Understanding Store-Operated Calcium Signal Transduction

项目摘要 Ca2+信号的显著空间和时间精度精细地控制着大量的细胞功能。 Ca2+信号反映了高度协调的Ca2+传感器和Ca2+通道的功能。之间的相互作用 ER中的Ca 2+传感器STIM蛋白和PM中的奥赖Ca 2+通道在Ca 2+信号产生中至关重要。的 两种蛋白质在ER-PM连接处物理偶联，介导"钙库操作"的Ca2+信号，在 控制基因表达、分泌、运动和生长。它们在Ca2+信号传导中的作用是至关重要的， STIM或奥赖蛋白可引起一系列疾病，包括严重的联合免疫缺陷、肌肉萎缩、 张力减退、自身免疫、皮肤发育不良和外分泌缺陷。STIM/奥赖表达的失调与 与心血管和气道重塑、神经退行性疾病、免疫力改变和癌症有关。我们 已经对STIM和奥赖蛋白的分子特性和细胞组织进行了大量定义。我们现在 转向纳米级的ER-PM交界处的环境中，他们的运作。连接Ca2+是关键， 产生"局部" Ca2+信号和维持"全局" Ca2+振荡， 穿透细胞核使用分子探针的概要，成像技术，基因缺失的细胞 系统和动物敲除模型，我们的工作是针对三个主要领域：（1）确定关键 疾病相关的STIM和奥赖亚型的差异，重点关注广泛表达的 缺乏关键Orai结合结构域的STIM2的STIM2.1剪接变体;使用超分辨率STED 通过显微镜和TIRF/FRET成像，我们探索了一种模型，通过该模型，STIM2.1破坏了奥赖的交联 通道亚型的连接，并确定聚类如何组织连接Ca2+信号 机械. (2)利用基因编码的钙离子通道，确定钙离子通道的微生理环境。 并将标记在STIM和奥赖蛋白上的Ca 2+探针应用于光遗传学，以监测高度限制的 交界处的Ca2+微环境;使用这些测量来探索STIM介导的奥赖聚集 通道控制局部连接Ca2+信号，改变InsP3R和SERCA泵的接近度， 调节全球钙振荡的产生。(3)了解钙库操纵的Ca2+信号是如何 转录转导，探索平滑肌和B细胞中的"STIM 2表型"如何靶向 STIM1缺失的动物与STIM1/STIM2.1表达水平和连接Ca2+信号有关，STIM1- 调节的奥赖聚集控制特定NFAT亚型激活并确定转录组变化 与STIM类型特异性Ca2+信号产生有关。我们的研究解决了我们在知识上的关键差距， 连接Ca2+如何调节局部和全局Ca2+信号的产生。STIM/Orai介导的核心作用 细胞生理学中钙库操纵的Ca2+信号，STIM/奥赖突变引起的广泛功能障碍， STIM/奥赖亚型之间的深层功能差异，以及亚型表达的异常改变 在重大疾病状态下，所有这些都将STIM-Orai接口定义为关键治疗靶点。

Project Summary The remarkable spatial and temporal precision of Ca2+ signals finely controls a vast array of cellular functions. Ca2+ signals reflect the function of highly coordinated Ca2+ sensors and Ca2+ channels. The interaction between Ca2+ sensor STIM proteins in the ER and Orai Ca2+ channels in the PM, is crucial in Ca2+ signal generation. The two proteins physically couple in ER-PM junctions, mediating “store-operated” Ca2+ signals, essential in controlling gene expression, secretion, motility and growth. Their role in Ca2+ signaling is critical, and defects in STIM or Orai proteins cause a spectrum of disorders including severe combined immunodeficiency, muscular hypotonia, autoimmunity, skin dysplasia, and exocrine defects. Dysregulation of STIM/Orai expression is closely linked to cardiovascular and airway remodeling, neurodegenerative disorders, altered immunity, and cancer. We have defined much on the molecular properties and cellular organization of STIM and Orai proteins. We now turn toward the nanoscale ER-PM junctional environment in which they operate. Junctional Ca2+ is critical in generating both “local” Ca2+ signals and in sustaining “global” Ca2+ oscillations that extend across the cytoplasm and penetrate the nucleus. Using a compendium of molecular probes, imaging technology, gene-deleted cellular systems, and animal knockout models, our work is directed toward three major areas: (1) Identifying critical differences in the disease-related STIM and Orai isoforms, focusing on operation of the widely expressed STIM2.1 splice variant of STIM2 that lacks the critical Orai-binding domain; using super-resolution STED microcopy and TIRF/FRET imaging we explore a model by which STIM2.1 disrupts the cross-linking of Orai channel subtypes within junctions, and determine how clustering organizes the junctional Ca2+ signaling machinery. (2) Defining the micro-physiological environment of Ca2+ entry junctions, utilizing genetically encoded and optogenetically applied Ca2+ probes tagged onto STIM and Orai proteins to monitor the highly restricted junctional Ca2+ micro-environment; using such measurements to explore how STIM-mediated clustering of Orai channels controls local junctional Ca2+ signals, modifies the proximity of InsP3Rs and SERCA pumps, and modulates the generation of global Ca2+ oscillations. (3) Understanding how store-operated Ca2+ signals are transcriptionally transduced, exploring how the “STIM2 phenotype” in smooth muscle- and B cell-targeted STIM1-deleted animals is related to STIM1/STIM2.1 expression levels and junctional Ca2+ signals, how STIM- modulated Orai clustering controls specific NFAT subtype activation, and determining transcriptome changes related to STIM type-specific Ca2+ signal generation. Our studies address the critical gap in our knowledge of how junctional Ca2+ tunes the generation of local and global Ca2+ signals. The central role of STIM/Orai-mediated store-operated Ca2+ signals in cell physiology, the widespread dysfunction caused by STIM/Orai mutations, the deep functional distinctions between STIM/Orai subtypes, and the extraordinary alteration of subtype expression in major disease states, all define the STIM-Orai interface as a pivotal therapeutic target.