Name: monitoring endoplasmic reticulum calcium homeostasis using a gaussia luciferase sercamp
Uploaded: 2015-09-06T14:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

This work was supported by the Intramural Research Program at the National Institute on Drug Abuse. We thank Doug Howard, Chris Richie, Lowella Fortuno, and Josh Hinkle for their contributions to developing this method.

1. 体外试验 :从一个稳定的SH-SY5Y细胞检测SERCaMP发布<ol><li>板的SH-SY5Y-GLUC-ASARTDL（SERCaMP）在组织培养物以每表面面积的平方厘米150,000个细胞处理的平板。为96孔板，例如，种子50,000个细胞每孔（图1A）。生长SH-SY5Y细胞在DMEM（高葡萄糖，含有GlutaMAX，丙酮酸）+ 10％牛生长血清+ 1×青霉素/链霉素。 <ol><li>传代细胞的15倍（图1B）。更高的通道数尚未经过测试。 </li><li>返回细胞湿度培养箱在37℃下用5.5％的CO 2孵育过夜。 </li></ol></li><li>实验性治疗（S）前，以及通过将5微升培养上清到一个不透明的收集每一个基准样品寨96孔板。在收集，轻轻敲击板各方和漩涡，避免渐变效果。用粘合剂印章不透明板伊灵片，并储存于4℃直至酶法测定的时间。 注意:分泌GLUC-SERCaMP是在细胞培养基中非常稳定。我们以前报道GLUC活性约5-10％被损失了72小时培养后，在37℃（培养上的SH-SY5Y细胞）12。 </li><li>根据需要检查ER钙枯竭治疗的细胞（ 如环境 ，药物，遗传操作）。避免长时间暴露在外界环境（外控孵化器）作为pH值的变化会迅速发生在大气中的二氧化碳。添加HEPES到培养基中，在20毫米，来稳定pH值14，如果长时间操作是必需的。在培养液中15使用HEPES时，应避免暴露于光。 <ol><li>阳性对照:把细胞用100nM毒胡萝卜素（TG）或50μM环匹阿尼酸（CPA）4-6小时，在SH-SY5Y细胞的实验。在其它细胞系最大应答可能需要更长的孵育或不同concen化合物trations。 </li><li>阴性对照:来自父母的SH-SY5Y细胞收集培养基。在我们的经验中，背景发光该测定是从稳定的SH-SY5Y-GLUC-SERCaMP细胞基础分泌小于0.05％。 </li></ol></li><li>收集和条件培养基的储存器5微升样品在期望的时间点，如在步骤1.2中概述。 </li><li>在中期评估酶活性，如第5概述。 </li><li>检查细胞内的胰高血糖免疫印迹或发光测定法。 <ol><li>对于免疫印迹:裂解细胞在含有50mM Tris（pH值7.4），150 mM氯化钠，0.25％脱氧胆酸钠，1mM的EDTA，1％NP40改性RIPA缓冲液，和蛋白酶抑制剂。单独的上SDS-聚丙烯酰胺凝胶，并转移到0.20微米PVDF膜上。孵育膜GLUC抗体（1:2000稀释）过夜，在4℃。执行二抗孵化和膜洗涤根据用户定义的协议选择的检测试剂。 </li><li>供发光测定法（ 图2）:执行在冰上以下步骤: <ol><li>从组织培养板的孔中取出所有平台和冲洗用200μl冷PBS中。 </li><li>添加75微升含有50mM Tris（pH值7.5），150毫摩尔NaCl，1％NP40裂解缓冲液和蛋白酶抑制剂每个孔中。 </li><li>旋转板在定轨振荡器（120转），在4℃进行20分钟。 </li><li>轻轻吸取溶解物上下使用多通道移液器，避免产生气泡。将5微升裂解液为不透明板和评估，如第5条所列发光。 </li></ol></li></ol></li></ol> 2.在体外试验 :永生化细胞与SERCaMP的瞬时转染注意:我们已观察到，瞬时转染程序可诱导的细胞应激和钝随后GLUC-SERCaMP响应。下面的方法被开发，以尽量减少转EFF<html>学分在SH-SY5Y细胞。可能需要此过程（包括选择转染试剂），用于替代细胞系的优化。如果可能，建议使用分别在3和第1中概述的稳定细胞株或病毒转导的方法。 <ol><li>板SH-SY5Y细胞在100mm培养皿在4×10 6个细胞。这个菜将足以重新播种约300孔中（96孔板形式）以下转染过程。 </li><li>转染细胞用20微克质粒DNA（编码GLUC-SERCaMP）Xfect试剂和6微升Xfect试剂。规模的DNA和Xfect试剂相应的较大或较小的培养容器。 </li><li>返回细胞培养箱中培养48小时。 </li><li> Trypsinize细胞和补种到96孔板中，每孔60,000个细胞。按照第1列出后续步骤。 </li></ol> 3.在体外试验 :对病毒载体介导的表达GLUC-SERCaMP 帐篷"&gt;注意:腺相关病毒（AAV）载体包装16和纯化12和慢病毒的生产13先前已经报道。 <ol><li>使用下列引物和探针滴度GLUC-SERCaMP AAV:正向引物，5&#39;-CACGCCCAAGATGAAGAAGT-3&#39;;反向引物，5&#39;-GAACCCAGGAATCTCAGGAATG-3&#39;;探针（5&#39;-6-FAM / 3&#39;-BHQ-1标记的），5&#39;-TACGAAGGCGACAAAGAGTCCGC-3&#39;用于定量PCR。 <ol><li>通过线性化含有GLUC的质粒，并使用旋转柱（例如Machery-纳格尔NucleoSpin凝胶和PCR净化）纯化该DNA制备的标准曲线。通过在沿着一个质量梯状琼脂糖凝胶分离定量的DNA。从100微克/毫升，以PBS中的10 FG /毫升制备1点10稀释的DNA。计算的GLUC质粒DNA的拷贝/ ml，在每一个基于所述质粒的分子量的浓度。 </li><li>稀释所述AAV股在PBS（适当稀释将依赖于病毒制备的参数和根据经验确定）。 </li><li>使用以下条件运行在实时PCR系统进行PCR反应:95℃×5分钟，94℃×20秒，和60℃×1分钟的41个循环。利用标准曲线计算毫升拷贝/。 </li></ol></li><li>慢病毒滴度与P24伦蒂-X快速滴度试剂盒。解冻慢病毒等分冰和稀的SH-SY5Y介质中的p24调控蛋白。 <ol><li>稀释慢病毒，使得其将落入标准曲线（如1:20000或1:100）上。所需的稀释因子将基于所述慢病毒制剂参数而变化，并且必须凭经验确定。遵循制造商的指示对所有后续步骤。 </li></ol></li><li>板的细胞的96孔板。对SH-SY5Y细胞，在第1节中概述电镀程序可以遵循。对大鼠原代皮质神经元，细胞应隔离并接种于适当涂层板一小号先前描述的16。维持在补充有1×B27和500μM的L-谷氨酰胺Neurobasal培养基大鼠原代皮质神经元。执行中的一半交流隔日1次。 </li><li>转导细胞病毒。病毒转导的目标是实现低水平表达，如荧光素酶Gaussia提供健壮信号。 <ol><li> AAV转导SH-SY5Y细胞:转导以下电镀的一天。稀释的AAV至6.0×10 7的vg /微升在AAV稀释缓冲液（PBS + 0.5mM的MgCl 2的）。加入5微升稀释的AAV（3.0×10 8的vg;约6000 MOI）每96孔平板（图3A）良好。用10％漂白粉灭活病毒载体的浪费。 </li><li>大鼠原代皮层神经元的AAV转导:电镀转导后6-8天。 AAV稀释至4.0×10 6 VG /微升AAV稀释液。加入5微升稀释AAV（2.0×10 7 VG;约350 MOI）每井96孔板（图3B）。最优MOI应凭经验其它细胞类型来确定。用10％漂白粉灭活病毒载体的浪费。 </li><li> SH-SY5Y细胞的慢病毒转导:转导以下电镀的一天。稀慢病毒至20微微克/微升的p24中Hank氏平衡盐溶液（用滴定试剂盒浓度测定）。各5μl每孔的稀释的病毒添加（100皮克的p24，相当于125万​​的慢病毒颗粒，或〜1250 IFUs）96孔平板含有100微升体积。因此规模较大的格式。用10％漂白粉灭活病毒的浪费。 </li></ol></li><li>收集介质的预处理样品，并开始实验处理48小时（SH-SY5Y）或5-7天（大鼠原代皮质神经元）转导之后。 </li></ol> 4. 体内 SERCaMP分析注意:在开展任何动物的程序一定要通过您的机构，以获得适当的批准。都生存手术是有足够的麻醉无菌条件下进行。下面描述的所有程序已获得批准，并符合美国国立卫生研究院ACUC指引。 <ol><li>高压釜手术器械启动手术前（121℃，30分钟灭菌，20分钟干燥时间）。清洁的超声发生器紧随其后的所有程序，并在高压灭菌前所有仪器。保持在生存手术无菌条件。对于需要多个动物手术，擦拭用70％的酒精，ultrasonicate和胎圈之间的大鼠消毒手术器械。请参阅材料清单进行必要的手段。 </li><li>准备大鼠进行手术。这里我们使用雄性SD（180-200克）。 <ol><li>麻醉用异氟烷气体大鼠3分钟（4-5％异氟烷在1000毫升/分钟输送），然后加入甲苯噻嗪（8毫克/千克）和氯胺酮（80毫克/千克）的腹膜内注射。应用眼药膏，以防止角膜干燥。乙egin手术一旦老鼠是深度麻醉具体表现为以下尾巴或脚捏缺乏撤退反射的。 </li><li>剃略低于肋（下胸区域）到中间腹部区域腹部的区域。擦洗手术区三次，交70％的酒精和优碘擦洗。将大鼠在无菌手术单无菌手术区仰卧位。 </li></ol></li><li>稀的AAV-GLUC-SERCaMP至7.6×10 9的vg /毫升（最终浓度）。颠倒离心管充分混匀。 注意:病毒浓度的范围可以变化（ 图4）。 <ol><li>吸取105微升稀释AAV到无菌培养皿。使用30号针头收集病毒到注射器。 </li></ol></li><li>进行手术暴露肝脏和注入AAV-GLUC-SERCaMP。 <ol><li>在此之前做腹部切口，应用0.25％布比卡因的切口区域。使用手术刀，使下面的肋骨水平切口（AP近因2-3厘米）。生硬的解剖，从皮下组织分离结缔组织。 </li><li>切腹部肌肉，露出右内侧叶肝。 注:另外叶可根据最终应用注入。 </li><li>放置动物手术显微镜下，并调整以获得正确的内侧叶肝的视场。病毒注入到内侧叶实质内; 3个独立的站点，每个站点约33微升。 注:保留针组织5-10秒以下注射，以确保交付的整个注射量。 </li><li>分别缝合腹部肌肉和皮肤，用棉签添加抗生素软膏。将大鼠在恢复室，直到意识恢复了和大鼠能够保持直立的姿势。不要让老鼠（S）无人看管，直到它重新获得足够的意识，以保持胸骨斜卧。众议院单独直至切口愈合和缝线已被删除（7-14天）。 <br/&gt;注:根据美国国立卫生研究院手术后的指导方针，保持手术记录。用程序，日期，实验标识符，体重，和手术天注释笼卡。痛/窘迫，屎生产，活动和食物和水的消耗将被监测和记录3天后手术。术后镇痛通过加入对乙酰氨基酚到饮用水（450毫克/ 100毫升）提供，尽管我们不通常观察疼痛或痛苦的手术后的迹象。 </li></ol></li><li>注射后4-7天开始尾采血。用标签和前称量制备血液采集管。加入50微升肝素（1000单位/毫升），每管。 </li><li>地方大鼠在异氟烷麻醉室3分钟（4-5％异氟烷在1000毫升/分送到）。从腔室，并装在鼻锥（2-3％异氟烷以500cc /分钟提供）除去大鼠。如下尾部圆周率证明了缺乏撤退反射采血就可以开始，一旦老鼠被深度麻醉NCH​​。 <ol><li>用无菌剪刀尾（1-2毫米），切尖和滴采集血液进入预充肝素管。收集到的血液量达到大于2:1的血液肝素比（&gt; 100微升血液/ 50微升肝素）。采血卷可以基于实验设计进行调整。用湿棉签来止血粉适用于尾止血。 </li><li>库存血管在4℃，如果采集后续样品。剪刀擦拭用乙醇垫和珠集之间消毒。 </li><li>称重收集管和与肝素调整，以获得2:1的比例（血液:肝素）。这一步将正常化每种样品（表1）在肝素的量。 </li><li>离心管在2000×g离心5分钟，在4℃。转移上清液（血浆）到新的管中并储存在-80℃直至荧光素酶检测（第5部分）的时间。 注:样品达72小时之前，酶测定储存在4℃具有m上发光（图5A）inimal效果。至多3个冻融血浆样品的周期对发光没有影响（ 图5B）。 </li></ol></li><li>毒胡萝卜素施用（阳性对照）: <ol><li>通过在乙醇中稀释至2.5毫克/毫升的最终浓度制备毒胡萝卜素。注入毒胡萝卜素以1mg / kg腹腔进入下腹。 注意:thapsigargin触发增加凝血酶诱导的血小板凝聚17，并且可以从尾更难使采血。 </li></ol></li><li> Gaussia荧光素酶测定: <ol><li>解冻血浆样品在冰上。 注意:对于纵向研究，解冻并在同一天的所有时间点的样品（使用基板的单一制剂）进行发光测定。 </li><li>将10微升的血浆，以不透明的围墙板在部分每个血浆样品中5运行3-4技术重复描述测量酶活性。 </li></ol></li><li> Euthanasi一个 注意:SERCaMP技术的优点是，以纵向显示器的ER钙的能力。取决于实验参数和端点分析，动物是用适合的端点分析，并根据机构ACUC准则措施安乐死。 </li></ol> 5.发光检测<ol><li>通过在酸化的甲醇（30微升10当量盐酸至3毫升甲醇中）稀释所述化合物至20mM制备腔肠素（CTZ）储备液。准备一次性使用等分并储存在-80℃。 </li><li>准备上试验当天工作衬底。 <ol><li>用于体外测定 ，稀释腔肠至8微米的PBS，例如添加10微升20mM的CTZ库存到25毫升的PBS（图6A，B）。 </li><li> 对于体内测定法，腔肠素稀释至100μM的PBS，500mM的抗坏血酸，5mM的NaCl洗涤。 </li></ol></li><li>孵育制备的CTZ为至少30分钟在室温下开始测定前。 注意:该步骤通常包括在Gaussia萤光素酶测定法有快速基板衰减在制备后第一30分钟的报告。我们没有观察到这种效果在我们的系统（图6C）;然而，我们通常包括温育步骤，因为我们已发现对测定没有任何负面影响。 </li><li>使用读板器，其能够监测生物发光的，并配有一个基片喷射器。总理的线条与底物。 注意:通过行中的初始衬底可以容易降解。为了避免人为的低读数早期样品，测量实验样品之前注入20-30井空（装上读卡器空盘）。 </li><li>注入100微升底物为好，摇中速，持续5秒，并测量发光。为百特协同II板读取器，集成的光发射超过0.5秒（体外样品）和5秒（ 在体内的样品），用于读出步骤。优化平板阅读器参数理想的检测性能。 注:快速的信号衰减（相比，焕发出其他的荧光素酶观察动力学）Gaussia荧光素酶的展品闪烁动力学。 GLUC的闪光灯动力学是由血清细胞培养基中（图7A）的影响，突出在控制了横跨样品介质组合物的重要性。由于发光的迅速衰减加入底物（闪光动力学）后，注射和读取步骤必须均匀对于所有样品之间的时间。该读板器应设置为衬底到一个井注入，等待一个固定时间 （如我们使用5秒摇步骤），并读那么好。加入底物到整个板读数之前构成显著挑战除非衬底可以被同时加入到所有孔中。如果喷射器不可用，该问题可以部分通过孵育板规避为衬底阿迪之间10分钟化和测量（从而避免了衰减曲线的陡峭部分）（图7B）。 </li><li>重复注射，等待时间，并宣读了每口井的上板的步骤。 </li></ol>

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The authors have nothing to disclose.

这个协议强调了在体外和 GLUC-SERCaMP的体内效用并监控ER钙耗竭。虽然蛋白修饰产生SERCaMP似乎推广到其他记者蛋白12，我们选择了Gaussia荧光素酶是因为其强健（200-1000倍以上）生物发光相对于其他的荧光素酶18。我们证明跨越递送至原代大鼠皮质神经元，SH-SY5Y细胞，和大鼠肝100倍的剂量范围GLUC-SERCaMP病毒的检测的毒胡萝卜素诱导的GLUC-SERCaMP释放（图3和4）。我们先前描述的表达GLUC-ASARTDL一个稳定转染的细胞系的产生，并表明少至20个细胞，5×10 4个未标记的细胞群可以报告诱导响应12中的毒胡萝卜素。这里，我们表明，稳定细胞系可一致报告的Tg致响应出到15代，最analyz版，表明记者的随时间持续表达不影响它的能力被释放响应于ER钙耗竭（ 图1）。我们的数据表明，GLUC-SERCaMP主要的细胞的内质网内一直保持到ER钙耗竭的一个时间时，它被分泌。在"正常"条件下，存在低基础分泌，允许建立基线质网钙稳态12。以下的ER钙耗竭由毒胡萝卜素，以相反的方向细胞内外发光变化的水平。这可以表示为一个比率，以指示在传感器（图2）的稳态分布的偏移。重要的是，作为SERCaMP释放通过KDEL受体表达12进行调制，释放的大小可根据细胞类型而有所不同。我们设想，代&#39;ASARTDL&#39;交替KDEL状羧基端序列可被要求达到加厚在一个特定的细胞或组织类型发作SERCaMP响应。 对于体外试验 ，细胞外GLUC-SERCaMP的水平将随时间累积由于分泌的记者的稳定性;我们报道的近似5-10％的活性降低72小时12之后。因此，之前发起药物或基因的挑战ER钙稳态交换介质可能是必要的，以减少由于传感器积累背景信号。与此相反，GLUC-SERCAMP 在体内的半衰期为3.5-4.7分钟12表示在等离子体信号代表最近发布的记者进入循环血液。一旦血液是离体和加工成血浆，然而，GLUC-SERCaMP是非常稳定的（图5）。 为描述的方法，介质或血浆被转移到不透明的96孔板酶测定之前。两个重要的因素，使用GLUC为基础的记者时需要考虑s为酶的闪光动力学和腔肠基板的破裂。最适合于运行胰高血糖酶试验正常化加入底物和发光测量之间的时间酶标仪配备注射器。腔肠素的化学性质使它容易发生衰减，这就排除了比较在衬底制备后不同的时间测量的原始发光的值。折叠的样品间的差异，但是，被保持（图6），从而允许控制和实验样品之间的相对差被跟踪过的实验的持续时间（ 图9）。 调查渐进性疾病时，监测体内 ER钙波动的能力是有利的。而其它的方法，如荧光染料细胞质，适合用于急性在体外研究中，一个SERCaMP记者是首次允许纵向ER钙监控。欧- [R协议概述AAV-GLUC-SERCaMP直接肝注射。我们检测GLUC-SERCaMP流通不成问题动物的稳定水平56天注射后（最新的时间点测试，到目前为止），并预测再实验是可能的12。 AAV载体的体积小，测量在直径约20纳米，并造成最小的生物安全要求19。规避AAV单链基因组的转化成双链DNA，AAV-SERCaMP被打包为AAV血清型-1双链载体20。 AAV血清型-1已经显示有效地转导大鼠肝脏19,21;然而，我们的注射技术的需要注意的是整个身体前往其他组织潜在的病毒。虽然载体直接注射到肝，我们的方法所提出不能辨别SERCaMP释放源，因此，它是可能的组织比肝脏其他可能有助于GLUC-SERC放大器的信号。未来的遗传操作会限制言论自由目标的组织类型。例如，组织特异性的Cre驱动线交叉的一个依赖于Cre的GLUC-SERCaMP将允许ER钙经由等离子体采样组织特异性监控。 所描述的在体内的技术使用，由于记者的稳健性低的病毒滴度。 GLUC-SERCaMP释放可以从病毒且具有7.6×10 7的vg至7.6×10 9的vg（图4）被检测到。这个范围是低4-400倍报道在利用肝脏19的萤火虫荧光素酶为基础的病毒注射以前的工作。在较高浓度，我们观察到可检测的表达随时间的，这是可能的损失，由于动物的转基因22的免疫应答。病毒的效价更高应在可能的情况，由于信号损失的增加的可能性避免。比呈现低滴度也不会连接测试。该协议概述AAV-GLUC-SERCaMP注射到肝脏;类似的方法在理论上对于其他类型的组织是可行的。如病毒浓度和血清型参数必须有足够的表达在其它组织进行优化。 由于记者的稳健性，小体积的血液（100-150微升）可以从尾剪报收集，允许重复集合整个实验。这对于GLUC-SERCaMP释放的响应于毒胡萝卜素，其中我们观察到毒胡萝卜素施用接着返回至基线水平（图4）后高的水平纵向表征有用。适当的处理之后的血液收集血浆样品也很重要。因此，我们已经表明，SERCaMP之前酶测定（图5）是在血浆中稳定贮存于4℃多达72小时。此外，血浆样品可以经历至少三个冷冻/吨山楂周期对发光的影响最小（图5）。在实践中，我们存储所有样本在-80℃下，避免前评估酶测定不必要冻融循环，并分析所有样品用于实验的同时。 ER钙耗竭牵涉于多种疾病的发病机理。它经常被认为是阻碍细胞功能，导致未折叠蛋白反应（UPR）的激活的上游事件。普遍定期审议是由ER用来重建稳态条件5的适应性反应。内质网应激慢性状态超过UPR的能力，以恢复平衡，最终导致细胞死亡。 ER应激及ER钙失调疾病，如1型糖尿病，糖尿病性肾病，神经变性疾病和心血管dieases 5中观察到。钙失调和疾病的发病机制之间的关系，但是，difficULT划定。该SERCaMP技术具有跟踪这个过程结束的动物的寿命，从而提供洞察疾病的发生和发展的潜力。此外，设计成防止或修正的ER钙失调可以通过使用SERCaMP的来评估潜在疗法的评价。最后，查明GLUC-SERCaMP释放发生疾病提供了机会，设计并采用分泌的治疗性蛋白质或多肽作为SERCaMPs作为疾病调节基因治疗的一种手段。

内质网（ER）中的许多细胞的能力的功能，包括蛋白质折叠，蛋白质分泌，脂质稳态，和细胞内信号1。中央对正常的雌激素受体 ​​的功能是保持管腔钙离子浓度在约5000倍于细胞质2-4找到。这种能量密集型过程由萨尔科/内质网钙ATP酶（SERCA），一个泵，移动钙离子进入内质网调节。从ER钙的流出主要由兰尼（碱受体）和肌醇三磷酸（IP3受体）受体介导的。由于许多ER过程是依赖于钙，破坏商店会导致内质网应激和最终的细胞死亡。 ER钙失调已经在疾病，包括心肌病，糖尿病，阿尔茨海默氏观察到的，和帕金森氏5。由于这些疾病的进步性，已经挑战划定因果重发病机制和改建在ER钙库之间lationship。多种技术已经允许在我们的ER钙动力学，包括染料和遗传编码钙指标（GECIs）的理解显著进展。低亲和性钙染料，当结合到钙离子在荧光其中增加，可以装载到细胞以检查亚细胞区室的高浓度的钙6。 GECIs，如D1ER和捕手允许与亚细胞定位7-9的更精确的控制监测钙的波动。近日，又有类GECIs称为钙测定细胞器包埋蛋白质指标（CEPIA）已被描述10。第三种方法结合遗传学和小分子化学是靶向酯酶染料加载（TED），其利用遗传编码羧酸酯酶（靶向ER）与基于酯的钙染料11。 虽然AFORementioned方法都有内在的优势和弱点，他们可以通过荧光急性测量提供了有价值的见解ER钙的动态。然而，它们不是最佳的为经常需要调查疾病进展的纵向研究。以制定一个方法过度延长的时期以监测钙动力学的目标，我们确定并开发出一种蛋白修饰以创建分泌的ER钙监测蛋白（SERCaMPs）12。 SERCaMP规避与其他方法相关的，通过提供一种微创方法反复询问ER钙库一些局限性。我们已经证实了羧基末端肽ASARTDL（丙氨酸 - 丝氨酸 - 丙氨酸 - 精氨酸 - 苏氨酸 - 天冬氨酸 - 亮氨酸）足以促进ER滞留;然而，导致减少在ER钙的条件下，肽序列是不再能保留ER localization和蛋白质分泌13。所述SERCaMP技术的基础是ASARTDL到一个分泌蛋白的羧基末端（例如Gaussia萤光素酶，或GLUC），使得分泌受ER钙耗竭触发，从而产生的ER钙失调12健壮记者的附属物。 GLUC-SERCaMP通过转基因方法中的表达使得生物流体包括细胞培养基和等离子体对作为内质网钙稳态的指标的变化GLUC活性进行分析。该方法具有渐进的改变在ER钙库在体外和体内的纵向研究应用。以下协议被写为用于使用GLUC基于SERCaMP研究ER钙稳态的概要，但协议可以作为替代记者SERCaMPs的指南。

<table><tbody><tr><td>1.5 ml tubes</td><td>Fisher&amp;nbsp;</td><td>02-682-550</td><td></td></tr><tr><td>10% NP-40 solution&amp;nbsp;</td><td>Pierce</td><td>28324</td><td>for intracellular GLuc assays</td></tr><tr><td>1 ml luer-lok syringes</td><td>Fisher</td><td>14-823-30</td><td></td></tr><tr><td>200 microliter filter tips</td><td>Rainin</td><td>RT-L200F</td><td></td></tr><tr><td>3-0 surgical sutures</td><td>Fisher</td><td>NC9598192</td><td></td></tr><tr><td>30 G needles</td><td>Fisher Scientific</td><td>14-821-13A&amp;nbsp;</td><td></td></tr><tr><td>Adhesive microplate sealing sheets</td><td>Thermo</td><td>AB-0558</td><td></td></tr><tr><td>Alcohol prep pads</td><td>Fisher</td><td>22-246-073</td><td></td></tr><tr><td>Anesthesia Auto Flow System</td><td>E-Z Anesthesia</td><td>EZ-AF9000</td><td></td></tr><tr><td>Animal recovery chamber</td><td>Lyon Vet</td><td>ICU-912-004</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>Betadine solution</td><td>Fisher</td><td>NC9386574</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>n/a</td><td></td></tr><tr><td>Bovine growth serum</td><td>Thermo</td><td>SH30541.03</td><td></td></tr><tr><td>Coelenterazine, Native</td><td>Regis Technologies</td><td>1-361204-200</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>Cutting needles 3/8 circle sutures</td><td>WPI</td><td>501803</td><td></td></tr><tr><td>Digital ultrasconic cleaner</td><td>Fisher Scientific</td><td>FS60D</td><td></td></tr><tr><td>DMEM high glucose, GlutaMAX, pyruvate</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>DNA mass ladder</td><td>Life Technologies</td><td>10496-016</td><td></td></tr><tr><td>Gaussia luciferase (recombinant protein)</td><td>Nanolight</td><td>321-100</td><td></td></tr><tr><td>Gaussia luciferase antibody (for WB, ICC, or IHC)</td><td>New England Biolabs</td><td>E8023S</td><td>1:2,000 (WB)</td></tr><tr><td>Germinator 500</td><td>CellPoint Scientific</td><td>DS-401</td><td></td></tr><tr><td>Gluc assay plates (96 well, opaque)</td><td>Fisher</td><td>07-200-589</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>Life Technologies</td><td>14175-095</td><td></td></tr><tr><td>Heparin</td><td>Allmedtech</td><td>63323-276-02</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Schein</td><td>29404</td><td></td></tr><tr><td>Ketamine</td><td>Henry Schein</td><td>995-2949</td><td></td></tr><tr><td>Kwik Stop Styptic powder</td><td>Butler Schein</td><td>5867</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>a452-4</td><td></td></tr><tr><td>Microfuge 22R Centrifuge</td><td>Bekman Colter</td><td>368831</td><td></td></tr><tr><td>Neosporin</td><td>Fisher</td><td>19-898-143</td><td></td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103049</td><td></td></tr><tr><td>Nikon Stereoscope</td><td>Nikon</td><td>SMZ745T</td><td></td></tr><tr><td>Nucleospin Gel and PCR Cleanup</td><td>Machery-Nagel</td><td>740609</td><td></td></tr><tr><td>P200 pipet</td><td>Rainin</td><td>L-200XLS+</td><td></td></tr><tr><td>p24 Lenti-X rapid titer kit</td><td>Clontech</td><td>632200</td><td></td></tr><tr><td>PCR film seal</td><td>Fisher</td><td>AB0558</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>ReFresh Charcoal Filter canister</td><td>E-Z Anesthesia</td><td>EZ-258</td><td></td></tr><tr><td>Scalpel blades, #10</td><td>Fine Science tools Inc</td><td>10010-00</td><td></td></tr><tr><td>SD rats 150-200 g</td><td>Charles River</td><td>Rats</td><td>rats ordered at 150-200 g.&amp;nbsp; Surgery 5 days after arrival</td></tr><tr><td>Small animal ear tags</td><td>National Band and Tag co</td><td>1005-1</td><td></td></tr><tr><td>Sterile surgical drapes</td><td>Braintree Scientific</td><td>SP-MPS</td><td></td></tr><tr><td>Synergy 2 plate reader</td><td>BioTek</td><td>n/a</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystems</td><td>4304437</td><td></td></tr><tr><td>Thapsigargin</td><td>Sigma</td><td>T9033</td><td>harmful to human health</td></tr><tr><td>Virapower lentiviral packaging mix</td><td>Life Technologies</td><td>K4975-00</td><td></td></tr><tr><td>Xfect Transfection reagent</td><td>Clontech</td><td>631318</td><td></td></tr><tr><td>Xylazine</td><td>Valley Vet</td><td>468RX</td><td></td></tr></tbody></table>

monitoring endoplasmic reticulum calcium homeostasis using a gaussia luciferase sercamp

内质网（ER）中含有的细胞内钙的最高水平，其浓度大约5000倍大于细胞质水平。严格控制ER钙是必不可少的蛋白质折叠，修改和贩运。扰动ER钙可导致解折叠蛋白质效应，一个三脚ER应激反应机制的激活，和在各种疾病有助于发病机理。在疾病发作和进展并监控ER钙改变的能力是在实践中在原则上重要，但具有挑战性。用于监测的ER钙目前可用的方法，如钙依赖性荧光染料和蛋白质，都提供了深入了解细胞中的ER钙动力学，但是这些工具不适合用于体内研究 。我们的实验室已经证明，一个修改Gaussia萤光素酶的羧基末端赋予记者的分泌响应于ER钙枯竭。该方法使用荧光素酶基，分泌的ER钙监测蛋白（SERCaMP），用于在体外和体内应用如本文所述。该视频强调了ER钙纵向监测肝注射，药理学处理GLUC-SERCaMP，血液采集和加工，检测参数。

该GLUC-SERCaMP方法通过取样外液允许对ER钙稳态评估。多个控件可以包括在实验设计，以提高结果的解释。首先，使用组成型分泌记者的（例如未经GLUC C-末端ASARTDL或"GLUC-否标签"）可以被用来评估对分泌途径（全球细胞分泌）和转基因表达实验处理的影响。例如，增加在两个GLUC-SERCaMP和GLUC-否标签的胞外水平将被认为是一个不明确的结果。可替换地，在增加GLUC-SERCaMP分泌与对应缺乏GLUC-否标签响应支持一个ER钙依赖性事件（图8）。 GLUC-SERCaMP的响应ER钙耗竭的分泌可通过测量细胞内GLUC进一步评估，不过这仅限于单一时间点作为裂解是必需的。诠释<html> racellular GLUC-SERCaMP将减少在响应于ER钙耗竭的，因为它逐渐分泌，和细胞外:胞内比（计算为每个单独的孔）将增加（图2B）。外:胞内比是有用的，以控制更改在转基因表达。 对ER钙库的药物调节剂可被用来确认ER钙与SERCaMP释放在新颖范例的贡献。丹曲林，一碱受体拮抗剂，已知以稳定的ER钙，这应该减少或抑制SERCaMP的释放响应于Tg的（图8B）。它建议，如果可能的话，要测试影响ER钙流（例如Xestospongin C，4-氯-间-甲酚）在新的实验范例采用SERCaMP另外的化合物。这些研究通常是在体内挑战，但也可以是在相应的组织培养模型可行的。 "&gt; 对于体外使用GLUC基SERCaMP研究，推荐计算相对于对照（多个）上各个板的响应。在&#39;治疗引起的反应可以通过跟踪变化与对照相对响应（评估在时间过程对于比较）板之间，这是不可取的多个板，以比较原始数据，由于衰变衬底，其可以导致改变的时间点（图6C）之间的原始值。一板内使相对比较，可以减少这种影响（图6D）。数据分析在体外的Tg诱导SERCaMP释放实验的一个例子是在图9。 对于体内使用GLUC-SERCaMP研究，数据应为每个独立的动物来评价的，每一个作为自己的"前处理"的控制动物。这是必要的，以说明可变性在转基因德尔ivery和动物（ 图4A）之间的表达。 SERCaMP响应的大小会受各种因素，其中许多都涉及到细胞的基线健康，并且可以由研究者来直接控制。为了最大SERCaMP响应，当务之急是要优化有利于基础ER钙稳态条件。这包括种子细胞以最佳密度和使用低病毒滴度。不同的细胞和组织类型可能有额外的要求，应根据经验确定。 <img alt="图1" src="/files/ftp_upload/53199/53199fig1.jpg" /> 图1:稳定的SH-SY5Y细胞系接种密度和传代次数的考虑（A）中的SH-SY5Y-GLUC-SERCaMP细胞以不同密度和培养40小时。分泌胰高血糖-SERCaMP测量与300纳米的TgØ治疗后4小时ř车辆控制（平均值±SD，N ​​= 6）。毒胡萝卜素诱导的增加GLUC-SERCaMP分泌被表示为每个细胞密度。 （B）的SH-SY5Y-GLUC-SERCaMP细胞传代次数2和15检查毒胡萝卜素诱导的分泌。将细胞用100nM的Tg处理2小时或4小时，并分泌GLUC活性标准化为赋形剂处理的对照组（平均±SD，n = 4时，通行因子无显著影响，双因子ANOVA）。虚线表示Y = 1， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53199/53199fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53199/53199fig2.jpg" /> 图2:酶测定细胞内GLUC-SERCaMP的测量（A）的细胞内和 （B）的胞外GLUC酶活性在大鼠原心病评估蒂卡尔神经元（转导AAV-GLUC-SERCaMP）。转导后六天，将细胞用60纳米毒胡萝卜素处理4小时。由于GLUC-ASARTDL累积在培养基，细胞内含量相应减少被观察到。 （C）的细胞外的比例:胞内GLUC活性可以计算为每个单独的井。 <img alt="图3" src="/files/ftp_upload/53199/53199fig3.jpg" /> 图3:AAV-GLUC-ASARTDL滴度与为SH-SY5Y细胞和大鼠原代皮质神经元毒胡萝卜素诱导的反应（A）中的SH-SY5Y细胞接种于96孔板中，每孔5×10 4个细胞，使其附着过夜。将细胞转导的感染的指示的复数（MOI），培养48小时，然后用300nM的Tg的或载体处理。测量发光的介质8小时后（平均值±SEM，n = 3时）。 * P &lt;0.05，MULtiple t检验（霍尔姆 - Sidak校正）。 （B）大鼠大脑皮质神经元被转上DIV8与AAV-GLUC-ASARTDL在指定的MOI（使用每孔逼近60,000个细胞的传导时间计算）。转导后五天，细胞用100nM的Tg的或在介质中的车辆控制和发光物8小时后，测量（平均值±SEM，N = 6）。 * P &lt;0.05，多重t-检验（Holm的-Sidak校正）。 <img alt="图4" src="/files/ftp_upload/53199/53199fig4.jpg" /> 图4:大鼠thapsigargin触发诱导下肝内注射了病毒滴度范围GLUC-SERCaMP释放到血液中（A）的原始发光值（n = 8），肝内注射不同的AAV-GLUC-ASARTDL滴度。毒胡萝卜素（1毫克/千克）注射（IP）给药第12天收集血液，在指定的时间点，并储存在-80＆＃176; C，直到检测的时间。 （ 二）从面板的标准化荧光值，表明大鼠毒胡萝卜素诱导SERCaMP反应（N = 8），表达不同量的AAV-GLUC-ASARTDL的。 <img alt="图5" src="/files/ftp_upload/53199/53199fig5.jpg" /> 图5:搬运和存储GLUC-SERCaMP血浆样品（体内）（A）的血浆从大鼠收集肝内表达GLUC-SERCaMP（10例），等分并储存在-80℃直到测定的时间。从-80℃取出并储存在4℃下之前发光测定指示的时间管（平均值±SD）。上GLUC酶活多次冷冻/血浆样品解冻的（B）的影响。血浆样品从大鼠收集肝内表达AAV-GLUC-SERCaMP（10）进行指定数目冻融循环，并测定发光（平均值±SD）。 <img alt="图6" src="/files/ftp_upload/53199/53199fig6.jpg" /> 图6:准备腔肠基板GLUC-SERCaMP测定（A）的培养基，从与300nM的Tg的（或载体对照）5小时处理过的SH-SY5Y-GLUC-SERCaMP细胞系收集的。在介质GLUC活性通过注射PBS +各种浓度腔肠素的评估（平均值±标准差，N = 6）。从面板A（B）发光值标准化为车辆在每个衬底浓度处理的对照，以评估毒胡萝卜素诱导的效应。 （C）的底物衰变在几个小时。从重组胰高血糖（0.1毫微克或0.02纳克）的已知量测量发光了准备基板后几个小时（平均值±SD，N ​​= 4）。 （D）当原始发光的重组GLUC随时间而减少由于衬底衰变，将样品中的倍数差异（如通过发光测定）得到维持。 <img alt="图7" src="/files/ftp_upload/53199/53199fig7.jpg" /> 图7:注意事项有关GLUC / CTZ反应动力学GLUC-SERCaMP分析（一）培养基改变GLUC / CTZ信号的衰减。 5微升含有GLUC-SERCaMP上清液具有变化的PBS和培养基的比例混合。将100μl的底物（15μMCTZ在PBS + 500mM的抗坏血酸）加入到50微升样品的该稀释​​如表中所概述。光发射监测在15分钟内。 （B）的5微升培养基，从SH-SY5Y-GLUC-ASARTDL细胞和100微升底物（8μMCTZ在PBS + 5mM的NaCl）中收集的溶液。发光被过加入底物（T = 0），并每30秒后立即测量<html> 15分钟（±SD，​​N ​​= 12）的过程。数据被绘制为相对于前30秒的时间间隔的百分比信号以评估衰减率。插图显示的原始发光的数据。 <img alt="图8" src="/files/ftp_upload/53199/53199fig8.jpg" /> 图8:100纳米毒胡萝卜素对胰高血糖的分泌ER钙枯竭的药理佐证（A）的影响进行了检查为GLUC-ASARTDL和GLUC -无标签控制（平均值±SD，N = 6）。 （二）稳定ER钙与丹曲林（碱受体拮抗剂）抑制的Tg诱导SERCaMP释放。细胞预先用丹曲林的指定浓度为30分钟或添加100nM的毒胡萝卜素之前16小时。在介质GLUC活性测定4小时后（平均值±标准差，N = 6）。 99fig9.jpg"/&gt; 图9:模拟代表性结果为GLUC-SERCaMP测定注意载体处理值可能板之间下降，由于衬底击穿（依赖于时间间隔读取各个 ​​板之间）。为了考虑到这种效果，具体的治疗效果被独立地计算每个板和对面板上这个比率进行比较。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>迄今</td><td>鼠</td><td>管质量（毫克） </td><td>肝素体积（ml） </td><td>肝素密度（g / ml）的</td><td>肝素质量（毫克） </td><td>管+肝素（毫克）质量</td><td>采血后的总质量（毫克） </td><td>采血质量（毫克） </td><td>血密度（克/毫升） </td><td>计算体积血（UL） </td><td>需要2肝素体积:1的比例</td><td> ML额外的肝素旋前加</td></tr><tr><td> 15年1月1日</td><td> SD男</td><td> 1135.9 </td><td> 50 </td><td> 1.117 </td><td> 55.83 </td><td> 1191.73 </td><td> 1317.70 </td><td> 125.97 </td><td> 1.05 </td><td> 119.97 </td><td> 59.98 </td><td> 9.98 </td></tr></tbody></table> 表1:血液采集表举例SERCaMP研究采血日志。

Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

monitoring-endoplasmic-reticulum-calcium-homeostasis-using-gaussia

Universidad San Sebastian

Research

JoVE Journal

Biology

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

13.1K 次观看.  National Institutes of Health. Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

视频: Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

科研

生物化学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

生物学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

<meta charset="utf8"></head><body>一种用于高通量筛选的测量 ER-线粒体接触的简单检测

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

<meta charset="utf8"></head><body>作者聚焦：ER-线粒体接触的调节和失调 — 对神经退行性疾病发病机制的影响

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜靶向 ca 2 + 指标对培养细胞中局部 Ca2 +信号的解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

神经科学

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

病毒感染的人肠道类器官单层的钙成像

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

作者聚焦：研究肠道上皮信号传导的病毒破坏 – 研究见解和未来方向 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

监测内质网钙稳态使用

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膜靶向 ca 2 + 指标对培养细胞中局部 Ca^{2 +}信号的解剖

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像