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

这项研究的广泛的长期目标是全面了解钙池操作的钙离子进入， 在许多类型的细胞中，持续生理Ca 2+信号传导的基础过程。它有特别的 在T细胞和其他免疫系统细胞的活化中的重要性，并且因此是免疫缺陷综合征的治疗靶点。 自身免疫性疾病最近的研究发现，钙池操作的Ca 2+内流在心肌细胞中起着重要作用。 各种实体瘤的发展、进展和侵袭性。关键的蜂窝控制器 2005年发现了钙库操纵的钙内流，STIM 1和STIM 2，钙通道ORAI 1及其 2006年鉴定了旁系同源物ORAI 2和ORAI 3。然而，只有有限的研究， 调节该通路的细胞蛋白质。 这一具体的建议来自一个全基因组RNAi筛选，该筛选鉴定了数十个以前的 未被识别的调控器的存储操作的钙离子进入，其中丝状蛋白septins 4和5。 已知隔蛋白丝可划分质膜的某些特化亚区， 作为信号蛋白的支架，但它们没有连接到Ca 2+信号。进一步 实验得出这样的结论，即隔膜蛋白支配ER-等离子体下STIM-ORAI通道簇的稳定性 膜连接septins和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。这些发现强调了了解奥赖在 信号簇和动态变化的ER-质膜连接在Ca 2+信号。 该项目将使用传统的共聚焦显微镜和先进的成像技术，包括TIRF 显微镜和单粒子跟踪，沿着明智的蛋白质工程，来剖析蛋白质和脂质 在ER-质膜连接处的重排和局部信号传导过程， STIM-ORAI信号。目的是（1）确定septins沿着其他蛋白质和局部脂质 微结构域，限制奥赖在ER-质膜连接处的运动，从而稳定STIM。 奥赖复合物;（2）检查ER-1中磷酸肌醇空间分布的动态变化。 Ca 2+信号传导过程中的质膜连接，以及这些变化对STIM-ORAI的重要性 （3）确定ER膜蛋白TRIM 59和TMEM 110的作用，它们是Ca 2+的调节剂 在RNAi筛选中鉴定的内流，与STIM 1一样，在Ca 2 + 信号装置. 这项研究将为生理Ca 2+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.