Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

研究进展 STIM2在SOCE和细胞功能调节中的作用：之前我们已经证明了STIM2在调节SOCE和细胞功能中的作用。我们发现STIM2有助于(I)STIM1聚集和(Ii)在相对较高的ER-Ca~(2+)下对Orai1进行STIM1门控，即可能不会引起STIM1反应的条件；(Iii)将STIM1 C末端重塑为活性构象。我们在本财年的主要发现如下： 1.当外源表达时，低亲和力的ER-Ca~(2+)感受器蛋白STIM2在ER-PM连接处表现出结构性聚集，当与Orai1共表达时，它将通道招募到这些连接处。与这种共定位一致，在基础条件下，STIM2的表达导致内源性Orai1的激活。目前，还没有关于内源性STIM2的信息，无论它是预先聚集的，还是在基础和低强度刺激下如何调节的。在本研究中，我们使用CRISPR/Cas9将mVenus插入内源性STIM2，在N端标记该蛋白，并建立了表达荧光标记的内源性STIM2的细胞系。内源性STIM2在未受刺激的细胞的内质网-质膜连接中预先聚集。虽然大多数STIM2群集是移动的，但也有一小部分固定的STIM2群集。重要的是，在静息细胞的内质网-质膜连接中检测到的不动簇，表明了由功能性IP3Rs介导并由STIM2的N端感知的局部ER-Ca~(2+)减少的位置。在没有添加激动剂的情况下，依赖于PLC的PIP2的结构性水解和PKA活性共同控制着IP3R的功能和STIM2的预聚簇。因此，在激动剂刺激和协调Orai1/STIM1的招募后，固定的STIM2簇增加。我们的数据表明，STIM2簇在内质网-PM连接处的固定是对IP3R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IP3R和STIM2之间一种新的关键通信机制，即在基础条件下将ER-钙释放与Orai1/STIM1通道的组装结合起来，然后在激动剂刺激细胞后放大该通道。 2.我们先前证明IR引起的唾液腺功能障碍与早期一过性线粒体Ca~(2+)和ROSmt升高有关。这项工作的目的是研究早期ROSmt升高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的疗效，方法是在IR前不久和IR后几天对小鼠进行治疗。我们报道，用线粒体靶向抗氧化剂MitoTEMPO治疗小鼠，无论是单次(15GY)或分次(5x3GY)剂量的照射后，唾液腺分泌几乎完全得到保护。从MitoTEMPO处理、照射的小鼠唾液腺细胞分离出的ROSmt、(钙离子mt)和激活的caspase-3的初始增加与照射但未处理的动物细胞相比显著减弱。重要的是，MitoTEMPO治疗阻止了辐射导致的STIM1下降，从而保护了对唾液分泌至关重要的储存操作的钙离子进入。综上所述，这些发现确定了由辐射引起的ROSmt最初的增加是持续性唾液腺功能低下的关键驱动因素。我们认为，线粒体靶向抗氧化剂MitoTEMPO在预防IR诱导的唾液腺功能障碍方面具有潜在的重要作用。 3.Orai1与STIM蛋白在内质网-质膜连接处的组装触发了钙离子内流。质膜PIP2以及PIP2结合蛋白SEPT4显著影响Orai1-STIM1的相互作用。虽然Septins和PIP2可以组织肌动蛋白细胞骨架，但目前尚不清楚肌动蛋白在连接处的状态是否有助于SOCE。我们在这里报道了肌动蛋白重塑调节STIM1的聚集。我们的发现表明，涉及CDC42、WASP/WAVE和Arp2的PIP2和SEPT4依赖的机制调节了肌动蛋白重塑为STIM1点周围的环状结构。内质网钙库耗尽后，CDC42在内质膜区域的定位增强。PIP2的耗尽或SEPT4的敲除减弱了Cdc42在内质网-PM区域的招募。重要的是，SEPT4或CDC42+Arp2的敲除会扰乱肌动蛋白的组织以及STIM1的聚集。因此，Orai1对STIM1斑点的募集、SOCE和NFAT易位到细胞核的作用都减弱。CDC42基因敲除不影响STIM1-C末端诱导的钙内流。综上所述，我们的发现揭示了PIP2和SEPT4通过协调ER-PM连接中的肌动蛋白重塑来影响Orai1/STIM1聚集。肌动蛋白的这种动态重组在SOCE及其下游的钙依赖效应器功能的调节中起着重要作用。

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.