这项工作得到了p53lab（BMSI、A*STAR）和JCO VIP赠款（A*STAR）的核心预算的支持。

HEK293细胞系用于协议，并在杜尔贝科的改良鹰介质中培养，补充10%胎儿牛血清，2米L-谷氨酰胺和100U/mL青霉素/链霉素。细胞在37°C的培养下，在潮湿 的环境中产生5%的二氧化碳。
1. 通过 eIF4E 对 eIF4F 复杂中断进行定量评估:eIF4G604-646 补充分析
<ol><li>eIF4E 的细胞培养和瞬态瞬变:eIF4G604-646 补充测定<ol>
<li>使用新鲜解冻的细胞，所有实验的通道少于20个。在第 1 天，使用标准细胞计数器确定细胞编号，并使用 Trypan 蓝色排除方法13计算总可行细胞。</li>
		<li>种子 6 井板与 0.9 - 1.2 x 106 的 HEK 293 细胞每井在 2 mL 的标准生长介质. 注意:为了在上述细胞系内实现最佳的转接效率，确保镀膜细胞在播种后的第二天达到70-90%的汇流。</li>
		<li>在第2天早晨，使用脂基转染试剂（见材料表）与 SubA-eIF4E 和 eIF4G604-646-SubB 质粒共同传输细胞（见下文所述）。<ol>
<li>在含有 125μL 的减少血清介质的管中稀释 9μL 脂质溶液，每次转染时不含酚红色（见 材料表），并在室温下孵育 5 分钟。</li>
			<li>通过在 125 μL 的减少血清介质中稀释每个质粒中的 3μg 来为每个输血管准备 DNA 的主混合。</li>
			<li>将 12 μL 的增强剂加入 DNA 主混合管中，混合良好，并立即添加 DNA:增强剂主混合到稀释脂体的每个管中，比例为 1:1。在室温下孵育15分钟。</li>
			<li>将DNA脂质复合物添加到每口井中，并在37°C的孵化器中孵化细胞，24小时使用5%的二氧化碳 。</li>
		</ol>
</li>
		<li>在第 3 天的早晨，用 1 mL 的 PBS 冲洗每个油井。</li>
		<li>在 37 °C 下，用 0.3 mL 的三氯苯丙辛在每口井中取出 PBS 并孵育细胞 5 分钟。</li>
		<li>通过添加2mL的减少血清介质，在每口井中不含0%FCS的酚红色，将受感染的细胞转移到15mL管中，中和三氯苯酚素。 注:酚红色会干扰路西法酶的活动:因此，细胞必须从这一步开始在中等处理，没有酚红色。</li>
		<li>在290 x g 下旋转细胞5分钟，吸气介质。在没有含有 0% FCS 的酚红色的情况下，在减少的血清介质的 2 mL 中补充细胞。按步骤 1.1 中描述的计数。</li>
		<li>种子在 96 个不透明板中传输了 HEK 293 个细胞，在 90μL 的介质中，每口油井的密度为 30，000 个细胞，没有含有 0% FCS 的酚红色。为了在同一实验中为3种不同的化合物获得3个技术复制品，60个板材的种子井，除了边缘的油井外，还有细胞。</li>
		<li>在播种受感染的细胞后，立即添加 10 μL 的 10% DMSO 复合溶液（参见步骤 1.2）。</li>
	</ol></li>
	<li>复合制备<ol>
<li>通过溶解 100% DMSO（v/v） 中的复利复利，准备 1 mM 复合库存解决方案。为了每个复合滴定有 3 个复制品，请使用 1 mM 复合库存溶液中的 8 μL。 注:所使用的1mM库存化合物的体积大于所需量。这样做是为了考虑管道错误（如果有）。</li>
		<li>执行库存复合溶液的 2 倍连续稀释，每个滴定点将 1 mM 库存中的 4μL 放入 4 μL 100% DMSO 中。 注意:对于完整的复合滴定，在 100% DMSO 中从起始库存执行 9 次连续稀释。如果需要测试多个化合物，请使用 96 井板和多通道移液器来促进这一步骤和随后的稀释。</li>
		<li>在 2 倍串行稀释的每个点添加 36μL HPLC 级无菌水，以在 10% DMSO （v/v） （即 100 μM 至 0.39 μM 10% DMSO 库存系列将导致最终解决方案为 10 μM 至 0.039 μM，添加到单元格时为 1% DMSO）。至于治疗控制，还要在HPLC级无菌水中制备10%的DMSO仅存量溶液。</li>
		<li>在 96 个不透明板中的细胞中加入 10μL 的 10 倍工作解决方案，以产生预期的最终浓度，剩余 DMSO 浓度为 1%（v/v），总容量为 100 μL，在 37 °C 时孵育 3 小时，其中 5% v/v 大气 CO2。</li>
	</ol></li>
	<li>路西法酶补充和可行性检测<ol>
<li>经过3小时的药物化，开始准备路西法酶基板试剂，将1卷基板与19卷稀释试剂相结合（见制造商的说明）。 注:路西法酶检测使用市售套件执行（参见 材料表）。</li>
		<li>使用多通道移液器立即添加 25 μL 的基板试剂。</li>
		<li>在室温下，在轨道摇床上以 350 rpm 摇动板 50 分钟。</li>
		<li>使用板阅读器评估发光。为此，将镜像读卡器设置为发光，将发射滤镜设置为 455。使用 6.5 mm 的测量高度，测量时间为 1 s。</li>
		<li>为了评估细胞的生存能力，添加 33 μL 的可生存性检测试剂，然后在室温下 15 分钟后再次测量发光，使用板阅读器。</li>
		<li>通过在发光上设置镜面读卡器，在 600 nm 上设置排放过滤器，用板阅读器评估发光。使用 6.5 mm 的测量高度，测量时间为 1 s。 注:根据制造商的说明，通过测量ATP在细胞裂解上的细胞内水平来评估可行性。在这种情况下，多路复用是可能的，因为可行性检测使用不同的路西法酶与不同的发射波长。</li>
		<li>使用数据通过将数据与 4 个参数拟合曲线方程相适应的曲线来确定每个化合物的 IC50 值: Y = （A-D）/（1+x/C）=B= D 在曲线拟合过程中必须限制数字值 D 和 A: D = Y 轴上的发光值，用于从 eIF4E:4G 补充检测中获得最小曲线无同位素或最低理论响应水平。 A = Y 轴上的发光值，用于从 eIF4E:4G 互补测定中预期的最大曲线无同位素或最大理论水平响应。 注意:最小响应的价值来自 1% DMSO （v/v） 控制处理，最大响应的价值来自高亲和力 mTOR 抑制剂的最大响应，这种抑制剂已经稳定下来，对细胞生存能力没有影响。</li>
	</ol></li>
</ol>2. 相关 eIF4E:eIF4G604-646 检测抑制与 eIF4F 在细胞中的复杂干扰
<ol><li>为了确认通过测定测量的信号与化合物 eIF4E-eIF4G 相互作用的物理中断相对应，第 1.1 步中描述的种子细胞。</li>
	<li>后天，用不含酚红色的1mL血清介质替换介质，用复合物孵育4小时。确保剩余 DMSO 浓度为 1%（v/v），总容量为 1 mL。 注意:为了轻松地允许与结果相关联，请在测量的补充测定滴定曲线的开头、中点和终点使用复合浓度。</li>
	<li>Lyse 细胞并执行 m7GTP 向下拉，以分离 eIF4F 和 eIF4E:4EBP1 复合体。关于如何执行m7GTP下拉实验的详细程序可以在其他地方找到14。</li>
	<li>通过西式污点分析检测 m7GTP 绑定 eIF4G、eIF4E 和 4EBP1 蛋白质水平，然后与 eIF4F 复杂干扰与补充检测信号抑制相关联。</li>
</ol>

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作者没有什么可透露的。

本文所述方法采用基于路西法酶的补充测定法，通过直接测量活细胞中的eIF4G-eIF4E相互作用，对eIF4F复合组件进行定量监测。我们提供了使用eIF4E-eIF4G补充系统的详细信息，也表明该系统在测量eIF4E-eIF4G相互作用16的药物诱导4EBP1介导分离方面非常准确。为了便于此检测的吞吐量，本文中描述的实验设置已设计为 96 井微板格式的使用。
为了获得最佳结果，在执行测定时应考虑两个关键步骤。首先，实验之间的转染效率应该保持相似。这可以通过使用低通道数细胞，并通过在播种当天严格计数细胞来确保。在进行DNA转化之前，还应评估细胞共性，因为如果细胞共性小于70-90%，则不建议用脂基转染试剂对细胞进行变质。其次，将补充分析与细胞生存性分析进行多重分析非常重要。某些化合物可能主要通过有害效应减少可行细胞的数量来影响路西法酶信号。因此，在 eIF4E:eIF4G604-646 补充测定后立即测量细胞的生存能力，以解决目标外和非特定影响非常重要。

基因表达控制在正确执行细胞程序（如生长增殖和分化）方面起着至关重要的作用。监管控制机制可以在基因转录水平或mRNA转化水平上进行。在过去十年中，越来越明显的是，通过调节启动过程而不是后来的拉长和终止步骤进行转化控制，可以精细地调节具有广泛生物功能的蛋白质特定子集的合成。
参与生存、抗自噬和抗凋亡反应的 mRNA 的翻译增加与几种癌症有关，并且与异常激活或过度表达翻译启动因子1有因果关系。
eIF4F 综合体是翻译启动的主调节器。通过将上限结构与 mRNA 的 5' 端绑定，eIF4F 正在推动初始 mRNA-里博索姆的招聘，进而提高翻译不力的真核素 mRNA2的 mRNA 翻译效率。据报道，许多癌症模型都存在异常激活RAS/MAPK或AKT/TOR通路的癌症模型，eIF4F的中介翻译，这表明癌细胞会调节eIF4F，以增强自身的抗肿瘤活性。通过抑制eIF4F复合形成来破坏这个进气回路，因此是一个非常有希望的治疗策略3，4。
eIF4F 综合体由 （i） eIF4E 组成， eIF4F 的帽绑定子单位，与 mRNA 的 5' UTR、（ii） eIF4A、RNA 六氯酶和 （iii） eIF4G 的上限结构相互作用，后者是与 eIF4A 和 eIF4E 相互作用的脚手架蛋白，最终招募了 40S 核糖核酸亚单位5。eIF4G 与 eIF4E 关联是功能 eIF4F 复合体组装的限速步骤，由 eIF4E 结合蛋白（4EBP、成员 1、2 和3）6负调节。通过由规范和非规范 eIF4E 绑定序列7、8、9（人类 eIF4E 上的 aa 604-646 区域）组成的界面与 eIF4G 绑定竞争，4EBP 可减少积极参与翻译和防止 eIF4F 复杂形成的 eIF4E 池。这些蛋白质-蛋白质相互作用的相互作用主要受4EBP的拉帕霉素（mTOR）介质磷酸化的哺乳动物目标的调节。在偏头痛刺激下，mTOR直接磷酸化4E-BP蛋白家族的成员，减少他们与eIF4E的关联，从而促进eIF4E-eIF4G相互作用和功能eIF4F复合体10的形成。
尽管在开发针对eIF4F复合完整性的化合物方面付出了巨大努力，但缺乏测量活细胞中eIF4E-eIF4G相互作用直接中断的检测结果，限制了对细胞活性命中化合物的搜索。我们应用了基于共聚苯乙烯模拟（例如，基于纳诺鲁克的补充测定）的路西法酶检测，通过 eIF4E-eIF4G 交互实时监控 eIF4F 完整性的状态。路西法酶补充蛋白系统由18kDa蛋白片段（SubA）和11氨基酸肽片段（SubB）组成，优化为最小的自结和稳定性11。一旦表示为与人类全长eIF4E和eIF4E相互作用域从人类eiF4G1（aa 604-646）的融合产品，两种相互作用的蛋白质将把SubA和SubB片段彼此接近，并诱导形成活性荧光酶，当细胞渗透基质存在时，最终将产生明亮的发光信号（图1）。我们已在其他地方报告了eIF4E:eIF4G604-646 补充系统16的建设和验证情况。
在这里，我们描述了如何应用 eIF4E:eIF4G604-646 补充系统（可应要求提供）来准确测量活细胞中 4EBP1 介质的 eIF4E-eIF4G 中断。此外，我们通过测量目前作为潜在的癌症治疗药物12的临床试验中的几种mTOR抑制剂的效果来证明它的效用。由于目标外效应通常掩盖药物特异性活动，我们还描述了如何通过细胞生存能力正交测量来扩展 eIF4E:eIF4G604-646 系统测量的多功能性。

<table><tbody><tr><td>293FT cells</td><td>Thermo Fisher Scientific</td><td>R70007</td><td></td></tr><tr><td>Cell culture microplate 96 well, F-Bottom</td><td>greiner bio-one</td><td>655083</td><td></td></tr><tr><td>Cell titer Glo 2.0</td><td>PROMEGA</td><td>G9241</td><td></td></tr><tr><td>Envision Multilabel Reader</td><td>PerkinElmer</td><td>not applcable</td><td></td></tr><tr><td>Finnpipette F2 Multichannel Pipettes 12-channels 30-300 ml</td><td>Thermo Fisher Scientific</td><td>4662070</td><td></td></tr><tr><td>Finnpipette F2 Multichannel Pipettes 12-channels 5-50 ml</td><td>Thermo Fisher Scientific</td><td>4662050</td><td></td></tr><tr><td>FUGENE6</td><td>PROMEGA</td><td>E2692</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/L3000015&quot; target=&quot;_blank&quot;&gt;L3000015&lt;/a&gt;</td><td></td></tr><tr><td>NanoBiT PPI Starter Systems</td><td>PROMEGA</td><td>N2014</td><td></td></tr><tr><td>Optimem I Reduced Serum Mediun, no phenol red</td><td>Thermo Fisher Scientific</td><td>11058021</td><td></td></tr><tr><td>Orbital shaker</td><td>Eppendorf</td><td>not appicable</td><td></td></tr><tr><td>&amp;gamma;-Aminophenyl-m&lt;sup&gt;7&lt;/sup&gt;GTP (C&lt;sub&gt;10&lt;/sub&gt;-spacer)-Agarose</td><td>Jena Bioscience</td><td>AC-155S</td><td></td></tr></tbody></table>

monitoring eif4f assembly by measuring eif4e-eif4g interaction in live cells

eIF4F复合物的形成已被证明是信号通路收敛的关键下游节点，这些信号通路经常在人类中经历致癌活化。eIF4F 是一个上限绑定复合体，涉及翻译启动的 mRNA - 相关招聘阶段。在许多癌症的细胞和临床前模型中，eIF4F 的放松管制导致参与癌症增殖和生存的特定 mRNA 子集的翻译增加。eIF4F 是一个由上限绑定子单位 eIF4E、六角酶 eIF4A 和脚手架子单元 eIF4G 构建的异质三元复合体。活性eIF4F复合物组装的关键在于eIF4E和eIF4G蛋白质之间的蛋白质-蛋白质相互作用。在本文中，我们描述了一个测量 eIF4F 组件的协议，该程序可监控活细胞中的 eIF4E-eIF4G 交互状态。eIF4e:4G 基于细胞的蛋白质-蛋白质相互作用检测还允许准确可靠地评估 eIF4F 复杂完整性的药物诱导变化。我们设想，这种方法可以应用于验证市售化合物的活性，或进一步筛选有效破坏eIF4F复合物形成的新化合物或方式。

为了验证eIF4E:eIF4G604-646 补充系统的灵敏度，使用mTOR抑制剂对eIF4F复合组件进行了4EBP1介停抑制评估。通过抑制4EBP蛋白家族的mTORC1激酶依赖磷酸化，mTOR抑制增强4EBP1与eIF4E的关联，因此，eIF4F拆解15。测试了两类机械上不同的mTOR激酶抑制剂:拉帕洛格（如拉帕霉素），它们是mTORC1的同位素抑制剂，但不是mTORC2和ATP基于竞争的抑制剂（例如PP242），它们专门用来抑制mTORC1和mTORC2激酶催化活性。
HEK293 细胞与 eIF4E:eIF4G604-646 补充系统（如第 1 步中描述的那样）发生变异。经过24小时的转染后，细胞被重新播种，并使用mTOR抑制剂PP242和拉帕霉素（如第1.2步所述）治疗。治疗四小时后，按照先前描述，对发光进行了评估，然后是细胞存活率。如图 2所示，PP242 产生对信号的剂量依赖抑制，计算的IC50为 0.72 ± 0.04μM，而 IC50 为 6.88 ± 0.88 μM 用于拉帕霉素（图 2A）。然后，板被多路复用，用于细胞生存能力测定（图2D）。这一分析表明，PP242和拉帕霉素都没有显著降低细胞存活率，证明eIF4E:eIF4G604-646 补充系统中发光的减少不是由于非特定细胞死亡，而是由于eIF4E:4G相互作用的中断。
m7GTP 拉下实验，通过孵育与开始相对应的化合物浓度的未传输细胞来执行， 图2A 中测量滴定曲线的中端和末点显示，4EBP1介质的内源性eIF4E-eIF4G交互中断与测量的eIF4E:eIF4G604-646 测定信号（图2B，2C）相关。与这些结果一致， 在 HEK 293 细胞（图 3A）测试的实验条件下，PP242 是总 4EBP1 磷酸化的更有力的抑制剂，而这两种抑制剂均对 mTOR 信号正常产生影响，拉帕霉素对 mTORC1 基板和 PP242 对 mTORC1 和 mTORC2（图 3B）的活性更强。
综合起来，这些结果表明，PP242比HK293细胞中的雷帕霉素更能有效破坏eIF4F复合形成，并进一步证明eIF4E:eIF4G604-646 系统能够准确测量活细胞中的eIF4F复合组装。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60850/60850fig1.jpg"> 图1:eIF4E:eIF4G604-646 互补系统。 示意图表示显示蛋白质 X （eIF4E） 和蛋白质 Y （eIF4G604-646）的相互作用如何使 SubA 和 SubB 融合彼此接近并重组活性红道酶。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60850/60850fig1large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60850/60850fig2.jpg"> 图2:4EBP1调解的eIF4F综合体中断。 （A） PP242 和拉帕霉素 eIF4E:eIF4G604-646 测定滴定建模 eIF4E 和 eIF4G 在变种 HEK 293 细胞中的相互作用。（B ， C）西方污点分析显示，在 HEK 293 提取物中，eIF4E、eIF4G 和 4EBP1 的内源性水平分别在培养细胞（浓度为 PP242 或 Rapamycin）后向下拉。（D） 在A中对细胞生存能力和发光进行多路复用的处理细胞进行评估。所有值均表示平均± SD （n=3）。这个数字已由16日修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60850/60850fig2large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60850/60850fig3.jpg"> 图3:mTOR抑制对4EBP1磷酸化的微分作用。 （A） 西方污点分析未经转染的 HEK293 细胞中 4EBP1 的磷酸化状态，经处理后显示浓度为 PP242 和 Rapamycin。（B） 用双MTORC1/2活性站点抑制剂PP242或mTORC1拉帕霉素的异体抑制剂处理的非转染性 HEK293 细胞中的AKT和S6磷酸化状态的西斑分析。测试版可视化为加载控制以及总 4EBP1、AKT 和 S6。这个数字已由16日修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60850/60850fig3large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>

Watch this Scientific Journal Video about Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells at JoVE.com

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

在这里，我们提出了一个协议，以测量eIF4E-eIF4G在活细胞中的相互作用，使用户能够评估药物引起的扰动eIF4F复杂动态的筛选格式。

通过测量活细胞中的 eIF4E-eIF4G 交互来监控 eIF4F 装配

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

monitoring-eif4f-assembly-measuring-eif4e-eif4g-interaction-live

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

3.0K 次观看.  A*STAR (Agency for Science, Technology and Research). 在这里，我们提出了一个协议，以测量eIF4E-eIF4G在活细胞中的相互作用，使用户能够评估药物引起的扰动eIF4F复杂动态的筛选格式。

视频: Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

科研

遗传学

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

生物化学

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

生物学

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

免疫与感染

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

通过测量活细胞中的 eIF4E-eIF4G 交互来监控 eIF4F 装配

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暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

用于分析依赖帽的翻译动力学的体外单分子成像测定

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

单细胞荧光显微镜定量 Efferocytosis 的研究

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

非洲爪蟾作为一种模型来确定翻译减值

高分辨率法监测IRF3的磷酸化依赖性激活

通过测量活细胞中的 eIF4E-eIF4G 交互来监控 eIF4F 装配

摘要

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暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

单细胞荧光显微镜定量 Efferocytosis 的研究

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

非洲爪蟾作为一种模型来确定翻译减值

高分辨率法监测IRF3的磷酸化依赖性激活

用于分析依赖帽的翻译动力学的体外单分子成像测定