Name: protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves
Uploaded: 2014-03-09T14:00:00
Description: Watch this Scientific Journal Video about Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves at JoVE.com

我们要感谢于尔根索尔的有益讨论和克里斯嘉莉的手稿的批判性阅读。该项目资助的东风集团和全宗DER chemischen工业公司（补助号SFB 1035项目A04至SS和做二十二分之一百八十七到RS）通过。

1。附设的转型构建农杆菌中<ol><li>附设构建的克隆<ol><li>使用含侧翼attB位站点寡核苷酸相应的模板扩增目的基因。执行使用校对聚合酶的PCR。适应的退火步骤来设计引物组合的长度，并根据片段大小的延伸步骤的长度。检查PCR产物通过琼脂糖凝胶电泳，并通过使用PCR清洁试剂盒纯化它。 </li><li>执行与所获得的片段即BP反应，并用BP重组酶的入门载体。混合15-150纳克的attB位-PCR产物用150纳克的条目...

<ol>
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Biol. 479, 189-202 (2009).</li><li>Weinthal, D., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein-protein+interactions+in+plant+cells+by+bimolecular+fluorescence+complementation+assay.">Imaging protein-protein interactions in plant cells by bimolecular fluorescence complementation assay.</a> Trends Plant Sci. 14, 59-63 (2009).</li><li>Ohad, N., Shichrur, K., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+protein-protein+interactions+in+plants+by+bimolecular+fluorescence+complementation.">The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.</a> Plant Physiol. 145, 1090-1099 (2007).</li><li>Ohad, N., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+bimolecular+fluorescence+complementation+(BiFC)+to+assay+protein-protein+interaction+in+plants.">Utilizing bimolecular fluorescence complementation (BiFC) to assay protein-protein interaction in plants.</a> Methods Mol. Biol. 655, 347-358 (2010).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Lee, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+a+cDNA+library+for+protein-protein+interactions+directly+in+planta.">Screening a cDNA library for protein-protein interactions directly in planta.</a> Plant Cell. 24, 1746-1759 (2012).</li><li>McFarlane, H. E., Shin, J. J., Bird, D. A., Samuels, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ABCG+transporters,+which+are+required+for+export+of+diverse+cuticular+lipids,+dimerize+in+different+combinations.">Arabidopsis ABCG transporters, which are required for export of diverse cuticular lipids, dimerize in different combinations.</a> Plant Cell. 22, 3066-3075 (2010).</li><li>Dunschede, B., Bals, T., Funke, S., Schunemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+studies+between+the+chloroplast+signal+recognition+particle+subunit+cpSRP43+and+the+full-length+translocase+Alb3+reveal+a+membrane-embedded+binding+region+in+Alb3+protein.">Interaction studies between the chloroplast signal recognition particle subunit cpSRP43 and the full-length translocase Alb3 reveal a membrane-embedded binding region in Alb3 protein.</a> J. Biol. Chem. 286, 35187-35195 (2011).</li><li>Gehl, C., Waadt, R., Kudla, J., Mendel, R. R., Hansch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+GATEWAY+vectors+for+high+throughput+analyses+of+protein-protein+interactions+by+bimolecular+fluorescence+complementation.">New GATEWAY vectors for high throughput analyses of protein-protein interactions by bimolecular fluorescence complementation.</a> Mol. Plant. 2, 1051-1058 (2009).</li><li>Qbadou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+chaperone+Hsp90+delivers+precursor+proteins+to+the+chloroplast+import+receptor+Toc64.">The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64.</a> EMBO J. 25, 1836-1847 (2006).</li><li>Schweiger, R., Muller, N. C., Schmitt, M. J., Soll, J., Schwenkert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtTPR7+is+a+chaperone+docking+protein+of+the+Sec+translocon+in+Arabidopsis.">AtTPR7 is a chaperone docking protein of the Sec translocon in Arabidopsis.</a> J. Cell Sci. , (2012).</li><li>Schweiger, R., S, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtTPR7+as+part+of+the+Arabidopsis+Sec+post-translocon.">AtTPR7 as part of the Arabidopsis Sec post-translocon.</a> Plant Signal Behav. 8, (2013).</li><li>Nelson, B. K., Cai, X., Nebenfuhr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolored+set+of+in+vivo+organelle+markers+for+co-localization+studies+in+Arabidopsis+and+other+plants.">A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.</a> Plant J. 51, 1126-1136 (2007).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+analysis+as+a+probe+of+protein+interactions+in+living+cells.">Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.</a> Annu. Rev. Biophys. 37, 465-487 (2008).</li><li>Koop, H. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+foreign+sequences+into+the+tobacco+plastome+via+polyethylene+glycol-mediated+protoplast+transformation.">Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.</a> Planta. 199, 193-201 (1996).</li></ol>

当规划一个附设实验几点应予以考虑。虽然对感兴趣的蛋白质没有结构信息是必需的，拓扑与跨膜蛋白质工作时是已知的。荧光蛋白必须驻留在同一个亚细胞区室或面对的膜，以允许相互作用的同一侧。自然地，分析这需要一个N-末端的定位序列的蛋白质时，只有一个C-末端标签可以考虑的。因为它是可能的标记干扰感兴趣的蛋白质，最好是预先测试的亚细胞定位，例如，通过表达GFP标记的蛋白的...

研究蛋白质复合物的形成和它们在植物细胞在体内的定位是必不可少的探讨蜂窝网络，信号和代谢过程。附设允许的蛋白质-蛋白质相互作用可视化直接在活体植物细胞1-5在他们的自然环境。 在附设接近的荧光蛋白引到一个重组荧光蛋白2非荧光性的N-和C-末端片段的互补。许多不同的荧光蛋白的片段已被用于检测蛋白质相互作用， 例如绿色荧光蛋白（GFP），其中由三种不同的残基6化学键形成的发色团。荧光蛋白可以在循环或β-链以产生两个非荧光片段可以被融合到感兴趣的两种蛋白质内减半。该测定可用于检测任何亚细胞compartme相互作用nt的任何需氧生长的生物体或细胞，可以进行基因修饰以表达融合蛋白。如果这两种蛋白进入细胞内的接近，荧光被重构，并且可以通过显微镜，而不添加外源荧光团或染料3来监控。 烟草（ 本氏烟 ）已被证明是方便的模型有机体可视化植物蛋白质的相互作用，因为蛋白质可以容易地通过利用烟叶与所产生的构建体农杆菌介导的转化来表达。农杆菌使用一个所谓的Ti质粒（肿瘤诱导）编码介导感兴趣的基因转导到植物细胞中的酶。附设是很好适用于水溶性以及对所有细胞区室中的膜蛋白，并已成功地用于在过去几年，以确定在体内相互作用的蛋白质，以及对分析相互作用位点内蛋白质7-9。待导入的基因表达，荧光蛋白的相互作用，可以直接在叶片可视化，这是适合于较大的蜂窝结构，如内质网（ER），在质膜或叶绿体。然而，为了监视在更精细的结构定位，例如，叶绿体被膜，最好是可视化的荧光在原生质体从转化的烟草叶中分离。一组含有任一C-末端或N-末端的荧光标记有用于在植物10的附设方法附设载体。在下文中描述的协议被用来研究细胞内的热休克蛋白90（HSP90）的相互作用与三十四肽重复含蛋白质对接Toc64和AtTPR7居住在叶绿体外封套和内质网，分别11-13（TPR）结构域。为了这个目的，HSP90融合到的CtSCFP（SCFP C）的erminal一部分。标签上还有N端融合到伴侣，以确保HSP90的C-末端MEEVD结合基序钳型TPR结构域的可访问性。与此同时，金星（金星N）的N-末端部分融合到含有蛋白质对接Toc64和AtTPR7分别TPR结构域的胞内域。作为阴性对照，我们克隆SCFP 的C可溶性C-末端部分完全驻留在细胞质中，因此适当的控制。 所研究的蛋白质的荧光标记要面对同样的细胞区室，让靠近，从而荧光信号的重建。来确定重构的荧光信号稠合到不同荧光标签标记的蛋白质可共转化来演示的相互作用的亚细胞定位的定位。融合到mCherrry一个ER标志物蛋白的同时转化案例位于AtTPR7 14急诊室的。叶绿素的自发荧光担任叶绿体标记的情况下Toc64的。通过这不仅Toc64和AtTPR7，分别与胞质HSP90分子伴侣的体内相互作用，可以直接在烟草叶片监测，但也相互作用的亚细胞定位可以进行调查。 附设很适合作为一种补充的方法来研究蛋白质 - 蛋白质相互作用的其它方法。相比于免疫共沉淀或体外下拉实验，例如，没有特异性抗体必须是可用于感兴趣的蛋白质，以及这些蛋白质没有被重组表达在体外 ，它可以是具有挑战性的，特别是对于膜蛋白。此外，也瞬时相互作用可以利用附设监视，因为蛋白质是由稠合的荧光标签15的相互作用捕获。

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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">3&#39;,5&#39;-Dimethoxy-4&#39;-hydroxyacetophenone</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:255px;">D134406</td>
			<td style="width:195px;">Acetosyringone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Cellulase, Onozuka-R10</td>
			<td style="width:147px;">Serva</td>
			<td style="width:255px;">16419</td>
			<td>from Trichoderma viridae</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Macerozyme R-10&#160;</td>
			<td style="width:147px;">Serva</td>
			<td style="width:255px;">28302</td>
			<td>from Rhizopus sp</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:293px;">GATEWAY, BP Clonase II, Enzyme Kit</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:255px;">11789-(020)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">GATEWAY, LR Clonase II, Enzyme Kit</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:255px;">11791-(020)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:255px;">27106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">NucleoSpin Gel and PCR Clean-up Kit</td>
			<td style="width:147px;">Macherey-Nagel</td>
			<td style="width:255px;">740609-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">pDEST-GWVYNE</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:293px;">pDEST-VYNE(R)GW</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:293px;">pDEST-SCYCE(R)GW</td>
			<td style="width:147px;"></td>
			<td style="width:255px;">Gehl C. et al, 2009, Molecular Plant</td>
			<td>Gateway-cloning (Invitrogen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;"></td>
			<td style="width:147px;"></td>
			<td style="width:255px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves

许多蛋白质​​相互作用的瞬时与其他蛋白质或集成到多蛋白复合物以发挥其生物学功能。双分子荧光互补（附设）是一种体内方法来监控在植物细胞中这种相互作用。在所提出的协议所研究的候选蛋白融合至荧光蛋白互补的两半，并在各自的结构是通过农杆菌介导的转化导入植物细胞。随后，将蛋白质瞬时表达在烟草叶和还原的的荧光信号可以在完整的细胞共聚焦激光扫描显微镜来检测。这允许相互作用本身不仅可视化，而且还蛋白复合物的亚细胞定位可以被确定。为此目的，含有荧光标记的标记基因可以一起附设构建体共表达，从而可视化的细胞结构，如叔他面内质网，线粒体，高尔基体或质膜。荧光信号可以直接在表皮叶细胞或单原生质体，可从转化的烟草叶容易地分离出被监控。附设非常适合于活细胞内研究在其自然环境中的蛋白质 - 蛋白质相互作用。然而，它必须被认为表达必须通过强启动子来驱动，而相互作用伙伴由于融合了相对大的荧光标记，这可能与交互机制干扰的修改。然而，附设是一个很好的补充方法，其它常用的方法研究蛋白质-蛋白质相互作用，如免疫共沉淀， 体外下拉测定法或酵母双杂交实验。

在这个例子中，我们使用了附设法来监测细胞内分子伴侣的HSP90的膜对接蛋白AtTPR7和Toc64的相互作用。 AtTPR7是二段易位子的一部分，并与细胞内分子伴侣，其可能传送分泌前蛋白进行翻译后转运到ER膜相互作用。同样，Toc64在叶绿体外膜由接收HSP90相关的叶绿体前蛋白的作用在翻译后导入。两种蛋白包括细胞内露出TPR结构域，其介导的相互作用与HSP90的C-末端MEEVD动机。 蛋白通过...

Watch this Scientific Journal Video about Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves at JoVE.com

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

蛋白质复合物在体内的形成可以通过双分子荧光互补可视化。合作伙伴的互动融合，以荧光标记的互补部件和瞬时表达烟叶，导致后两种蛋白质的接近改组后的荧光信号。

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

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Fluorescence Activated Cell Sorting of Plant Protoplasts

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

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Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

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微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Cell Electrofusion Visualized with Fluorescence Microscopy

荧光显微镜下细胞电融合的可视化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Imaging Protein-protein Interactions in vivo

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成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

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双分子荧光互补

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

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Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...