Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

研究进展 STIM 2在SOCE和细胞功能调节中的作用：以前我们证明了STIM 2在调节SOCE和细胞功能中的作用。我们发现，STIM 2促进（i）STIM 1聚类和（ii）在相对高的ER-Ca 2+下，即可能不会引起STIM 1反应的条件下，Orai 1的STIM 1门控;（iii）将STIM 1 C末端重塑为活性构象。我们于本财政年度的主要发现如下： 1.当外源性表达时，STIM 2，一种低亲和力的ER-Ca 2+传感器蛋白，在ER-PM连接中显示组成性聚集，当与Orai 1共表达时，它将通道募集到这些连接中。与这种共定位一致，STIM 2的表达导致内源性Orai 1在基础条件下的激活。目前，还没有关于内源性STIM 2的信息，无论是预聚还是在基础和低强度刺激下如何调节。在这项研究中，我们使用CRISPR/Cas9将mVenus敲入内源性STIM 2，在N末端标记蛋白质，并生成表达荧光标记的内源性STIM 2的细胞系。内源性STIM 2在未刺激细胞的ER-PM连接处预聚簇。虽然大多数STIM 2群集是移动的，但有少量非移动的STIM 2群集。 重要的是，在静息细胞的ER-PM连接中检测到的不动簇表示由功能性IP 3R介导并由STIM 2的N-末端感测的局部ER-Ca 2+减少的位置。在不存在添加的激动剂的情况下，组成型PLC依赖性PIP 2水解和PKA活性协调控制IP 3R功能和STIM 2的预聚簇。因此，在激动剂刺激和协调Orai 1/STIM 1的募集后，不动的STIM 2簇增加。我们的数据表明，在ER-PM交界处的STIM 2簇的固定，IP 3R介导的局部ER-Ca 2+储存释放的早期反应，是启动SOCE的关键检查点。 总之，我们的数据揭示了IP 3R和STIM 2之间的一种新的和关键的通信机制，该机制将ER-Ca 2+释放与基础条件下Orai 1/STIM 1通道的组装偶联，然后在激动剂刺激细胞后扩增。 2.我们以前证明，IR诱导的唾液腺功能障碍与线粒体Ca 2+和ROSmt的早期短暂增加有关。这项工作的目的是检查ROSmt的早期上升在辐射诱导的唾液腺功能丧失中的作用。我们测试了mito-ROS清除剂，MitoTEMPO，治疗小鼠前不久，并在几天后IR。我们报告说，治疗小鼠的直肠靶向抗氧化剂，MitoTEMPO，导致几乎完全保护唾液腺分泌后，无论是单一的（15戈伊）或分次（5 × 3戈伊）剂量的辐射。从MitoTEMPO处理的、辐射的小鼠中分离的唾液腺细胞与来自辐射但未处理的动物的细胞相比显示出ROSmt、Ca 2 +mt和活化的半胱天冬酶-3的初始增加的显著减弱。重要的是，MitoTEMPO治疗防止了辐射诱导的STIM 1减少，从而保护了对唾液分泌至关重要的钙池操作的Ca 2+进入。 总之，这些发现确定了ROSmt的初始增加，这是由辐射诱导的，作为持续性唾液腺功能减退的关键驱动因素。我们认为，有针对性的抗氧化剂，MitoTEMPO，可以在预防IR诱导的唾液腺功能障碍的潜在重要性。 3.钙库操纵的钙进入（SOCE）是由Orai 1与ER-PM连接处的STIM蛋白组装触发的。质膜PIP 2以及PIP 2结合蛋白SEPT 4显著影响Orai 1-STIM 1相互作用。虽然septins和PIP 2可以组织肌动蛋白细胞骨架，但尚不清楚连接处内肌动蛋白的状态是否有助于SOCE。我们在此报告，肌动蛋白重塑调节STIM 1集群。我们的研究结果表明，PIP 2和SEPT 4依赖性机制涉及CDC 42，WASP/WAVE和ARP 2调节肌动蛋白重塑成一个环状结构周围的STIM 1斑点。在ER-质膜区域中的CDC 42定位在ER-Ca 2+库耗尽后增强。PIP 2缺失或SEPT 4敲低减弱了CDC 42向ER-PM区的募集。重要的是，SEPT 4或CDC 42 + ARP 2的敲低破坏了肌动蛋白的组织以及STIM 1簇集。因此，Orai 1募集到STIM 1斑点，SOCE和NFAT易位到细胞核都被减弱。STIM 1-C末端诱导的Ca 2+内流不受CDC 42敲低的影响。总之，我们的研究结果表明，PIP 2和SEPT 4通过协调ER-PM连接内的肌动蛋白重塑来影响Orai 1/STIM 1聚类。肌动蛋白的这种动态重组在调节SOCE和下游Ca 2+依赖性效应器功能中具有重要作用。

Research Advances Role of STIM2 in SOCE and Regulation of Cell Function: Previously we demonstrated the role of STIM2 in regulating SOCE and cell function. We showed that STIM2 facilitates (i) STIM1 clustering and (ii) STIM1-gating of Orai1 at relatively high ER-Ca2+, i.e. conditions that likely do not elicit a STIM1 response; (iii) remodeling STIM1 C-terminus into the active conformation. Our key findings in this fiscal year are as follows: 1. When exogenously expressed, STIM2, a low affinity ER-Ca2+-sensor protein, displays constitutive clustering within ER-PM junctions and when co-expressed with Orai1, it recruits the channel to these junctions. Consistent with this co-localization, expression of STIM2 leads to activation of endogenous Orai1 under basal conditions. Currently, there is no information about endogenous STIM2, whether it is pre-clustered or how it is regulated under basal and low intensity stimulations. In this study, we used CRISPR/Cas9 to knock-in mVenus into endogenous Stim2, tagging the protein at the N-terminus, and generated cell lines expressing fluorescently labeled endogenous STIM2. The endogenous STIM2 is pre-clustered in the ER-PM junctions of unstimulated cells. While majority of the STIM2 clusters are mobile, there is a small population of immobile STIM2 clusters. Importantly, the immobile clusters, detected in ER-PM junctions of resting cells, denote locations of local ER-Ca2+ decrease that is mediated by functional IP3Rs and sensed by the N-terminus of STIM2. In absence of added agonist, constitutive PLC-dependent PIP2 hydrolysis and PKA activity concertedly control IP3R function and pre-clustering of STIM2. Consequently, immobile STIM2 clusters increase following agonist stimulation and co-ordinate recruitment of Orai1/STIM1. Our data suggest that immobilization of STIM2 clusters in ER-PM junctions, an early response to IP3R-mediated local ER-Ca2+ store release, is a critical check-point for initiation of SOCE. Together, our data reveal a novel and critical mechanism of communication between the IP3R and STIM2 that couples ER-Ca2+ release with assembly of Orai1/STIM1 channels in basal conditions which is then amplified after agonist stimulation of cells . 2. We previously demonstrated that IR-induced salivary gland dysfunction is associated with an early transient increase in mitochondrial Ca2+ and ROSmt. The goal of this work was to examine the role of the early rise in ROSmt in radiation-induced loss of salivary gland function. We tested the efficacy of mito-ROS scavenger, MitoTEMPO, by treating mice shortly before and on several days after IR. We report that treatment of mice with the mitochondrial-targeted antioxidant, MitoTEMPO, resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5x3 Gy) doses of irradiation. Salivary gland cells isolated from MitoTEMPO-treated, irradiated, mice displayed significant attenuation of the initial increases in ROSmt, (Ca2+mt, and activated caspase-3 as compared to cells from irradiated, but untreated, animals. Importantly, MitoTEMPO treatment prevented radiation-induced decrease in STIM1, consequently protecting store-operated Ca2+ entry which is critical for saliva secretion. Together, these findings identify the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We suggest that the mitochondrially targeted antioxidant, MitoTEMPO, can be potentially important in preventing IR-induced salivary gland dysfunction. 3. Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.