间细胞器间拴系和钙转运的光遗传平台 周围的细胞器。该项目的成功实施将提供新的机会 实现内质网钙稳态和细胞器间钙信号的无创性和精确化调控 具有很高的时空精度，从而对包括内质网在内的细胞生理进行远程控制 压力、能量代谢、自噬和mTOR信号。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干预方法 与内质网钙信号的异常有关。

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.