Nanoscale regulation of store-operated calcium entry through STIM-ORAI signalling

这项研究广泛的长期目标是完全理解存储操作的钙离子内流，a 在许多类型的细胞中，持续的生理钙信号的基础过程。它有特殊的 在T细胞和其他免疫系统细胞的激活中的重要性，因此是治疗的靶点 自身免疫性疾病。最近的研究发现，商店操作的钙离子内流在 实体瘤各种实体肿瘤的发展、进展和侵袭性的关键蜂窝控制器 2005年发现了钙离子内流，STIM1和STIM2，钙通道ORAI1和STIM2。 ORAI2和ORAI3是在2006年确定的。然而，只有有限的研究是关于 调节这一途径的细胞蛋白质。 这一具体建议源于全基因组RNAi屏幕，该屏幕识别了数十个以前的 未被识别的商店操作的钙离子内流的调节器，其中包括丝状蛋白Septins 4和5。 已知Septin细丝可以区分质膜的某些特化亚区，并能 作为信号蛋白的支架，但它们还没有与钙信号联系起来。进一步 实验得出结论，Septins控制着STIM-ORAI通道簇在ER-血浆中的稳定性 膜结合部。间隔蛋白与质膜脂质磷脂酰肌醇的局部重排 4，5-二磷酸起着关键作用。这些发现突显了理解ORAI的稳定性的必要性 Ca~(2+)信号转导过程中的信号簇和ER-质膜连接的动态变化。 该项目将使用传统的共聚焦显微镜和包括TIRF在内的先进成像技术 显微镜和单粒子跟踪，以及明智的蛋白质工程，以剖析蛋白质和脂肪 控制和调制内质网-质膜连接的重排和局部信号过程 STIM-ORAI信号。其目的是(1)确定间隔蛋白以及其他蛋白质和局部脂质是如何 微域，抑制内质网-质膜连接的ORAI运动，从而稳定STIM- Orai复合体；(2)研究肌醇磷脂在内质网中空间分布的动态变化。 钙信号转导过程中的质膜连接及其对STIM-ORAI的重要性 信号传递；以及(3)确定内质网膜蛋白TRIM59和TMEM110的作用，它们是钙离子的调节因子 在RNAi屏幕上发现的内流，与STIM1一样，在钙离子过程中移动到内质膜连接 发信号。 这项研究将为生理钙信号的调节提供重要的见解，并可能揭示 自身免疫性疾病和癌症治疗干预的新靶点。

The broad longterm objective of this research is a complete understanding of store-operated Ca2+ entry, a process that underlies sustained physiological Ca2+ signalling in many types of cells. It has particular importance in the activation of T cells and other immune system cells, and hence is a therapeutic target in autoimmune diseases. Very recent research has uncovered an important role for store-operated Ca2+ influx in the development, progression, and invasiveness of a variety of solid tumors. The key cellular controllers of store-operated Ca2+ influx, STIM1 and STIM2, were identified in 2005, and the Ca2+ channel ORAI1 and its paralogues ORAI2 and ORAI3 were identified in 2006. There have been only limited studies, however, on cellular proteins that regulate the pathway. This specific proposal arises from a genome-wide RNAi screen that identified dozens of previously unrecognized regulators of store-operated Ca2+ entry, among them the filamentous proteins septins 4 and 5. Septin filaments had been known to demarcate certain specialized subregions of the plasma membrane and to serve as scaffolds for signalling proteins, but they had not been connected to Ca2+ signalling. Further experiments led to a conclusion that septins govern the stability of STIM-ORAI channel clusters at ER-plasma membrane junctions. A local rearrangement of septins and the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate plays a critical role. These findings highlight the need to understand the stability of ORAI at signalling clusters and the dynamic changes at ER-plasma membrane junctions during Ca2+ signalling. The project will use conventional confocal microscopy and advanced imaging techniques including TIRF microscopy and single-particle tracking, along with judicious protein engineering, to dissect the protein and lipid rearrangements and local signalling processes at ER-plasma membrane junctions that control and modulate STIM-ORAI signalling. The aims are (1) to determine how septins, along with other proteins and the local lipid microdomain, constrain ORAI movements at ER-plasma membrane junctions and thereby stabilize the STIM- ORAI complex; (2) to examine dynamic changes in the spatial distribution of phosphoinositides in the ER- plasma membrane junction during Ca2+ signalling, and the importance of these changes for STIM-ORAI signalling; and (3) to define the role of the ER-membrane proteins TRIM59 and TMEM110, regulators of Ca2+ influx identified in the RNAi screen, that, like STIM1, move to ER-plasma membrane junctions during Ca2+ signalling. This research will provide important insights into the regulation of physiological Ca2+ signals, and may uncover new targets for therapeutic intervention in autoimmune disease and cancer.