我们的工作一直支持由美国心脏协会（大中华区东南亚联盟）开始赠款援助0765185B，艾尔莎美国帕迪基金会的研究经费，韦恩州立大学校内的启动基金和心血管研究所伊希斯倡议的奖助学金。这种方法在体外 CFTR的大分子复杂的装配最初率先博士AP那仁（田纳西大学健康科学中心）。

1。标记细菌中的重组融合蛋白表达和纯化<ol><li>放大定义CFTR的一览表2，MRP2的，MRP4，β2受体，和NHERFs（全长或PDZ1过或PDZ2域地区的C-尾（最后50-100个氨基酸，包含在C-末端的PDZ结构图案） ）通过PCR方法。 </li><li> PCR产物克隆到pGEX4T-1载体（如GST-NHERFs的，GST-MRP4 CT），GST融合蛋白的MBP融合蛋白的质粒pMAL-C2载体（如的MBP-β2受体的CT，MBP-CFTR的CT ），pET30-S融合蛋白（如他 - S-CFTR的CT，他的S-PDZK1）。 </li><li>变换在蛋白酶缺陷E.大肠杆菌菌株（大肠杆菌BL21-DE3），以减少可能出现的重组蛋白的降解。 </li><li>在37℃培养过夜增长在卢里亚Bertani培养基含有适当的抗生素（如氨苄青霉素或卡那霉素）（pH值7.0）。在1:10稀释过夜培养，另2小时增长在37°C。在杜奇与0.5-1米M为接下来的4小时在30℃经IPTG在8000离心沉淀细胞×G为10分钟，在4°C。 </li><li>蔗糖缓冲液裂解细胞（含50 M M的Tris-HCl，pH值8.0，1 M M 1 M M的EDTA，PMSF，10％的蔗糖）溶菌酶（1毫克/毫升），0.2％的Triton X-100和蛋白酶抑制剂。从1升文化起源的细胞沉淀用20毫升。 </li><li>在4°C，在30分钟摇床混合</li><li>自旋为30分钟，在4°C，在20000×G收集上清。 </li><li>到上层清液，加入1毫升以下的树脂/琼脂糖珠（50％蔗糖缓冲泥浆）： <ul><li> GST融合蛋白谷胱甘肽琼脂糖珠。 </li><li>爪珠-S的融合蛋白。 </li><li> MBP融合蛋白的直链淀粉树脂。 </li></ul></li><li>组合为4小时，在4°Ç在摇床。 </li><li>洗珠重新悬浮在1X PBS（15毫升），混合2公里N，纺纱800×2分钟，弃去上清Ğ。六次重复此步骤。 </li><li>从珠，通过各自的洗脱缓冲液（2毫升，每1毫升珠洗脱缓冲液）洗脱蛋白质。 <ul><li> GST融合蛋白的洗脱缓冲：25 M M的Tris-HCl（pH值8.0），140 M M氯化钠和20 M M谷胱甘肽。 </li><li>他的融合蛋白的洗脱缓冲液：20 M M的Tris-HCl（pH值8.0）M氯化钠，500米和200米M咪唑。 </li><li> MBP融合蛋白的洗脱缓冲液：20 M M的Tris-HCl（pH值8.0），200米M氯化钠，1 M M M M的EDTA，1数码地面电视，和10 M M麦芽糖。 </li></ul></li><li>透析1X PBS对2大号洗脱蛋白质在4°C间（以避免可能的蛋白质降解）。改变PBS每4小时的四倍。蛋白作为小等分1 Centricon滤波器（截止10,000兆瓦，Millipore公司）和存储集中在-80°C。 </li><li> DET貂皮Bradford法蛋白浓度。评估通过SDS-PAGE使用BSA作为标准蛋白质量。如果蛋白质的完整性是不能令人满意的，如二次净化凝胶过滤或离子交换程序可以使用。 （例如，我们用一个G-75琼脂糖凝胶柱进一步纯化GST融合蛋白）。 </li></ol> 2。细胞培养和细胞裂解液的制备<ol><li>文化幼仓鼠肾细胞（BHK），稳定过表达的CFTR WT和的CFTR his10或BHK细胞，瞬时过表达旗一览表2-WT或旗一览表2ΔSTL，Madin 4197犬肾（MDCK细胞细胞），稳定过表达MRP2的鹰的最低限度的基本培养基（MEM）的5％CO 2含10％小牛血清，青霉素/链霉素聚苯乙烯瓶在37°C间。 </li><li>文化人类胚胎肾293细胞（HEK293细胞），稳定过表达的CFTR WT的CFTR his10的在杜尔贝科“的改良Eagle培养基（DMEM培养基）在37°C 5％的CO 2与10％小牛血清和青霉素/链霉素聚苯乙烯瓶补充。 </li><li>裂解细胞裂解液（PBS - 0.2％TRITON-X-100的补充含有M M phenylmethylsulphonyl氟化物，1微克/毫升抑肽酶，1微克/毫升亮肽素，1微克/毫升胃蛋白酶抑制剂，蛋白酶抑制剂的混合物）;使用500μL裂解液，每60毫米培养皿，1000μL裂解液，每100毫米的培养皿。 </li><li> 20分钟摇滚细胞裂解液在4°C，并删除15分钟在16000×g下离心的不溶性物质，在4°C。确定由Bradford法蛋白浓度。 </li></ol> （3） 在体外一个CFTR含的高分子复合体大会（CFTR的PDZK1-MRP4） <ol><li>到200μL裂解缓冲液（PBS - 0.2％的Triton-X的100蛋白酶抑制剂），添加20μg纯化GST-MRP4-C终端50 AA（CT50）融合蛋白。 </li><li>加入S-PDZK1融合蛋白纯化的各种款项（0-40微克）。 </li><li>上旋转22°1-2 C H搅拌机混合的两种蛋白质。 </li><li>加入20微升谷胱甘肽珠（50％浆料）的蛋白质混合物，继续拌匀，另1小时。这一步也被称为成对结合。 </li><li>在此期间，准备HEK293细胞裂解过度旗，CFTR（WT）如上所述，在第2步）。 </li><li>裂解液洗两次复杂。旋转800×G为每洗1分钟，小心吸出上清后，每洗。谨慎使用不吸在底部的珠子。 </li><li>添加上述准备HEK293细胞裂解珠，轻轻混合为3小时（或隔夜）在4°C。 </li><li>洗珠广泛裂解液三个步骤3.6）倍。 </li><li>洗脱的蛋白质使用30μL的样品缓冲液（5×）：0.6米<html>的Tris-HCl（pH值6.8），50％的甘油，2％的SDS，0.1％溴酚蓝;含5％β-巯基乙醇。 </li><li>孵育在37°C间水浴10-15分钟，和自旋为5000×30秒Ğ沉淀珠。 </li><li>加载到4-15％凝胶洗脱液。 </li><li>运行约30-40分钟的SDS-PAGE。 </li><li>蛋白条带转移到PVDF膜，为1.5小时。 </li><li>座中的TBS-吐温膜含有5％非脂乳。 </li><li>酝酿的初级抗体（如抗CFTR的抗体，R1104）膜在4°C间，过夜。 </li><li>膜用TBS-吐温洗5分钟，六次。 </li><li>在22°C孵育45分钟二次抗体（如羊抗鼠HRP标记的第二抗体）膜</li><li>膜用TBS-吐温洗5分钟，六次。 </li><li>通过的ECL蛋白带可视化。 </li><li>代表性的数据， 如图1所示。 </li></ol>jove_title“&gt; 4代表结果。 如图1所示的CFTR含大分子信号在体外组装的复杂的例子。 MRP4的C-末端50个氨基酸（MRP4-CT50），，PDZK1，全长度的CFTR（ 图1， 底部 ）之间形成一个大分子复合物。形成复合物剂量依赖性增加，越来越多的中介蛋白质，PDZK1（ 图1， 底部 ）18。 <img alt="图1" src="/files/ftp_upload/4091/4091fig1.jpg" /> 图1图示（ 顶部 ）的大分子复杂的检测。大分子复合物在体外检测与三种蛋白质在剂量依赖性（ 底部 ）18（GST-MRP4-CT50，他- S-PDZK1，旗CFTRwt）。 <table border="1"><tbody><tr><td></td><td><strong&gt;的CFTR的NHERF1-β2 AR（见14） </td><td> CFTR的NHERF2-LPA的2（注释15） </td><td> CFTR的PDZ蛋白MRP的2（注释17） </td><td> CFTR的PDZK1-MRP4（注释16） </td></tr><tr><td> 亲和力珠 </td><td>直链淀粉树脂</td><td> S - 蛋白琼脂糖</td><td>直链淀粉树脂</td><td>谷胱甘肽琼脂糖</td></tr><tr><td> 纯化的蛋白质-1 </td><td> MBP的β-2受体的CT </td><td> - S-CFTR的CT </td><td> MBP-CFTR的CT </td><td> GST-MRP4 CT </td></tr><tr><td> 纯化的蛋白质-2 </td><td> GST-NHERF1 </td><td> GST-NHERF2的</td><td>的GST-PDZ蛋白</td><td> - S-PDZK1 </td></tr><tr><td> 纯化蛋白3（或细胞裂解） </td><td> CFTR基因-WT的CFTR his10的的 （染BHK的HEK细胞裂解） </td><td>旗-LPA的2-WT或旗一览表2ΔSTL（染BHK细胞裂解物） </td><td> MRP2的（MDCK细胞裂解） </td><td>纯化旗的CFTR WT或的CFTR his10（或细胞裂解物） </td></tr><tr><td> 抗体 </td><td>抗-CFTR的IgG </td><td>反旗酶的</td><td>抗-MRP2的IgG </td><td>反旗酶的</td></tr></tbody></table> 表1总结各种CFTR的含大分子复合物在体外组装。

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+study+of+intermolecular+and+intramolecular+interactions+regulating+CFTR+function.">Methods for the study of intermolecular and intramolecular interactions regulating CFTR function.</a> Met. Molecul. Med. 70, 175-186 (2002).</li><li>Li, C., Roy, K., Dandridge, K., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+assembly+of+cystic+fibrosis+transmembrane+conductance+regulator+in+plasma+membrane.">Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane.</a> The Journal of biological chemistry. 279, 24673-24684 (2004).</li><li>Li, C., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CFTR+Interactome+in+the+Macromolecular+Complexes.">Analysis of CFTR Interactome in the Macromolecular Complexes.</a> Met. Molecul. Med. 741, 255-270 (2011).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemokine+receptor+CXCR2+macromolecular+complex+regulates+neutrophil+functions+in+inflammatory+diseases.">A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.</a> J. Biol. Chem. , (2011).</li></ol>

没有利益冲突的声明。

在这个协议中，我们展示了一个方法， 在体外装配，检测包含使用纯化蛋白（或蛋白质碎片）和/或细胞裂解大分子信号复合物的CFTR报告以前16-19,29,30。为了达到最佳效果临界点以下，在筹备过程中，需要特别注意： <ul><li>重要的是，洗脱缓冲液的pH值调整到8.0后加入还原型谷胱甘肽的纯化GST融合蛋白，如步骤1）中所述时。此外还原型谷胱甘肽后，pH值可低到3.0。如果PH值未调整?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> pGEX4T-1载体</td><td> GE医疗集团</td><td> 28-9545-49 </td><td>原Amersham Biosciences公司</td></tr><tr><td>质粒pMAL-C2载体</td><td> New England Biolabs公司</td><td></td><td></td></tr><tr><td> pET30载体</td><td>电解二氧化锰化学品</td><td> 69077-3 </td><td>前Novagen公司</td></tr><tr><td>谷胱甘肽琼脂糖珠</td><td> BD公司</td><td> 554780 </td><td></td></tr><tr><td>直链淀粉树脂</td><td> New England Biolabs公司</td><td> E8021S </td><td></td></tr><tr><td>爪珠</td><td> Clontech公司</td><td> 635501 </td><td></td></tr><tr><td>还原型谷胱甘肽</td><td> BD公司</td><td> 554782 </td><td></tD&gt; </tr><tr><td>咪唑</td><td>费舍尔</td><td> BP305-50 </td><td></td></tr><tr><td>麦芽糖</td><td>费舍尔</td><td> BP684-500 </td><td></td></tr><tr><td> S - 蛋白琼脂糖</td><td>电解二氧化锰化学品</td><td> 69704-3 </td><td>前Novagen公司</td></tr><tr><td>反旗酶的</td><td>西格玛</td><td> A8592 </td><td></td></tr><tr><td>抗-CFTR的IgG </td><td>定制</td><td> R1104 </td><td>单克隆抗体识别抗原的CFTR AA 722-734 </td></tr><tr><td>抗-MRP2的IgG </td><td> Chemicon公司国际</td><td> MAB4148 </td><td>现在部分的微孔</td></tr></tbody></table> 表2。特定的试剂和设备。

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

囊性纤维化跨膜电导调节器（CFTR），主要位于上皮细胞的顶膜的氯离子通道，起着至关重要的作用，在跨上流体稳态1-3。 CFTR基因有牵连的两个主要疾病：囊性纤维化（CF）4和第5分泌性腹泻。在CF的CFTR氯离子通道的功能活动，合成或降低。这种疾病会影响美国的约1 6 2500白种人。过度CFTR的活动也被牵连毒素诱导的分泌性腹泻（如霍乱毒素和热稳定大肠杆菌肠毒素）刺激cAMP或cGMP生产在肠道内的7例。 越来越多的证据表明CFTR和其他蛋白质的数目越来越多，包括运输，离子通道，受体，激酶，磷酸酶，信号之间的物理和功能相互作用的存在ING分子和细胞骨架的元素，CFTR和其结合蛋白之间相互作用已被证明要严格调节在体外和体内8-19 CFTR介导的跨膜离子转运。在这个协议中，我们只注重方法，援助在CFTR的羧基末端，它拥有一个蛋白质结合基序之间的相互作用的研究[简称为PSD95/Dlg1/ZO-1（序）序]，和PDZ结构域所指的脚手架蛋白，其中包含一个特定的绑定模块组。到目前为止，几个不同的PDZ骨架蛋白已绑定到CFTR的羧基末端与各种亲缘关系，如NHERF1，NHERF2，PDZK1，PDZK2，CAL（CFTR的相关配体），Shank2，把握20-27。内CFTR的PDZ结构图案是公认的PDZ骨架蛋白是在过去四年在C端（即1477-DTRL-1480在人类的CFTR）20个氨基酸。有趣的是，CFTR的，可以绑定多个PDZ结构域都NHERFs和PDZK1，尽管有不同的亲和力22。这方面的CFTR的具有约束力的多价已被证明是功能性的意义，这表明，PDZ结构骨架蛋白可能促进CFTR的形成大分子信号为特定/选择性和高效率的信号复合物在细胞16-18。 已开发多个生化分析研究，CFTR涉及蛋白质的相互作用，如免疫共沉淀，下拉法，成对结合法，比色法成对结合实验，和复杂的大分子组装检测16-19,28,29 。在这里，我们重点对1的PDZ基序依赖CFTR含大分子体外 ，这是我们的实验室研究蛋白质或域的域的相互作用，涉及的CFTR 16-19,28,29广泛用于复杂装配的详细程序。

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

囊性纤维化跨膜电导调节器（CFTR），上皮细胞的氯离子通道，已与多种蛋白质相互作用和调节重要的细胞过程的CFTR序序介导的相互作用，其中已经有据可查。这个协议描述我们开发组装的PDZ-依赖的CFTR大分子信号复合物的方法<em&gt;在体外</em&gt;。

依赖的PDZ-CFTR的大分子信号复合物的体外分析

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

in-vitro-analysis-pdz-dependent-cftr-macromolecular-signaling

Research

JoVE Journal

Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

10.3K 次观看.  Wayne State University School of Medicine. 囊性纤维化跨膜电导调节器（CFTR），上皮细胞的氯离子通道，已与多种蛋白质相互作用和调节重要的细胞过程的CFTR序序介导的相互作用，其中已经有据可查。这个协议描述我们开发组装的PDZ-依赖的CFTR大分子信号复合物的方法在体外。

视频: In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

科研

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, ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.