检测<em>原位</em>蛋白质 - 蛋白质复合物的<em>果蝇</em>幼虫神经肌肉接头使用接近结扎分析
Summary
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。
Abstract
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.
Introduction
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>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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of Material/Equipment | Company | Catalog Number | Comments/Description |
| Forceps (fine #5) | Almedic | A10-704 | |
| Sylgard Disc | World Precision Instruments | SYLG184 | Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting. |
| Minutien Pins (0.0125 mm tip diameter) | Fine Science Tools | 26002-10 | |
| Microdissection Scissors (ultra fine) | Fine Science Tools | 15200-00 | |
| Platform Slides | Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a <20mm gap in between for sample mounting. | ||
| w1118 | Bloomington Drosophila Stock Center | 3605 | |
| hts01103 | Bloomington Drosophila Stock Center | 10989 | Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal. |
| 1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0 | NaH2PO4 (Caledon Laboratories – 8180-1), Na2HPO4 (Caledon – 8120-1), NaCl (Caledon – 7560-1) | ||
| Bouin's Solution | Sigma-Aldrich | HT10132 | |
| 4% PFA (Paraformaldehyde): 4% PFA in 1x PBS | PFA (Anachemia Science – 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution. | ||
| 1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton | Triton X-100 (Sigma-Aldrich – T8787) | ||
| 1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT | BSA (Bioshop Canada – ALB001). Store at 4 °C. | ||
| mouse anti-Dlg (Discs large) | Developmental Studies Hybridoma Bank | 4F3 | Use at a 1:10 dilution in 1% BSA. |
| rabbit anti-HtsM (Hu-li tai shao) | Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA. | ||
| rabbit anti-Pak (p21-activated kinase) | Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA. | ||
| mouse anti-Wg (Wingless) | Developmental Studies Hybridoma Bank | 4D4 | Use at a 1:5 dilution in 1% BSA. |
| goat anti-Hrp (Horseradish peroxidase) | Jackson ImmunoResearch | 123-065-021 | Use at a 1:200 dilution in 1% BSA. |
| FITC-conjugated donkey anti-goat | Jackson ImmunoResearch | 705-095-003 | Use at a 1:200 dilution in 1% BSA. |
| Duolink In Situ PLA Probe anti-mouse MINUS | Sigma-Aldrich | DUO92004 | |
| Duolink In Situ PLA Probe anti-rabbit PLUS | Sigma-Aldrich | DUO92002 | |
| Duolink In Situ Detection Reagents Red | Sigma-Aldrich | DUO92008 | |
| 1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4 | Sigma-Aldrich | DUO82049 | Tris (Caledon Laboratories – 8980-1), NaCl (Caledon – 7560-1), Tween 20 (Fisher Scientific – BP337) |
| 1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5 | Sigma-Aldrich | DUO82049 | Tris (Caledon Laboratories – 8980-1), NaCl (Caledon – 7560-1) |
| 0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5 | Sigma-Aldrich | DUO82049 | Tris (Caledon Laboratories – 8980-1), NaCl (Caledon – 7560-1) |
| Duolink In Situ Mounting Medium with DAPI | Sigma-Aldrich | DUO82040 |
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