Name: quantitative immunofluorescence to measure global localized translation
Uploaded: 2017-08-22T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Immunofluorescence to Measure Global Localized Translation at JoVE.com

我们感谢这份手稿的批判性解读博士 Rachid Mazroui （拉瓦尔大学、 加拿大魁北克省）。我们感谢它们的技术援助研究中心细胞成像单元。M.法国霍是全宗德初中 1 研究学者研究杜魁北克-健康 (FRQ S)。这项工作被支持由加拿大卫生研究院 （授权号卫生研究院，拖把 286437 到 M.É。霍）。

1。 Puromycilation 条件测定 注： 这种技术描述了用于评估期间 MRC 5 细胞粘附过程 5 本地化的翻译的方法。Puromycilation 可以做任何单元格中，是重要的是优化 puromycilation 条件为特定的单元格行使用，因为治疗条件是不相同的嘌呤霉素浓度和所需的每个单元格行孵育时间。若要显示这些条件如何定义，三个示例细胞株 （即， HeLa、 MRC-5 和 Huh 7） 患者的嘌...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+puromycin+action:+fate+of+ribosomes+after+release+of+nascent+protein+chains+from+polysomes.">Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes.</a> Proc Natl Acad Sci U S A. 70, 3866-3869 (1973).</li><li>Goodman, C. A., Hornberger, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein+synthesis+with+SUnSET:+a+valid+alternative+to+traditional+techniques.">Measuring protein synthesis with SUnSET: a valid alternative to traditional techniques.</a> Exerc Sport Sci Rev. 41, 107-115 (2013).</li><li>David, A., 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=Nuclear+translation+visualized+by+ribosome-bound+nascent+chain+puromycylation.">Nuclear translation visualized by ribosome-bound nascent chain puromycylation.</a> J Cell Biol. 197, 45-57 (2012).</li><li>Fallini, C., Bassell, G. J., Rossoll, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+muscular+atrophy:+the+role+of+SMN+in+axonal+mRNA+regulation.">Spinal muscular atrophy: the role of SMN in axonal mRNA regulation.</a> Brain Res. 1462, 81-92 (2012).</li><li>Costa-Mattioli, M., 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=Translational+control+of+hippocampal+synaptic+plasticity+and+memory+by+the+eIF2alpha+kinase+GCN2.">Translational control of hippocampal synaptic plasticity and memory by the eIF2alpha kinase GCN2.</a> Nature. 436, 1166-1173 (2005).</li><li>Costa-Mattioli, M., Sossin, W. 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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=Why+cells+move+messages:+the+biological+functions+of+mRNA+localization.">Why cells move messages: the biological functions of mRNA localization.</a> Semin Cell Dev Biol. 18, 171-177 (2007).</li><li>Ephrussi, A., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing:+the+bicoid+morphogen+gradient+matures.">Seeing is believing: the bicoid morphogen gradient matures.</a> Cell. 116, 143-152 (2004).</li><li>Mardakheh, F. 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R., 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=Premature+translational+termination+products+are+rapidly+degraded+substrates+for+MHC+class+I+presentation.">Premature translational termination products are rapidly degraded substrates for MHC class I presentation.</a> PLoS One. 7, e51968 (2012).</li><li>Schneider-Poetsch, T., 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=Inhibition+of+eukaryotic+translation+elongation+by+cycloheximide+and+lactimidomycin.">Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin.</a> Nat Chem Biol. 6, 209-217 (2010).</li><li>Jacobs, J. P., Jones, C. M., Baille, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+human+diploid+cell+designated+MRC-5.">Characteristics of a human diploid cell designated MRC-5.</a> Nature. 227, 168-170 (1970).</li><li>Gandin, V., 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=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J Vis Exp. (87), (2014).</li></ol>

最近的技术进步有允许参与平移上调机制更好地理解或对特定基因的生物学过程，如神经元的发育、 药物反应和转移的镇压。这里所述的符合成本效益方法允许翻译事件在细胞研究 RNA 结合蛋白是如何调节等细胞黏附、 迁移和侵袭转移过程可视化。
虽然已发展了许多方法来评估de novo蛋白质翻译，他们都共享相同的策略，拉长多肽包含标记与检出的基团修饰的氨基酸。...

翻译由不同的细胞功能的调节，促使许多的研究团队，开发新的工具和方法来确定亚细胞定位的 mRNA 翻译和调节的蛋白合成1，2 ，34。这些最近的技术进步允许更好地了解涉及翻译上调或特定基因生物学过程，如神经元的发育、 药物反应和转移5 的镇压的机制 ，，67，8。然而，这些方法大多数需要昂贵或有害试剂和可能不到大多数实验室可用的特定设备。因此，一个具有成本效益的方法，允许翻译事件快速评定了专门绕过这些潜在的问题。此方法检测特定细胞过程中发生的急性平移调制，并且还允许本地化的翻译用共聚焦显微镜。
这里描述的方法被用于监视在亚细胞隔离舱称为传播启蒙中心 (SIC)5的本地化的翻译。结果是在本地化在新生粘附复合物的种子细胞中发现的瞬态结构。虽然结果与粘附复合物是不同的他们的命运被息息相关。的确，结果也会逐渐消失后粘着复杂成熟粘附网站粘附的初始阶段。我们发现已知的具体控制 （例如， Sam68，FMRP 和 G3BP1） mRNA 翻译的 RNA 结合蛋白和 Rna 被内这些丰富的 polyadenylated 结构5。我们使用此处所述的方法，表明 SIC 相关的 mRNA 翻译作为检查点允许调节种子细胞巩固细胞粘附。这种基于嘌呤霉素纳入的方法可以考虑翻译测定 （日落） 的表面传改编的版本。最初开发用来衡量全球蛋白质合成率使用非放射性标记的氨基酸，蛋白质 puromycilation 提供可视化de novo蛋白质合成9的有效途径。这种方法依赖于嘌呤霉素，阻止通过过早链终止在核糖体10翻译抗生素的内在行为。事实上，嘌呤是结构上类似于 tyrosyl-tRNA，允许将纳入拉长肽链通过肽键的形成。但是，嘌呤霉素绑定到肽链阻止新的肽键形成与下一个氨酰-tRNA，嘌呤霉素以来非水解酰胺键而不是在转运中找到的水解酯键。因此，嘌呤霉素纳入拉长多肽产生过早披露的众多截断的 puromycilated 多肽对应积极翻译 mRNA9，11，12.
使用此方法，有可能评估活动翻译内短时间寡妇 (例如， 5 分钟) 期间使用针对嘌呤霉素对 5 分钟时间，辅以抗生素的细胞特异性抗体的细胞粘附在不同的时间点在细胞粘附过程5。这种测定方法的精度依赖于针对 puromycilated 基团的高度特异性抗体。Puromycilated 多肽免疫荧光法检测提供新翻译的 mRNA，也可以使用共焦显微成像系统的精确地量化一般亚细胞重新分区。
因此，此方法提供了大量的实验室研究平移监管机制所涉及的过程，如神经元造粒6，，1314，相关选项15、 形态 mRNA 本地化和翻译期间发展16，17。它也非常适合于快速生物事件，如细胞迁移、 粘连或入侵，或简单地评估可能诱发平移变化5，的药物治疗期间的本地化或条块分割的翻译研究7,18.总体而言，此方法允许为本地化或控制翻译事件的可视化快速、 精确，并且具有成本效益的方式。

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsine</td>
			<td>wisent</td>
			<td>325-043 EL</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr>
			<td>Puroycin antibody 12D10</td>
			<td>EMD millipore</td>
			<td>MABE343</td>
			<td>western blot dilution 1:25000 
			Immunofluoresence dilution 1:10000</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>cell signaling technology</td>
			<td>7076</td>
			<td>western blot dilution 1:8000</td>
		</tr>
		<tr>
			<td>Western Lightning Plus-ECL</td>
			<td>Perkin Elmer</td>
			<td>NEL104001EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG (H+L), F(ab&#39;)2 Fragment (Alexa Fluor 488 Conjugate)</td>
			<td>cell signaling technology</td>
			<td>4408</td>
			<td>immunofluoresecence dilution 1:400</td>
		</tr>
		<tr>
			<td>CF568 Phalloidin</td>
			<td>biotium</td>
			<td>00044</td>
			<td>immunofluoresecence dilution 1:400</td>
		</tr>
		<tr>
			<td>Cyclohexmide</td>
			<td>Sigma</td>
			<td>C1988-1G</td>
			<td>50&#181;g/ml final concentration</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>final concentration 1&#181;g/ml</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>bio-Basic</td>
			<td>PJ593</td>
			<td>2.5&#181;g/ml to 10&#181;g/ml</td>
		</tr>
		<tr>
			<td>Ibidi &#181;-Dish 35 mm, high, ibiTreat</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>MRC-5 cells</td>
			<td>ATCC</td>
			<td>CCL-171</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Huh-7 cells</td>
			<td colspan="2">from Dr. Mazroui (Universit&#233; Laval)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fv1000</td>
			<td>olympus</td>
			<td></td>
			<td>confocal imaging system</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td></td>
			<td></td>
			<td>http://fiji.sc</td>
		</tr>
		<tr>
			<td>PBS (Phosphate BuffeRed Saline)</td>
			<td>bio-Basic</td>
			<td>PD8117</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37% Solution</td>
			<td>bio-Basic</td>
			<td>C5300-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>bio-Basic</td>
			<td>TB0198</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Bioreagents</td>
			<td>BP9702-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Fisher Bioreagents</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative immunofluorescence to measure global localized translation

调节 mRNA 翻译机制被参与不同的生物学过程，如胚线发育、 细胞分化和器官，以及多种疾病。众多的出版物有令人信服地表明具体机制严格管制 mRNA 翻译。兴趣翻译诱导调控蛋白表达的增加导致了发展的研究并遵循de novo蛋白质合成中 cellulo的新方法。然而，这些方法大多数都复杂，使其成本高昂和经常限制的数量可以进行研究的 mRNA 指标。这份手稿提出要求只基本试剂和荧光共焦成像系统测量并可视化 mRNA 翻译中任何细胞株在不同条件下发生的变化的方法。此方法是最近用来显示本地化的翻译中贴壁细胞的亚细胞结构在短短的时间，从而提供了可视化de novo翻译短期间各种生物的可能性过程或更改进行验证，在翻译活动中对特定刺激的反应。

要准确地观察使用嘌呤霉素纳入翻译事件，它关键是确定每个单元格的行的最佳条件，因为每个显示11，不同嘌呤霉素纳入动力学 (图 1)9 ，，1218。因此，若要验证嘌呤霉素纳入，有必要治疗所需单元格行与标准谱的嘌呤浓度增加 （1.0、 2.5、 5.0、 7.5、 1...

Watch this Scientific Journal Video about Quantitative Immunofluorescence to Measure Global Localized Translation at JoVE.com

Quantitative Immunofluorescence to Measure Global Localized Translation

这份手稿描述了一种方法来可视化和量化本地化的翻译事件亚细胞的车厢内。提出这份手稿的做法需要基本的共焦成像系统和试剂，并且是快速和具有成本效益。

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Research

JoVE Journal

Biochemistry

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

科研

生物化学

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

通过并行反应监测进行无处不在的链分析

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

光学镊子研究翻译调控中的RNA-蛋白质相互作用

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

强大的单颗粒冷冻电子显微镜 （cryo-EM） 处理工作流程，具有冷冻 SPARC、RELION 和 Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...