钙离子是一种高度时空特异性的信号分子，参与调节细胞众多生理功能。作为动物细胞中最大的钙库，内质网在细胞复杂的钙信号调控系统中发挥着核心的作用。内质网中的钙离子在细胞的精确调控下保持动态平衡，即为内质网钙稳态。内质网钙稳态失衡，会导致细胞功能紊乱，并与免疫相关疾病、心血管疾病、神经退行性疾病以及癌症等密切相关。解析内质网钙稳态调控机制，对理解这些疾病的发病机理以及寻找有效的治疗方法具有重要意义。然而，由于以往内质网钙指示剂存在着灵敏度较低等问题，导致有关内质网钙稳态调控因子的研究结果较为零散，亟待对内质网钙稳态调控因子进行系统解析。此外，相关的内质网钙水平操纵工具相对匮乏，而当前主要使用的药物工具特异性差，在应用中具有较大的局限性，因此有待开发精准可逆的内质网钙水平操纵工具。

本文结合超灵敏的比值型内质网钙指示剂与全基因组编辑技术对内质网钙稳态的调控因子进行了系统的筛选和验证，成功地建立了一个包含66个基因的内质网钙水平负调控因子库。根据该调控因子库的GO富集分析结果，本文选择了深入研究神经元发育过程中内质网钙稳态的调控。钙成像的结果表明，小鼠神经母细胞瘤Neuro-2a细胞分化及果蝇神经元发育过程中，内质网钙水平显著降低。通过生信分析及qPCR验证，在负调控因子库中筛选出了在神经元发育过程中显著上调表达的关键基因腺苷酸环化酶9（adenylate cyclase 9，AC9）。在果蝇神经元中特异性地敲减AC9，发育成熟后的神经元内质网钙水平不再降低。进一步的机制探究表明，AC9调控内质网钙水平并不依赖于cAMP途径，而是通过作用于质膜钙通道Orai1蛋白的C端，抑制其介导的钙池操纵的钙内流（store-operated Ca²⁺ entry，SOCE）。负调控因子库中的锚定蛋白Yotiao，也能通过与AC9的互作，作用于Orai1的C端。两者均通过抑制SOCE间接减弱了内质网钙库的重填，从而降低内质网钙水平。

基于SOCE通路在内质网钙稳态调控中的关键作用，本文选择以Orai1蛋白为对象，开发钙信号的人为操纵工具。通过引入具有高度时空特异性的光遗传学技术，合作设计构建了一种光激活的钙通道LOCa3（light operated calcium channel），实现了对SOCE通路的人为干预，并应用LOCa3成功缓解了阿尔茨海默病模型果蝇爬行能力的衰退。此外，本文还通过将Orai1蛋白重定位于内质网膜，结合光激活的STIM1工具，开发出首个能够直接调控内质网钙水平的光遗传学工具，实现了对内质网钙水平的精准可逆操纵。

综上，本文利用新型超灵敏的内质网钙指示剂，结合全基因组编辑技术，系统筛选并鉴定了内质网钙稳态调控因子，并对其中在神经元发育过程中起显著调控作用的因子做了深入的机制探究。此外，本文还设计开发出多种光遗传学钙信号工具，实现了对内质网钙水平及相关钙信号的人为操纵。本研究不仅丰富了对内质网钙稳态调控网络的认识，而且为钙信号相关疾病的治疗提供了潜在的靶点和策略。

Highly spatiotemporal calcium (Ca²⁺) signaling plays crucial roles in regulating numerous physiological functions. As the largest calcium store in animal cells, ER Ca²⁺ levels maintain dynamic homeostasis under precise regulation. Its disturbance is strongly linked to the onset and progression of human diseases, including immune diseases, cardiovascular diseases, neurodegenerative diseases, and cancer. Hence, elucidating the regulatory mechanisms of ER Ca²⁺ homeostasis is pivotal for deciphering the pathogenesis of these diseases and devising effective therapies. The lack of sensitive ratiometric ER Ca²⁺ indicators, nevertheless, hinders the systematic investigation of ER Ca²⁺ modulators and the underlying mechanisms. Furthermore, limitations in tools for manipulating ER Ca²⁺ levels highlight the pressing need for developing precise and reversible methods in this field.

 In this study, capitalizing on two ultra-sensitive ratiometric ER Ca²⁺ indicators combined with CRISPR/Cas9-based genome-wide screening, we systematically screened the regulatory factors of ER Ca²⁺ homeostasis and identified and a library of 66 factors capable of reducing the ER Ca²⁺ content. Based on the GO enrichment analysis of these regulatory factors, we chose to investigate the regulation of ER Ca²⁺ homeostasis during neuronal development. Through Ca imaging, this study revealed a notable decrease in ER Ca²⁺ levels during this process. In the negative regulatory factor library, this work pinpointed adenylate cyclase 9 (AC9) whose expression was significantly upregulated during neuro-2a cells differentiation and Drosophila brain development. Specific knockdown of AC9 in Drosophila neurons results in the cessation of ER calcium decrease upon neuronal maturation, indicating that AC9 is essential for reducing ER Ca²⁺ levels in neuronal development.

Mechanistically, the AC9-mediated production of cAMP is not essential for its ability to reduce ER Ca²⁺ content. Instead, AC9 inhibits store operated calcium entry (SOCE) by acting on the C terminus of Orai1, ultimately causing attenuation of ER Ca²⁺ level. The interacting protein of AC9, Yotiao, is also identified as a negative regulator of ER Ca²⁺ levels. It may function upstream of AC9, also acting on the C-terminus of Orai1. Both AC9 and Yotiao indirectly reduce ER Ca²⁺ levels by inhibiting SOCE to hinder the refilling of ER Ca²⁺ stores.

Based on the critical role of the SOCE pathway in regulating ER Ca²⁺ homeostasis, this study focused on the Orai1 protein to develop optogenetic tools for manipulating calcium signals. By inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel, a light-switchable optogenetic Ca²⁺ signaling tool, LOCa3 (light-operated calcium channel) was constructed. Applying this tool to the Drosophila brain alleviated the decline in climbing ability in a Drosophila model of Alzheimer's disease. Additionally, by relocating the Orai1 protein to the ER membrane and combining with a light-activated STIM1 tool, the first optogenetic tool capable of directly regulating ER Ca²⁺ levels was developed. This tool enabling precise and reversible manipulation of ER Ca²⁺ levels.

In summary, this study employed novel, highly sensitive ER Ca²⁺ indicators combined with genome-wide editing technology to systematically screen and validate the regulatory factors of ER Ca²⁺ homeostasis. It also conducted in-depth mechanistic analyses of the factors with significant regulatory effects. Additionally, this work developed optogenetic tools to manipulate intracellular Ca²⁺ signals. This research not only enriches our understanding of the complex network of ER Ca²⁺ homeostasis regulation but also provides potential targets and strategies for the treatment of related diseases.