Name: adenofection: a method for studying the role of molecular chaperones in cellular morphodynamics by depletion-rescue experiments
Uploaded: 2016-09-16T15:00:00.000+00:00
Duration: 12 min 34 s
Description: Watch this Scientific Journal Video about Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments at JoVE.com

This work was supported by the Canadian Institutes of Health Research (Grant no 7077), and by the Bellini Foundation and Roby Fondazione.

1.中等和解决方案的准备（所有无菌过滤） <ol><li> C2C12小鼠肌肉细胞（分化的研究） <ol><li>制成500毫升之生长培养基中对C2C12细胞培养维护:补充有10％FBS和2mM L-谷氨酰胺的DMEM高糖。 </li><li>对C2C12分化制备100ml分化培养基（DM）:补充有2％马血清的DMEM高糖。 </li></ol></li><li> HeLa细胞（在有丝分裂细胞研究） <ol><li>制成500毫升之αMEM对于海拉RFP-H2B细胞培养维持和实验:αMEM补充有10％FBS和2mM L-谷氨酰胺（αMEM10％）。 </li><li>制成500毫升之αMEM-减去（无脱氧核糖核苷/核糖核苷），用于海拉RFP-H2B细胞同步:αMEM-减去补充有10％FBS和2mM L-谷氨酰胺（αMEM减10％）。 </li><li>准备20毫升αMEM无酚红的海拉-RFP-H2B升IVE细胞采集:αMEM无酚红补充有10％FBS和2mM L-谷氨酰胺。 </li><li>准备100毫米胸苷:涡旋1毫升水2 O溶解24.2毫克，37℃。过滤消毒，并保持在4℃。 </li></ol></li><li> 共同的解决办法 <ol><li>制备0.05％胰蛋白酶/ EDTA:0.05％胰蛋白酶，0.625毫摩尔EDTA于1×磷酸盐缓冲盐水（PBS）中。过滤消毒和储存等分在-20℃。一旦解冻，等分保持在4℃。 </li><li>制备HBS2x:280毫摩尔NaCl，50毫米的HEPES，1.5毫摩尔的 Na 2 HPO 4。调节pH恰好之间7.01-7.05用10N的NaOH。过滤消毒，并保持在4℃。 </li></ol></li></ol> 2.海拉-RFP-H2B细胞纤维连接蛋白和电镀细胞培养板的涂层注意:在此之前的实验中，每个操作器应设置的最佳细胞电镀条件的细胞达到一个适当的密度，因为变化可以OC每个机械手和每个不同的细胞线之间CUR。 <ol><li>在实验的前一天，展开的HeLa-RFP-H2B细胞，以确保它们的增长上电镀的天指数期之内。使10厘米板块连续2 1/3稀释从80％汇合1×10厘米板开始。 注:计划在实验板的数量。计算每个条件为重复，一个用于短期活细胞成像，一个用于蛋白提取物，以确定击倒和外源蛋白的表达通过Western印迹分析的效力。计划一个附加板，以确定前，病毒转导的细胞数。 </li><li>事先用10微克/毫升纤连蛋白与细胞电镀，涂层玻璃底菜肴。 1ml的1加100稀释的1mg / ml的纤连蛋白？35毫米（稀释在无菌1×PBS中）菜以及覆盖整个表面，并在37℃，5％的CO 2下孵育1小时。 </li><li>在培育期间，预温αMEM补充的Wi第10％FBS（αMEM10％）和0.05％胰蛋白酶/ EDTA中，在37℃。 </li><li>孵育45分钟后，开始准备的细胞溶液。在无菌罩，吸从10cm平板培养基中，用1.5ml 0.05％胰蛋白酶/ EDTA轻轻漂洗两​​次，并留下0.5毫升胰蛋白酶/ EDTA的在最后愿望。 </li><li>孵育2-3分钟将细胞在37℃，5％ 的 CO 2。轻轻拍打在显微镜下板和观察，验证所有细胞已被分离。加入10毫升αMEM10％和吸管轻轻几次以分离细胞。算使用血球细胞悬液的10微升样品。 </li><li>从玻璃底菜吸纤维连接蛋白的解决方案。不要让纤连蛋白细胞接种之前干。 </li><li>板1.5×10每35mm培养皿5个细胞于2mlαMEM10％，并在37℃，5％的CO 2下孵育。后在显微镜下30-45分钟验证细胞以及国家环保总局额定的，因为它们在板的中心积聚的倾向。 </li><li>如果有必要，搅动板轻轻交叉方式重新分配细胞。板一次35毫米的钢板附加一些条件才能算之前，病毒转导的细胞数量。 </li><li>生长细胞直到第二天，以达到至少50％的细胞密度。的细胞密度在病毒转导效率显著降低低于50％的结果，增加每细胞的蛋白表达的变化，以及增加的细胞毒性。 注:使用纤连蛋白的玻璃涂层的菜肴改善细胞的生长和形态，促进有丝分裂细胞的正常发展。它通常可通过市售的明胶是便宜更换。 </li></ol> 3.腺病毒转导和内源蛋白通过siRNA转染在HeLa-RFP-H2B细胞击倒使用CAPI沉淀 注意！工作与病毒ING需要特殊的预防措施，并且一直在与病毒接触的所有材料的妥善处置。 注意！在我们的手中，CAPI沉淀往往有更多的不良影响，例如对涉及囊泡运输（ 例如 ，自噬）生物过程。因此，建议使用阳离子脂质转染试剂（见下文）和分析前等待至少48小时。 注:对照腺病毒携带不相关的基因（ 即，LacZ基因）或没有基因被用来达到使用重组腺病毒携带目的基因的最低量的最小的MOI在所有Adenofections（10-20 PFU /细胞）。 注意:此过程已被证明有助于在大细胞群体每个细胞正火表达。 <ol><li>细胞铺板后一天，计数细胞从额外镀盘的数目。吸出介质并用1ml 0.05％胰蛋白酶/ EDTA冲洗一次。加再次1毫升0.05％胰蛋白酶/ EDTA中，并在37℃，5％CO 2孵育，直到所有的细胞脱离。分离细胞使用1ml的吸管孔和计数使用血球每毫升的细胞数。 </li><li>确定转导的细胞所需的病毒的量在感染复数的兴趣点（POI）的蛋白质的每单元2噬斑形成单位（PFU），并每一个空载体的细胞18 PFU（MOI）（ 例如，LacZ基因）有一个总每个细胞20 PFU的。同时转导各杆状病毒（肌动蛋白和αTubulin，GF​​P和RFP标记分别）的4 PFU。使用以下adenofection协议转导的细胞。如在Fuchs 等人所述，使用病毒。14 </li><li>在无菌罩准备1.5毫升的塑料管各自的条件，并添加400微升温暖αMEM减10％。 </li><li>在冰上解冻的病毒的等分试样慢慢和，如果需要的话，稀释吨他病毒的股票，以吸取体积大于1微升，以尽量减少移液错误。 </li><li>每个条件计算出的病毒量加入含400微升αMEM减10％，每次1.5毫升的塑料管中，吹打轻轻拌匀。放置病毒库存立即回在-80℃，以保持它的活性。 </li><li>吸从细胞培养基中并轻轻吸取病毒混合逐滴。孵育细胞在37℃，5％的CO 2，并仔细搅动板无菌罩下每15分钟1小时，以及覆盖该病毒的细胞。使用介质的少量促进病毒与细胞的接触。 </li><li>孵育后，轻轻吸取1.6毫升αMEM减10％至各板，以获得为2ml的总体积，加入2毫胸苷开始单元同步，并孵育另外2小时的细胞在37℃，5％CO 2的。 </li><li>同时，准备转染混合followi纳克的CAPI转染方法（ 见图1）。如果没有的siRNA是必要的，通过无菌水取代的siRNA的量来执行空转染 。 </li><li>计算200微升混合物为每个条件，导致400微升每重复。下面的例子给出了为50nM的终siRNA的浓度，并以适于每个感兴趣的蛋白质。在1.5ml的塑料管，移液管加入50μl1M的氯化钙2和11微升20μM的siRNA导入139微升无菌的 H 2 O和通过涡旋混合。一个快速旋转后，小心加入逐滴加入200μlHBS2x（280毫米氯化钠，50毫米的HEPES，1.5毫米的 Na 2 HPO 4，pH值7.01-7.05）。 注:较小的液​​滴越小是析出物，从而得到更好的转染效率。轻轻空气喷射用200μl移液器混合三次。 </li><li>在室温下孵育30分钟的混合。缓慢加入逐滴加入200μl转染混合物到每个板搅拌交叉明智的。传送板在37℃，5％CO 2的16小时。 </li><li>第二天，冲洗细胞两次用2毫升温的HEPES（6.7毫米氯化钾，150mM的氯化钠，10mM的HEPES，pH值7.3）和加2mlαMEM减10％。具有四个以上的细胞板在一时间不继续进行，因为在温度变化影响的细胞周期的长度。 </li><li>可视化倒置荧光显微镜下的感染效率在20-40x（空气）的放大倍数，并在文档荧光和传输信道获取每个条件三个有代表性的图像。七小时后，在37℃，5％CO 2的添加2mM的胸苷并孵育另外16小时。 </li><li>第二天，转染和病毒感染后48小时，冲洗细胞两次用2毫升温的磷酸盐缓冲盐水（PBS），并在2毫升αMEM10％释放7小时的w / o酚红对活细胞成像或酚红蛋白的提取。 </li><li>收获细胞每个条件48小时转染后用于制备蛋白质提取物和Western印迹分析，以确定击倒的效率和内源性蛋白15的表达。 注意:使用抗体对感兴趣的蛋白质以及作为装载对照适当抗体。击倒的效率应通过加载控制细胞裂解物减少量（与对照siRNA转染的）来确定，以提供一个滴定曲线（即 1，½，¼，⅛）。 </li></ol> 4.腺病毒转导和内源蛋白通过siRNA转染中使用阳离子脂质转染试剂HeLa细胞击倒注意:在这里，我们提出了适于不涉及细胞同步实验的协议和/或当siRNA转染不能由CAPI的方法进行，例如，以避免在某些细胞林不希望的毒性作用上课。该协议还包括为了在合适的细胞密度工作adenofection后的细胞replating步骤。我们只测试了阳离子脂质转染试剂。 注意！有病毒的工作需要特殊的预防措施，并且一直在与病毒接触的所有材料的妥善处置。 <ol><li>板每35mm培养皿1.75×10 5个细胞在2.5mlαMEM10％，并在37℃下孵育，5％ 的 CO 2。板一次35毫米的钢板附加一些条件才能算之前，病毒转导的细胞数量。 </li><li>生长细胞直到第二天，以达到至少50％的细胞密度。在病毒转导效率的显著下降小于50％的结果的细胞密度，增加每细胞的蛋白表达的变化，以及增加的细胞毒性。 </li><li>细胞铺板后一天，计数细胞从额外镀盘的数目。吸气培养基并用1ml 0.05％胰蛋白酶/ EDTA冲洗一次。加再次1毫升0.05％胰蛋白酶/ EDTA中，并在37℃，5％CO 2孵育，直到所有的细胞脱离。单独的细胞以及用1毫升移液管和计数每ml的细胞数。 </li><li>确定病毒的转导的感染复数的兴趣点（POI）的蛋白质的每个细胞20-40空斑形成单位（PFU），并每一个空载体的细胞0-20 PFU（MOI）所需的量（ 例如， LacZ的）有一个总的每个细胞40 PFU的。 </li><li>在无菌罩准备1.5毫升的塑料管各自的条件，并添加400微升温暖αMEM10％。 </li><li>在冰上解冻的病毒的等分试样慢慢和，如果需要的话，稀释的病毒原液以吸取的体积大于1微升，以尽量减少移液误差。 </li><li>每个条件计算出的病毒量加入含400微升αMEM10％，每次1.5毫升的塑料管中，吹打轻轻拌匀。第广场病毒的股票立刻回在-80°C至保持其活性。 </li><li>吸从细胞培养基中并轻轻吸取病毒混合逐滴。孵育细胞在37℃，5％的CO 2，并仔细搅动板无菌罩下每15分钟1小时，以及覆盖该病毒稀释细胞。使用介质的少量促进病毒与细胞的接触。 </li><li>同时，准备下转染法的siRNA转染组合。如果没有的siRNA是必要的，由介质替换的siRNA的量无血清来执行空转染 。 <ol><li>下面的例子给出了为50nM的终siRNA的浓度，并以适于每个感兴趣的蛋白质。对于每个adenofection，准备一份1.5毫升含416纳米的siRNA在150微升培养基无血清（ 例如 ，6.25微升20μM的siRNA + 144微升无血清培养基）和一个1.5毫升塑料管containi塑料管NG 6.25微升阳离子脂质体转染试剂+ 144无血清培养基微升。 </li><li>通过上下吹打数次用200微升吸管混合各塑料管的内容。结合两管的内容和通过上下吹打数次用200微升吸管混合。在室温下孵育5分钟。 </li></ol></li><li>与病毒孵育后，轻轻吸取1.8毫升αMEM10％至各板，以获得2.2毫升总体积，并立即添加慢慢逐滴所述siRNA转染混合物，以每盘和搅拌交叉明智的。传送板在37℃，5％CO 2的24小时。 </li><li>第二天，再培养板上的细胞，从35个毫米的钢板成四个新35毫米板在2.5mlαMEM各占10％和孵育另外24小时，在37℃，5％ 的 CO 2。 </li><li>第二天，转染和病毒感染，由一个板收获细胞用于制备的每个条件后48小时蛋白质提取物和Western印迹分析，以确定击倒的效率和内源性蛋白15的表达。 注意:对于其余板块，继续进行细胞固定，使用所选的协议，并接受了样品与关注14抗体免疫荧光分析。 </li></ol>有丝分裂细胞和数据分析的5活细胞成像<ol><li>用配有加湿/ 5％CO 2 /调温室的倒置显微镜上执行分裂细胞短期活细胞成像实验。 注意:在此研究中，使用了旋转盘共聚焦显微镜（40X，0.75 NA），配备有EMCCD冷却电荷耦合照相机在-50℃下。 </li><li>到收购前，请检查室已经达到37℃，适宜的温度。 注意:这可能需要几个小时，这取决于微观系统。 </li><li>放置在培养皿中的MICR的Oscope腔室采集前1小时，以使媒体的适当的平衡，并避免焦点因温度变化漂流。监测细胞的有丝分裂的状态。此时，该细胞的10-15％应在有丝分裂（前期-前中期）的早期阶段。 </li><li>在平衡时间，如在现有实验确定设置的采集参数。典型地，曝光时间为.2-3秒的两个通道（488和594）为100％的激光强度和121-130灵敏度是在我们手中合适的参数。 注意:首先测试应确定最小激光强度和采集时间/间隔导致与最小光漂白的适当的分辨率，导致细胞损伤和扰动丝分裂进程。 </li><li>选择每种条件几个字段来获得细胞的显著数不超过采集间隔来分析。使用旋转盘共聚焦系统，一个典型的安装将在CLUDE四种不同的条件下，每板7场和2个颜色通道（488和594）与1.5-2分钟的时间间隔超过75分钟周期。 </li><li>选择是在进入有丝分裂的细胞，重新设置，一旦所有字段已被选择的焦点并尽可能快地开始采集。 </li><li>监视系统的稳定性为至少三个时间点，并在必要重新设置的焦点。 </li><li>的75分钟的第一个短线活细胞成像后，有丝分裂细胞的新字段可被选择以获得第二组的电影的增加所分析细胞的数量。 </li><li>确定良好定义的标准来分析所使用的细胞的有丝分裂的表型，这取决于荧光标记。在有丝分裂的缺陷可能包括延长在有丝分裂所花费的时间（从核击穿，直到后期），染色体错位，主轴摇摆，和皮层起泡14。 </li></ol> 6. LifeAct-TagGFP2腺病毒转导Differentiatin摹C2C12成肌细胞鼠注:adenofection协议也适用于难以感染小鼠C2C12细胞进行分化。 <ol><li>板2×10 5的C2C12细胞35毫米培养皿中生长培养基上的塑料或选择的基片上。 注:细胞分化是在gelatine-或基质胶涂层的菜肴改善。计划一个附加板，以确定前，病毒转导的细胞数。 </li><li>次日，将细胞应已达到会合的80％。用温热的PBS洗涤细胞两次，并加入2ml的分化培养基（DM）的诱导成肌细胞分化。 </li><li>第二天，标记为分化的第1天（D1），转导腺病毒LifeAct-TagGFP2肌细胞可视化在活细胞肌动蛋白细胞骨架。根据3.2的描述从附加板计数细胞数。计算考试下面LifeAct-TagGFP2腺病毒5 PFU /细胞和45 PFU /细胞AdLacZPLE 表1中描述，总病毒量是50 PFU /细胞。描述14用于病毒</li><li>在无菌罩准备1.5毫升的塑料管各自的条件，并添加400微升温暖的DM。 </li><li>在冰上解冻的病毒的等分试样慢慢和，如果需要的话，稀释的病毒原液以吸取的体积大于1微升，以尽量减少移液误差。 </li><li>每种条件的病毒颗粒的适量添加到含有400微升的DM每次1.5毫升的塑料管中并通过移液轻轻混匀。放置病毒库存立即回在-80℃，以保持它的活性。 </li><li>吸从菜介质，轻轻吸管混合病毒滴。孵育细胞在37℃，5％的CO 2，并仔细搅动板无菌罩下每15分钟，共1小时，以良好覆盖的病毒的细胞。 </li><li>孵育后，轻轻吸取1.6毫升DM到每个板，并继续我在37℃下ncubation另外2小时，5％ 的 CO 2。 </li><li>同时，准备继CAPI转染法空染混合物。计算每个条件200微升组合。使用下面的示例为400μl的混合物的总体积。 <ol><li>在1.5ml的塑料管，移液管加入50μl的1M的CaCl 2到150微升无菌的 H 2 O和通过涡旋混合。一个快速旋转后，小心加入逐滴加入200μlHBS2x（280毫米氯化钠，50毫米的HEPES，1.5毫米的 Na 2 HPO 4，pH值7.01-7.05）。轻轻空气喷射三次用200μl移液器混匀。 </li></ol></li><li>在室温下孵育30分钟的混合。慢慢地加入逐滴加入200μl转染混合物到每个板块和搅拌交叉明智的。传送板在37℃，5％CO 2的16小时。 </li><li>第二天，冲洗细胞两次用2毫升温的HEPES（6.7毫米氯化钾，150mM的氯化钠，10mM的HEPES，pH值7.3）和加2ml DM。可视化INFE在20-40X（空气）的放大倍数倒置荧光显微镜下ction效率，并在文档荧光和传输信道获取每个条件三个有代表性的图像。 </li><li>根据所需的实验设置，请分化成肌管了好几天。细胞可以是固定的，并随后进行免疫荧光分析或活细胞成像的研究可以进行。 </li></ol>

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The authors have nothing to disclose.

在这里，我们描述的方法使得能够进行耗尽救援实验，这是适用于那些对影响的化学计量和蛋白质复合物和大分子结构的动力学的蛋白质的过表达特别敏感细胞生物学过程的功能分析。有丝分裂的细胞分裂是涉及在细胞中的整体结构的最引人注目的和引人注目的变化微调细胞地貌的一个极端的例子。使用adenofection用市售BacMam试剂相结合，引进低，但检测的量的肌动蛋白和微管蛋白标志物细胞成像?...

在哺乳动物细胞中基因表达的功能失活的金标准解剖蛋白质的功能。新开发的基础上，利用位点特异性核酸酶如锌指核酸酶的基因组编辑的技术和聚集定期相互间隔短回文重复序列（CRISPR）/ CAS9现在允许的有针对性的基因缺失和突变1,2-细胞系的产生。这些新的方法应该彻底改变我们正在研究蛋白质的功能和我们人类疾病的遗传学的理解方式。在一些情况下，然而，长期或完整基因敲除是不希望的，并可能会引起二次电池的补偿机制。遗传修饰的细胞系的产生，也可与原代细胞培养用有限增殖的能力，或当一大组在各种细胞类型的突变的筛查寻求处理时限制。这经常需要用于确定小区b的依赖性对蛋白质的结构要求iological过程。为此，通过RNA干扰，能够在各种细胞背景短暂枯竭救援实验可逆击倒仍然是一个简单而强大的方式来执行的利率3蛋白的结构与功能分析。但是，这种方法的主要缺点是难以实现高效的沉默和重新引入的在接近生理水平的兴趣或它的变体的蛋白质中的大多数细胞群。这是至关重要的，以使全面研究试图关联在单个细胞（亚效等位基因表型）与那些在小区基于人口的分析可见，例如在蛋白质 - 蛋白质相互作用的水平可见的作用效果。 使用传统的转染方法，人们很难达到一个人口众多的细胞外源性蛋白质的同质和低表达。重组病毒的细胞转导像腺病毒往往使外源蛋白质的更标准化的表达。然而，腺病毒的摄取是由CAR受体，其是在非人类细胞中不存在或仅在某些人类细胞类型弱表达限定。此外，腺病毒的细胞进入激活信令调节细胞形状和粘附4-6通路。研究细胞地貌的调节机制时，这显然是不理想的。当我们进行了伴侣复杂，BAG3-HSPB8的功能分析，在细胞分裂和肌动蛋白，我们正面临着这个问题。开拓性的工作压力7,8期间描述的蛋白质的质量控制和自噬这个伴侣复合作用。大部分这些研究，然而，依赖于蛋白表达，假定分子伴侣被应力过程中通常上调。这已经离开开放是否BAG3的问题，在与HSPB8复杂，可以向表达个分裂的细胞的正常运行ESE伴侣像许多癌细胞类型9。特别是，无论是伴侣复合有助于控制有丝分裂的进展非常感兴趣，因为HSPB伴侣和细胞骨架的动态10之间的连接出现基于肌动蛋白结构的重塑。为了解决这个问题，我们寻求发展为耗尽救援实验一种有效的方法，将不与有丝分裂进展或细胞形态干扰，并且这将保留蛋白质稳态避免大分子复合物的动力学的二次扰动调节细胞形状的改变。因此理想情况下，应同时进行的感兴趣的基因的耗尽加回。 腺病毒的复合物与阳离子聚合物或脂质的使用已被描述为促进在体外和体内 11,12基因转移。例如，磷酸钙（CAPI）出现以形成沉淀腺病毒增强通过CAR-独立通路13病毒绑定条目。事实上，我们发现，基于腺病毒的细胞转导和转染结合的阳离子化合物可增强耗尽救援实验的效率。这允许我们通过3-降低病毒的量至20倍，这取决于从更广泛的窗口的细胞系和感兴趣的基因，并从中受益，以便在大多数附近内源水平来调整外源蛋白的表达的对细胞形态的影响降到最低的兴趣的细胞群。在这种情况下，我们也可以实现内源蛋白质表达（&gt; 75％）的高效率击倒。我们特此通过步骤描述了方法步骤，并提供证据表明，由热休克蛋白家族的应激诱导的分子伴侣的不变的水平所评估蛋白质稳态不显著扰动，使得该方法适于生理RO的功能分析按时间推移视频显微镜分子伴侣乐。该协议是适合于细胞的同步程序，并有丝分裂进展过程中使用可商购的杆状病毒为低水平荧光标记的，与正常的肌动蛋白为基础的和主轴动力学干扰最小的共表达。我们进一步显示的方法中，这是适用于"硬转导"小鼠C2C12细胞，对成肌细胞分化无显著影响到体外肌管的通用性。

<table><tbody><tr><td>C2C12 Mouse Myoblasts</td><td>ATCC</td><td>CRL-1772</td><td></td></tr><tr><td>Adenovirus custom design</td><td>Welgen</td><td>Custom design</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>CellLight&amp;reg; Actin-GFP, BacMam 2.0</td><td>Thermo Fisher</td><td>C10582</td><td></td></tr><tr><td>CellLight&amp;reg; Tubulin-RFP, BacMam 2.0</td><td>Thermo Fisher</td><td>C10614</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM), High Glucose</td><td>Thermo Fisher</td><td>11965-092</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Fisher</td><td>12483-020</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141</td><td></td></tr><tr><td>Glass bottom dishes, 35 mm</td><td>MatTek Corperation</td><td>P35G-1.5-20-C Case</td><td></td></tr><tr><td>HeLa-RFP-H2B</td><td>Kind gift of Dr Sabine Elowe, Qu&amp;eacute;bec, Canada</td><td></td><td>Klebig C &lt;em&gt;et al.&lt;/em&gt; 2009</td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-1</td><td></td></tr><tr><td>Horse Serum, New Zealand</td><td>Thermo Fisher</td><td>16050-122</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher</td><td>25030081</td><td></td></tr><tr><td>Lipofectamine&amp;reg; RNAiMAX Transfection Reagent</td><td>Thermo Fisher</td><td>13778-150</td><td></td></tr><tr><td>Minimal Essential Medium (MEM) Alpha</td><td>Wisent</td><td>310-101-CL</td><td></td></tr><tr><td>Minimal Essential Medium (MEM) Alpha without Desoxyribonuleosides/Ribonucleosides</td><td>Thermo Fisher</td><td>12000-022</td><td></td></tr><tr><td>Minimal Essential Medium (MEM) Alpha without Phenol Red</td><td>Thermo Fisher</td><td>41061-029</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Biobasic</td><td>S0404</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>BP358-10</td><td></td></tr><tr><td>OptiMEM</td><td>Thermo Fisher</td><td>11058-021</td><td></td></tr><tr><td>rAV&lt;sup&gt;CMV&lt;/sup&gt;-LifeAct-TagGFP2</td><td>IBIDI</td><td>60121</td><td></td></tr><tr><td>siRNA duplexes</td><td>Dharmacon</td><td>Custom design</td><td></td></tr><tr><td>Thymidine</td><td>Sigma</td><td>T9250</td><td></td></tr><tr><td>Trypsine 2.5%</td><td>Thermo Fisher</td><td>15090-046</td><td></td></tr></tbody></table>

adenofection: a method for studying the role of molecular chaperones in cellular morphodynamics by depletion-rescue experiments

细胞过程，例如有丝分裂和细胞分化是通过细胞形状的变化在很大程度上依赖于细胞骨架结构的适当改造管辖。这涉及到更高阶的大分子结构的在给定时间和地点的组装拆卸，这个过程是引起的蛋白质的过表达干扰特别敏感。方法，可以保留蛋白质稳态和维持近到正常细胞形态是高度理想的是确定感兴趣的蛋白在广泛的细胞过程的功能的贡献。基于RNA干扰瞬态枯竭救援实验是分析蛋白质的功能和结构要求强大的方法。然而，从它的生理水平最小偏差靶蛋白的重新引入是一个真正的挑战。在这里，我们描述了开发研究的分子伴侣的作用的方法称为adenofection的在分裂细胞的正常d动作伙伴和肌动蛋白重塑的关系。 HeLa细胞耗尽BAG3与siRNA双链体靶向3&#39;UTR区。 GFP标记BAG3蛋白同时再引入使用耦合到转染试剂重组腺病毒的细胞的&gt; 75％。启用Adenofection表示在耗尽BAG3在HeLa细胞中接近生理水平BAG3-GFP蛋白，在不存在应激反应。观察内源性热休克蛋白分子伴侣蛋白动态平衡的主要胁迫诱导监管的水平没有影响。此外，通过添加杆状病毒驱动的荧光标记物的表达在细胞转导转染时，我们可以通过时间推移显微解剖的有丝分裂细胞动力学与正常的有丝分裂进展的最小扰动的分析。 Adenofection也适用于难以感染小鼠细胞，并适合于成肌细胞的diff功能分析erentiation成肌管。因此adenofection提供了一个通用的方法来进行参与依赖于高阶骨架动力学敏感的生物过程的蛋白的结构与功能分析。

使用阳离子脂质BAG3-GFP的质粒DNA的转染HeLa细胞与异质表达相关，一些细胞显示的蛋白质的勉强可检测的水平和其他轴承很高BAG3水平（ 图2A）。在这些细胞中，蛋白稳态损耗由BAG3-GFP的积累成核周聚集体（ 图2A，箭头）证明。与此相反，用携带BAG3-GFP腺细胞转导显示更均匀的低表达和BAG3-GFP的精确定位（ 图2B，单独感染）。值得注意的是...

Watch this Scientific Journal Video about Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments at JoVE.com

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Adenofection:用于研究分子伴侣在细胞地貌被耗尽，救援实验中的角色方法

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

adenofection-method-for-studying-role-molecular-chaperones-cellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.6K 次观看.  Centre de recherche sur le cancer de l'Université Laval. We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level. 

视频: Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

体外 使用分析、下拉和伴侣测定表征组蛋白伴侣

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

DNA帘法破译组蛋白组装的分子机制

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

细胞内复性检测

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

生物学

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Adenofection:用于研究分子伴侣在细胞地貌被耗尽，救援实验中的角色方法

摘要

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此视频中的章节

的pH依赖性活性的检测

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

体外使用分析、下拉和伴侣测定表征组蛋白伴侣

DNA帘法破译组蛋白组装的分子机制

细胞内复性检测

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定

耦合检测监控蛋白复性

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

使用卡诺哈布迪炎埃莱甘斯筛选组织特异性查佩龙相互作用

Adenofection:用于研究分子伴侣在细胞地貌被耗尽，救援实验中的角色方法

摘要

探索更多视频

此视频中的章节

的pH依赖性活性的检测

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

体外 使用分析、下拉和伴侣测定表征组蛋白伴侣

DNA帘法破译组蛋白组装的分子机制

细胞内复性检测

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定

耦合检测监控蛋白复性

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

体外使用分析、下拉和伴侣测定表征组蛋白伴侣

使用卡诺哈布迪炎埃莱甘斯筛选组织特异性查佩龙相互作用