Name: detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay
Uploaded: 2015-01-20T13:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

我们感谢布鲁明顿果蝇库存中心提供飞股票。我们也感谢发展研究杂交瘤细胞银行和林恩博士库利（耶鲁大学）提供抗体。特别感谢以AhHyun侑为她的手稿帮助。这项工作是由来自加拿大自然科学和工程研究理事会（克里格），威廉和Ada伊莎贝尔钢铁基金（克里格），和健康研究加拿大学院（哈登）的资助。

1.体壁准备注意:3龄幼虫体壁的制备（用于NMJs其支配体壁肌肉的研究）中的如先前在Brent 等 10，或德兰和Budnik 11,12所述进行，但具有一些修改。 <ol><li>解剖<ol><li>提高苍蝇种群和杂交在25℃下，使用标准方法13 5〜6 ​​天。 </li><li>挑选使用细镊子小瓶或瓶爬三龄幼虫。 </li><li>洗在含有磷酸盐缓冲盐水（PBS），以除去任何食物颗粒的小皮氏培养皿的幼虫。 </li><li>将一个单一的幼虫到SYLGARD盘，沉浸在一个几滴冰冷的PBS。用冰冷的PBS将有助于眩晕使其更容易操纵幼虫。整个夹层，确保该制剂总是浸没在PBS中，以防止其干燥。 </li><li>定位LARVA其背侧朝上使得两个气管道是在解剖显微镜（ 图2A）下可见。使用镊子抓住一个minutien销，销的幼虫向下在靠近气孔（ 图2B）的后端。与其他针，刺透角质层在靠近口钩的前端。轻轻舒展幼虫出纵向，则针下来（ 图2B）。 </li><li>采用显微剪，捏近销后端来创建一个小口。切口应肤浅不够的，只是通过角质层。 </li><li>放置剪刀的底部叶片的尖端插入切口，沿着两个气管束（ 图2C）之间的背中线的整个长度切割。切割时避免损坏腹体壁肌肉点剪刀片轻微向上。 </li><li>做一个小切口水平稍微先于后eriorly放置销（ 图2C）。使另一个类似的切口略微后方的前方放置的销（ 图2C）。切口应该只通过角质层。 注:从步骤1.1.7和1.1.8结合起来应该像一个三切口" <img fo:content-width="0.09in" src="/files/ftp_upload/52139/52139_letter_I.jpg" width="15px" /> "已完成时， 也就是说 ，在幼虫体的背侧的左和右挡板应来制造。 </li><li>认真清理内脏的镊子。加入数滴有力PBS将有助于取代器官出幼虫体内，从而使其更容易将其删除。避免戳幼虫的身体，因为它会造成对身体的损害墙上的肌肉。 </li><li>打出幼虫体内开放，针角向下（ 图2D）。当寄托，舒展身体墙面水平和垂直方向形成均匀的张矩形（SEE 图2G为形状），小心不要撕破体壁肌肉中的过程。 </li><li>完成删除任何剩余的内脏（ 图2E）。 </li></ol></li><li>固定和透<ol><li>沉浸在固定体墙壁几滴布安的解决方案。孵育在冰上15分钟。另外，使用4％多聚甲醛（PFA）作为替代固定剂;温育30分钟。 </li><li>漂洗三次，磷酸盐缓冲溶液用Triton（PBT）。 </li><li>用细镊子，小心地取出针和体壁由自己的角落转移到硅化0.65毫升的离心管。 </li><li>存储体墙壁PBT在4°C，直到准备PLA。为了达到最佳效果，开始一两天夹层内的免疫染色体墙壁。 注意:为了节省试剂，并确保所有体壁在测定期间一视同仁，不同基因型可以放置成一个单一的管。基因型可以通过切割主体壁的角部不一样（ 见图2F的例子）来区分。 </li></ol></li></ol> 2.免疫组化 。如前面布伦特等 14德兰和Budnik 11所述进行免疫染色的三龄幼虫体墙，但也有一些改动11,14:注意。 注:执行所有步骤在室温和温和搅拌，除非另有说明。 <ol><li>闭塞<ol><li>与PBT，每次10分钟洗身壁三次。 </li><li>用1％牛血清白蛋白（BSA）的1小时阻断。 </li></ol></li><li>染色<ol><li>孵育体壁与小鼠和兔的初级抗体对两种蛋白质的利益为2小时，在室温下，或过夜，在4℃（稀释在1％BSA）。在这种情况下，使用1点10小鼠抗了Dlg和1:250的兔一NTI-HtsM 15,16。针对未在小鼠或兔制成标记抗体也被包括- 例如 ，使用1:200的山羊抗-HRP来描绘神经元膜。 </li><li>与PBT，每次10分钟洗三次。 </li><li>用荧光标记的二抗孵育，以检测标志物（稀释在1％BSA）处理2小时，在室温下，或过夜，在4℃。作为PLA信号后可视化用红色荧光团，另一个荧光团必须被用来检测标记物- 例如 ，使用1:200的FITC缀合的抗山羊检测山羊抗HRP抗体。由于使用的光敏感的试剂，保持在黑暗中的管从这时开始。 注意:所使用的试剂盒可用于PLA将在小鼠和兔升高初级抗体之间进行的，以可视化下共聚焦显微镜红色通道的信号。如果需要的话，其他的试剂盒可允许测定用表面进行RY抗体提出在其他物种，并且在其他信道上可视信号。 </li></ol></li></ol> 3.接近结扎分析注:执行所有步骤在室温和温和搅拌，除非另有说明。 <ol><li>解放军探头<ol><li>与PBT，每次10分钟洗身壁三次。 </li><li>孵育与PLA探针（1:5稀释的每个中的1％BSA）2小时，在37℃。在这种情况下，使用40微升PLA探针抗小鼠MINUS，40微升PLA探针抗兔PLUS，或120微升的1％BSA的，以确保为5-10体壁适当浸泡和混合。高达1:25稀释的PLA探针仍然可导致足够的信噪比（ 即 ，对于这个实验）。 </li></ol></li><li>结扎<ol><li>每次5分钟洗身体的墙壁用洗涤缓冲液A的两倍。 </li><li>孵育结扎溶液（1:40稀释连接酶的连接缓冲液）进行1小时的吨37℃。在这种情况下，使用5微升连接酶，40微升5连接缓冲液和155微升的高纯度水，以确保为5-10体壁适当浸泡和混合。 </li></ol></li><li>放大<ol><li>每个2分钟洗身体的墙壁用洗涤缓冲液A的两倍。 </li><li>孵育在37℃下扩增溶液（1:80稀释聚合酶的扩增缓冲液）处理2小时。在这种情况下，使用2.5微升聚合酶，40微升5扩增缓冲液和157.5微升的高纯度水，以确保为5-10体壁适当浸泡和混合。 </li></ol></li><li>成像准备<ol><li>每10分钟，洗身壁用洗涤缓冲液B的两倍。 </li><li>与0.01X洗涤液B一次，持续​​1分钟洗净。 </li><li>平衡在几滴为至少30分钟的安装溶液的安装之前，或过夜储存于4℃。 </li><li>用细镊子，小心身体墙壁转移到一个平台幻灯片瓦特第i个他们的角质层朝下。定位体壁中的行数和在内部封固的一个或两个压降相同的方向。将22毫米×40mm的盖玻片上的准备，注意不要产生气泡，然后用密封透明指甲油的幻灯片。 </li><li>储存在黑暗中在-20℃下将载玻片直到准备共焦成像。 </li></ol></li></ol>

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作者什么都没有透露。

这份报告表明解放军如何应用到果蝇幼虫NMJ。该试验于幼虫体壁肌肉制剂执行用于检测内源蛋白质 - 蛋白质相互作用存在于NMJ的目的。利用这种技术，了Dlg和HTS示出为接近彼此，从而在一个复杂的存在，特别是在突触后区域27。为了支持这一结果，先前的研究已经提供了如下的数据的关联的证据:1）和了Dlg HTS的免疫反应性分布在幼虫NMJs的突触后区重叠，2）了Dlg和HTS形成复合体的基础上共免疫沉淀实验涉及整个成年飞裂解物，和3）了Dlg和HTS在上皮和突触结8相互作用。了Dlg和朴，以前在其他研究中发现了一个互动之间也观察到PLA信号的突触后区域，从而提供进一步的克雷德NCE采用此法在NMJ 15,20,21的。有趣的是，百和的Wg之间解放军导致没有明显的信号，即使他们的免疫反应分布重叠在NMJ。这个结果表明，并非所有的，显示重叠免疫反应的蛋白质的分布将产生一个PLA信号，因此表明PLA提供在检测比传统的共定位研究蛋白质 - 蛋白质相互作用的更高的分辨率。 多种变化，以原来的PLA协议作出以优化测定的幼虫NMJ（数据未显示）9,25。首先，它被确定的1％BSA是一个更好的封闭剂为主体壁，而不是提供封闭液。第二，以产生一个强有力的信噪比体内壁的，适当的浸泡和混合在反应溶液是至关重要的。甲五到十分体壁最小的200微升在0.65 ml离心管被认为suitab乐与解放军探头，连接酶或聚合酶孵化时，虽然卷可以处理更多的身体墙壁时，相应地增加。信号强度也通过在30分钟以上的推荐次数的增加，反应时间增加。第三，进行聚乳酸的探针系列稀释试验来优化试剂保护和信号强度之间的平衡。对于本报告中进行的，高达1:25稀释的实验 - 从推荐1:5稀释 - 仍然可以产生一个合理的信噪比。注意结扎和扩增反应的最优化进行了不执行。最后，由于聚乳酸可以是微小变化敏感，强烈建议不同基因型的单个实验的被放置在一个单一的管，以便控制和实验测定期间同等对待。基因型可以通过切割主体壁的角部不同来区分。 Severa的升方法用于检测果蝇蛋白质-蛋白质相互作用，包括酵母双杂交筛选，免疫共沉淀和FRET。但是，如何这些方法对解放军和可视化原位蛋白质-蛋白质相互作用的能力比较？酵母双杂交可以检测直接在酵母中表达果蝇蛋白质之间的结合，并且它在高通量全基因组的屏幕被使用。虽然许多已确定的相互作用是生物学相关的检测往往会产生误报。此外，假阴性可以发生于多种原因:蛋白质融合到转录因子结构域可具有结合干扰，许多相互作用需要不发生在酵母翻译后修饰，和细胞核（其中所述测试发生）可不是一个合适的环境，某些交互以适当地形成。它与内源性蛋白成交在家乡ENVIR这些问题都没有遇到与PLAonment。可用于检测内源蛋白之间的相互作用的一种方法是免疫共沉淀。涉及果蝇裂解物实验可以揭示在其中发生相互作用的生命周期的组织和阶段。然而，如裂解物形成涉及破碎细胞以提取蛋白质，在其中相互作用发生在组织中的细胞，以及其亚细胞定位，不能评估 - 不像在PLA。此外，共免疫沉淀检测蛋白质复合物的结合，不能区分该复合内蛋白质在接近彼此。有方法如FRET供Drosophilists允许的小区内蛋白质 - 蛋白质相互作用的可视化。然而，FRET涉及融合到荧光标记的转基因蛋白的表达，不反映内源性蛋白的行为在解放军。 尽管它有优点，也有一些limitat离子在使用PLA。一个这样的限制是针对感兴趣这是在不同物种所作的蛋白质的初级抗体的可用性。这个问题可以很容易地使用转基因标记蛋白的规避，尽管他们可能不是真正代表内生互动。另一个问题是，聚乳酸可能产生假阴性， 例如 ，当两个初级抗体在空间上阻碍彼此或当初级抗体之一的表位涉及蛋白质-蛋白质相互作用位点。此外，虽然聚乳酸检测的蛋白质;这些都是接近彼此，它不直接和间接的蛋白质 - 蛋白质相互作用区分，不像在下拉测定法和酵母双杂交筛选。此外，内源蛋白质之间PLA不会识别哪些域是负责的相互作用。因此，其它的蛋白质 - 蛋白质相互作用测定法仍然是需要的充分表征的相互作用molecularl年。 研究人员利用超分辨率显微镜来绘制果蝇幼虫NMJ的空间架构开始，随着正在实现几十纳米的26项决议。 PLA，其可比分子的分辨率，应该提供一个低成本，在技术上简单补充这些研究，将在建立的许多蛋白质​​在NMJ的组织的详细视图帮助。

果蝇光盘大（DLG）是支架蛋白，帮助协调的大蛋白质复合物的装配在质膜的特定位点的膜相关鸟苷酸激酶家族的保守构件。最初鉴定为肿瘤抑制蛋白，了Dlg用作上皮apicobasal极性1,2,3的重要决定因素。 DLG还担任以谷氨酸运动神经元的神经肌肉接头（NMJ）幼虫发育期间4的一大棚架模块。 DLG扮演的幼虫NMJ不同的角色，其多效依靠其与多种蛋白质5,6联系起来的能力。一个这样的蛋白是胡立泰邵（HTS），同源到主要被问候的调节肌动蛋白细胞骨架血影7描述自己的角色哺乳动物adducins。先前已表明了Dlg和HTS可以形成彼此基于体外复杂</EM&gt;涉及整个成年果蝇裂解物8共免疫沉淀实验。这些结果的一个缺点，但是，是它们不表示其中这个复杂的形式。通过使用免疫组化，和了Dlg HTS的分布观察幼虫NMJs的突触后膜重叠，但他们在该地区8复杂？由于最近的研究显示，进一步这里详述，接近结扎测定（PLA）是用来专门寻找一个在了Dlg和HTS之间的关联就地在幼虫NMJ 27。 PLA是一种使用主要集中在细胞和组织培养，可以检测原位 9蛋白质-蛋白质相互作用相对较新的技术。在该试验中，对两种蛋白的兴趣初级抗体与一对种特异性的第二抗体来检测，称为PLA探针，其缀合到寡核苷酸（ 图1A，B）。如果这两个蛋白s为在（内几十纳米的IE）靠近彼此，附加的PLA探针之间的距离可以通过增加两个连接器的寡核苷酸（ 图1C）的杂交来桥接。在这个构象中，连接器的寡核苷酸的自由端靠得足够近，以使彼此接触，以及一个闭合环状DNA分子可以在原位结扎（ 图1D）来形成。该环状DNA分子用作原位滚环扩增，这是由偶联到PLA探针（ 图1E）的寡核苷酸的一个引物的模板。所得扩增，concatemeric DNA产物中的序列然后可以可视化用荧光标记的，互补的寡核苷酸探针（ 图1F）。由于扩增的DNA保持附着到一个PLA探针，蛋白质 - 蛋白质相互作用withi的亚细胞定位NA组织可以很容易地确定。 几种方法通常用于检测蛋白质-蛋白质相互作用包括例如共免疫沉淀体外技术，下拉测定法和酵母双杂交筛选，并在如荧光共振能量转移（FRET）和双分子荧光互补体内技术（附设）。的体外技术的缺陷在于，它们不识别，其中相互作用是内源性存在的，而在上述的体内技术包括融合蛋白的人工表达，可能无法反映其内源性的对应的本机的行为。 PLA的一个主要优点是，它能够在组织内确定的内源蛋白相互作用物是在靠近彼此并可能形成复合物的亚细胞定位的，以产生一个信号是可比的FR所需亲密度ET和附设。 PLA可以检测具有高特异性和灵敏度的相互作用由于抗体识别和DNA扩增的耦合。因此，该测定法可以产生在泪点中，揭示了相互作用的确切位置的形式的离散，明亮的信号。此外，几乎不可见的抗原可被检测到。最后，PLA是一种相对简单的技术来执行，这需要不超过一个标准的免疫程序来完成。因此，PLA提供了技术优势，常常困扰着长的制备时间和广泛的故障排除其它的蛋白质 - 蛋白质相互作用测定。 此协议演示PLA如何可以适用于果蝇幼虫NMJ用于检测原位的内源蛋白质-蛋白质相互作用的目的。这里，聚乳酸的是幼虫体壁肌肉制剂哪里了Dlg和HTS被示为在一个复杂的确实存在于NMJs的突触后区域进行。解放军过去一直没有被用于研究幼虫NMJ，并有目前只有少数已经使用这个实验果蝇组织中发表的论文。希望解放军进一步暴露在果蝇社会将导致作为一个额外的工具，它越来越多地使用，以补充其他更常用的蛋白质-蛋白质相互作用分析。

<table><tbody><tr><td>Forceps (fine #5)</td><td>Almedic</td><td>A10-704</td><td></td></tr><tr><td>Sylgard Disc</td><td>World Precision Instruments</td><td>SYLG184</td><td>Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (&lt;em&gt;e.g.&lt;/em&gt; use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60 x 15 mm Petri dish lid when dissecting.</td></tr><tr><td>Minutien Pins (0.0125&amp;nbsp;mm tip diameter)</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Microdissection Scissors (ultra fine)</td><td>Fine Science Tools</td><td>15200-00</td><td></td></tr><tr><td>Platform Slides</td><td></td><td></td><td>Glue two 22 x 22 mm&lt;sup&gt;2&lt;/sup&gt; coverslips onto a microscope slide with clear nail polish, leaving a &amp;lt;20 mm gap in between for sample mounting.</td></tr><tr><td>w&lt;sup&gt;1118&lt;/sup&gt;</td><td>Bloomington Drosophila Stock Center</td><td>3605</td><td></td></tr><tr><td>hts&lt;sup&gt;01103&lt;/sup&gt;</td><td>Bloomington Drosophila Stock Center</td><td>10989</td><td>Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.</td></tr><tr><td>1x PBS (Phosphate Buffered Saline): 3&amp;nbsp;mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 7&amp;nbsp;mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 130&amp;nbsp;mM NaCl, pH 7.0</td><td></td><td></td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (Caledon Laboratories - 8180-1), Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (Caledon - 8120-1), NaCl (Caledon - 7560-1)</td></tr><tr><td>Bouin&#039;s Solution</td><td>Sigma-Aldrich</td><td>HT10132</td><td></td></tr><tr><td>4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td><td></td><td></td><td>PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td><td></td><td></td><td>Triton X-100 (Sigma-Aldrich - T8787)</td></tr><tr><td>1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td><td></td><td></td><td>BSA (Bioshop Canada - ALB001). Store at 4 &amp;deg;C.</td></tr><tr><td>mouse anti-Dlg (Discs large)</td><td>Developmental Studies Hybridoma Bank</td><td>4F3</td><td>Use at a 1:10 dilution in 1% BSA.</td></tr><tr><td>rabbit anti-HtsM (Hu-li tai shao)</td><td></td><td></td><td>Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td></tr><tr><td>rabbit anti-Pak (p21-activated kinase)</td><td></td><td></td><td>Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td></tr><tr><td>mouse anti-Wg (Wingless)</td><td>Developmental Studies Hybridoma Bank</td><td>4D4</td><td>Use at a 1:5 dilution in 1% BSA.</td></tr><tr><td>goat anti-Hrp (Horseradish peroxidase)</td><td>Jackson&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; ImmunoResearch</td><td>123-065-021</td><td>Use at a 1:200 dilution in 1% BSA.</td></tr><tr><td>FITC-conjugated donkey anti-goat</td><td>Jackson&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; ImmunoResearch</td><td>705-095-003</td><td>Use at a 1:200 dilution in 1% BSA.</td></tr><tr><td>Duolink In Situ PLA Probe anti-mouse MINUS</td><td>Sigma-Aldrich</td><td>DUO92004</td><td></td></tr><tr><td>Duolink In Situ PLA Probe anti-rabbit PLUS</td><td>Sigma-Aldrich</td><td>DUO92002</td><td></td></tr><tr><td>Duolink In Situ Detection Reagents Red</td><td>Sigma-Aldrich</td><td>DUO92008</td><td></td></tr><tr><td>1x Wash Buffer A: 0.01&amp;nbsp;M Tris, 0.15&amp;nbsp;M NaCl, 0.05% Tween 20, pH 7.4</td><td>Sigma-Aldrich</td><td>DUO82049</td><td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td></tr><tr><td>1x Wash Buffer B: 0.2&amp;nbsp;M Tris, 0.1&amp;nbsp;M NaCl, pH 7.5</td><td>Sigma-Aldrich</td><td>DUO82049</td><td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td></tr><tr><td>0.01x Wash Buffer B: 2&amp;nbsp;mM Tris, 1&amp;nbsp;mM NaCl, pH 7.5</td><td>Sigma-Aldrich</td><td>DUO82049</td><td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td></tr><tr><td>Duolink In Situ Mounting Medium with DAPI</td><td>Sigma-Aldrich</td><td>DUO82040</td><td></td></tr></tbody></table>

detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

在野生型的第三龄幼虫NMJs，的DLG为主发现在I型谷氨酸突触小结的突触后膜，用了Dlg免疫水平是在式Ib的终扣更加明显比类型是终扣（ 图3A）4。 HTS是整个肌肉存在，但集中于与HTS免疫反应水平出现等于在两个I型终扣突触后区域，并且还发现了突触前（ 图3A&#39;）8,17。注意了Dlg和HTS的分布在突触后区域（ 图3A&#39;&#39;）8很大程度上重叠。 如果要判断了Dlg和HTS存在于一个复杂的NMJ，PLA两种蛋白质之间的幼虫体壁肌肉的准备工作完成。对于该测定，小鼠抗了Dlg抗体，其检测在N-末端和兔抗HtsM抗体的第二PDZ结构域其检测在C-末端MARCKS同源域被用来15,16。在野生型，PLA了Dlg和HTS之间的信号，观察具体地说在NMJ（ 图3C-C&#39;&#39;）。所述信号大多局限在圆周向I型终扣，将其周围限定由HRP的突触前膜，从而表明了Dlg和HTS非常靠近彼此并可能形成复合物，在突触后的区域。结果被确定为具体的作为我们的阴性对照，涉及缺乏HTS免疫HTS 01103突变NMJs（ 图3B-B&#39;&#39;），没有表现出可观察到的聚乳酸的信号（ 图3D）8,17,18。此外，聚乳酸进行不添加兔抗HtsM抗体不产生信号（数据未显示）。所述终扣的高放大视图显示了Dlg和HTS免疫严重重叠在突触后区域（ 图3E）的，而这两种蛋白之间PLA导致谨慎泪点指示该总了Dlg和HTS蛋白的一个子集是在复杂的（ 图3F）。 进一步测试PLA的可靠性研究幼虫NMJ的工具，我们还评估了丝氨酸/苏氨酸的p21活化激酶，白，以及其他突触蛋白19之间的相互作用。先前免疫共沉淀实验表明，Pak是自由曲线复杂，其中了Dlg也是构件20的构件。在野生型NMJs，白定位于突触后密度（ 图4A，C）中，在那里它需要了Dlg定位15,21。白和了Dlg的免疫反应性分布的两种蛋白质产生了阳性信号特别在NMJ（ 图4B）之间重叠在突触后区域（ 图4A&#39;&#39;），和聚乳酸。这些结果表明，这两种蛋白是在C<html>失去相互接近，并有可能形成在该区域的复合物。我们测试了白复合物结合另一突触蛋白是无翅（WG）， 果蝇 Wnt配体，这在NMJ 22起着许多作用。在野生型，Wg的富集在I型终扣的突触前和突触后两侧，但也存在在整个肌肉细胞质（ 图4C&#39;）23泪点。有趣的是，虽然白和的Wg的免疫反应性的分布部分地重叠在突触后区域（ 图4C&#39;&#39;），聚乳酸两种蛋白质之间产生没有可观察的信号（ 图4D）。因此，白和Wg的不靠近彼此在NMJ。这个结果表明，并非所有示出通过传统的共定位实验重叠免疫反应的蛋白质将产生一个PLA信号，并作为一个额外的阴性对照。 ENT"FO:保持together.within页="总是"&gt; <img alt="图1" src="/files/ftp_upload/52139/52139fig1highres.jpg" width="700px" /> 图1:接近结扎分析示意图。 （A）的初级抗体结合于感兴趣的两种蛋白质使用标准免疫程序。（B）中的第一抗体，然后用一对种特异性的第二抗体来检测，称为PLA探头，它被缀合的寡核苷酸。（℃）如两种蛋白质都在靠近彼此，附加的PLA探针之间的距离可以通过两个附加连接的寡核苷酸杂交来桥接。（D）在本形态中，连接寡核苷酸可以在原位结扎形成闭合环状DNA分子（E） 在原位的环状DNA分子用作模板用于滚环扩增，这是由OLI之一引gonucleotides缀合到PLA探针。（F）中扩增的DNA产物中的序列，然后用荧光标记的，互补的寡核苷酸探针检测出来。请注意，这个数字是基于Weibrecht 等 24。 <a href="/files/ftp_upload/52139/52139fig1highres.jpg" target="_blank">请点击此处查看图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52139/52139fig2highres.jpg" width="700px" /> 图2:三龄幼虫体壁准备。 （AF）表示体壁夹层的原理图。（A）将单幼虫到SYLGARD盘，其背面朝上，使两个气管大片都是可见的。（B）引脚幼虫下来的前，后两端与minutien销，拉伸幼虫出纵向的过程中。（C） </strong&gt;的制作，在幼虫体内的背侧与显微剪刀三切口。切口应类似于一个&#39;&#39;我&#39;&#39;完成时（D）打出幼虫体内开放，针角下来，水平和垂直拉伸体壁的过程中，形成一个均匀的张矩形。（E）清洁出图中所示的任何剩余的内部器官。（F）是的不同的方式来切断体壁的拐角，以便区分不同基因型的例子。（G）的视图的拉伸体壁的准备完成时。该照片是用数码相机安装在解剖显微镜。在F组比例尺为1毫米。请注意，这个数字是基于德兰和Budnik，2010 12。 <a href="/files/ftp_upload/52139/52139fig2highres.jpg" target="_blank">请点击此处查看图的放大版本。</a> 图3:了Dlg和HTS存在于复杂的幼虫NMJs的突触后区（AB&#39;&#39;）了 Dlg（红色）和HTS（绿色）中的野生型的分布和HTS突变NMJs（AA&#39;&#39;）在野外。 -type NMJs，的DLG主要发现于Ⅰ型终扣的突触后膜，用了Dlg层次更加明显的Ib型比终扣。 HTS是整个肌肉本，而且集中围绕突触后区域，具有HTS水平是在I型终扣相似。了Dlg和HTS的重叠分布在突触后的区域。（BB&#39;）NMJs突变的HTS缺乏HTS免疫反应。需要注意的是HTS 01103突变的影响NMJ分支8。（CD&#39;&#39;）了 Dlg和HTS之间的PLA（红）对野生型执行， 高温超导突变体NMJs。 HRP是用来标记神经元膜（绿色）。示出的是几个终扣。（CC&#39;&#39;）在野生型高倍率的观点，PLA信号在NMJ具体观察到。所述信号大多局限在圆周向I型终扣的突触前膜，这表明了Dlg和HTS非常接近，彼此存在于复杂的突触后的区域。（D）的 NMJs突变为HTS显示没有可观察到的聚乳酸的信号。 （EF）显示的是单突触小结的高倍率的意见。（E）在突触后区域HTS和了Dlg免疫严重重叠。了Dlg和HTS结果在不同的泪点之间（F）的聚乳酸指示该蛋白质的一个子集是在一个复杂的。在面板B&#39;标尺&#39;代表40微米（AB&#39;）;在面板D&#39;标尺&#39;代表10微米（CD&#39;&#39;）; SCAL在F组Ë条代表5微米（EF）。所有显示的NMJs来自肌肉中的6/7腹节4影像拍摄与使用尼康A1R激光扫描共聚焦显微镜与NIS-Elements软件合并协议栈，并与Adobe Photoshop处理。 <a href="/files/ftp_upload/52139/52139fig3highres.jpg" target="_blank">请点击此处查看的放大版数字。</a> <img alt="图4" src="/files/ftp_upload/52139/52139fig4highres.jpg" width="700px" /> 图4:白中存在与了Dlg一个复杂的，但不是WG，在幼虫NMJs的突触后区域（AD）的图中所示是一个几终扣高倍率次（AA&#39;）百（绿）和了Dlg的分布（。红色）的野生型NMJs。朴定位于突触后密度。需要注意的是白和多元学习津贴的免疫反应分布在突触后区重叠。（B）PLA Between乐和了Dlg（红色）在野生型NMJs执行。 PLA信号在NMJ具体观察到，这表明这两种蛋白是在靠近彼此在这个区域。（CC&#39;&#39;）中的野生型NMJs分布朴（绿色）和Wg的（红色）的。 WG富集在两个突触前和突触后的NMJ的两侧，但也存在在整个肌肉泪点。需要注意的是白的免疫反应分布也观察到的Wg在突触后区部分重叠。（D）白及的Wg（红色）与中国人民解放军野生型NMJs执行。没有具体的PLA信号被观察到，这表明这两种蛋白是不靠近彼此在NMJ尽管它们重叠分布。在D组比例尺代表5微米（AD）。所有NMJs受神经支配的肌肉6/7的腹节4影像拍摄与使用尼康A1R激光扫描共聚焦显微镜与NIS-Elements软件合并栈，和亲cessed与Adobe Photoshop， <a href="/files/ftp_upload/52139/52139fig4highres.jpg" target="_blank">请点击这里查看图的放大版本。</a>

Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

此协议演示如何接近结扎检测试剂盒可用于检测原位蛋白质-蛋白质相互作用的果蝇幼虫神经肌肉接头。利用这种技术，光盘大和胡立泰邵示，以形成一复合的突触后区域，预先通过共免疫沉淀鉴定的关联。

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

detection-situ-protein-protein-complexes-at-drosophila-larval

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

13.1K 次观看.  Simon Fraser University. 此协议演示如何接近结扎检测试剂盒可用于检测原位蛋白质-蛋白质相互作用的果蝇幼虫神经肌肉接头。利用这种技术，光盘大和胡立泰邵示，以形成一复合的突触后区域，预先通过共免疫沉淀鉴定的关联。 

视频: Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

科研

生物化学

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

发育生物学

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

检测信号效应复合物BMP4的下游使用原位解放军，接近结扎分析

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

生物学

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

神经科学

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

研究内质网和线粒体相互作用通过<em&gt;原位</em&gt;接近连接分析在固定细胞

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

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