这项工作得到了波兰克拉科夫国家科学中心（NCN）第2016/23/D/NZ3/01314号资助。

1. 表达质粒的产生
<ol><li>使用拓扑预测工具检查目标蛋白质的膜拓扑结构。</li>
	<li>设计克隆策略，以便一旦融合蛋白插入高尔基体膜，较大和较小的片段将面对细胞质。如果，如这里所呈现的情况，目标蛋白质的N-和C-末端都是细胞质定向的，则以八种可能的方式标记蛋白质（见 图1B）。如果一种或两种感兴趣蛋白质的N-或C-末端是发光定向的，则将其从标记中排除。</li>
	<li>通过遵循标准克隆方案将感兴趣的基因亚克隆到适当的表达载体中（见 材料表）。 注意:建议测试所有可能的方向，因为某些标记选项可能由于距离不足、方向欠佳或空间限制而不起作用。</li>
</ol>2. 将表达质粒瞬时转染到细胞中
<ol><li>通过胰蛋白酶消化收获贴壁HEK293T细胞培养物，并将细胞重悬于专用的完整生长培养基中。将细胞（2 x 104 / 100μL /孔）接种到透明底部，白色侧96孔板上。调整孔的总数，以适应所有测试的组合和对照，包括重复。 注意:尝试仅使用板的内部60孔，以尽量减少热偏移并避免过夜蒸发。强烈建议使用聚-D-赖氨酸包被的板，例如 材料表中指示的板，否则细胞可能会在随后的洗涤步骤中分离。</li>
	<li>在标准条件（37°C，5%CO 2）下培养细胞过夜。</li>
	<li>第二天，用在1.1点获得的表达质粒的所需组合转染细胞。<ol>
<li>对于每种构建体，将无血清培养基中的表达质粒稀释至6.25ng / μL。</li>
		<li>以适当的脂质与DNA比例加入基于脂质的转染试剂，并根据制造商的说明进行孵育。</li>
		<li>将8μL脂质:DNA混合物加入指定的孔中。通过轻轻旋转混合板的内容物。这导致在50 ng /孔下转染两种表达结构。</li>
	</ol>
</li>
	<li>在标准条件（37°C，5%CO 2）中培养细胞20-24小时。 注意:将细胞培养更长的时间可能会导致更高水平的融合蛋白表达，这可能会促进片段之间的非特异性关联。</li>
</ol>3. 介质交换
<ol><li>第二天，在每个孔中用100μL无血清培养基替换调节培养基。确保细胞在培养基交换时没有脱落。 注意:此步骤应在加入呋马嗪工作溶液前2-3小时完成。血清戒断可最大限度地减少呋喃嗪自发光引起的背景。</li>
</ol>4. 呋马嗪工作液的制备
<ol><li>在测量之前，将1体积的呋喃嗪与19体积的稀释缓冲液（20倍稀释）混合。 注意:要制备的呋马嗪工作溶液的总体积取决于要分析的单个孔的数量（呋马嗪工作溶液以1:5的比例加入细胞培养基中，因此，向先前充满100μL无血清培养基的每个孔中加入25μL呋喃嗪工作溶液）。</li>
	<li>将呋马嗪工作溶液加入指定的孔（25μL/孔）。用手或使用轨道振荡器（例如，在300-500 rpm时15秒）轻轻混合板。</li>
</ol>5. 测量发光度
<ol><li>将板插入发光酶标仪。<ol>
<li>对于要在37°C下进行的实验，在指示的温度下平衡板10-15分钟。</li>
	</ol>
</li>
	<li>选择要分析的孔。</li>
	<li>读取集成时间为 0.3 s 时的发光。需要时，继续监测发光长达 2 小时。</li>
</ol>6. 数据分析
<ol><li>计算所有测试和对照组合的平均值和标准偏差。</li>
	<li>使用具有多重比较的单因素方差分析数据分析数据。</li>
	<li>通过将感兴趣的组合获得的平均发光除以为相应负对照获得的平均发光来计算折叠变化值。评估结果。 注:这里提出的数据分析方法假设，如果为测试组合获得的发光在统计上显着高于为相应控制组合获得的发光，并且同时，这两个值的比率超过10，则可以声称相互作用。</li>
</ol>

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作者没有什么可透露的。

在这里，我们提供了一个详细的方案，能够使用分裂荧光素酶互补测定法演示高尔基体驻留的III型膜蛋白（如NSTs）之间形成的异源复合物。所提出的数据分析和解释方法涉及将感兴趣的蛋白质组合获得的发光与为相应对照组合获得的发光相关联，其由与较大片段融合的目标蛋白质之一和与较小片段融合的细菌来源的控制蛋白组成。后者在哺乳动物细胞的细胞质中表达;因此，将其用作参考需要分析其相互作用的蛋白质的N和/或C末端位于高尔基体膜的细胞质侧。
该方案的关键步骤是质粒设计，接种要转染的细胞，转染本身，培养基交换，制备呋马嗪工作溶液并将其添加到细胞中。质粒设计应使两个荧光素酶片段都面向细胞质，否则对照组合将不起作用，并且将缺少参考RLU值。要转染的细胞应按照方案中的规定进行接种，否则转染效率可能次优。我们建议使用具有聚-L-赖氨酸涂层表面的多孔板，以便在交换培养基时支持细胞附着。最后，应新鲜制备呋喃嗪工作溶液并立即加入细胞中。添加后，应尽快执行读出。
为避免无结果，应始终包括阳性和阴性对照。应始终采用阳性对照，以便监测转染效率。生成和组合所有可能的融合蛋白非常重要，这些融合蛋白在拓扑上彼此相容以及与基于HaloTag的阴性对照相容。如果可能，应使用包含膜蛋白的阴性对照，该膜蛋白不与任何感兴趣的蛋白质相互作用。在这里，我们没有采用这种控制，因为它的发展仍在进行中。相反，我们通过共表达一种分析蛋白质的额外，未标记的副本来强制对感兴趣的复合物进行特定的反汇编。我们使用的表达载体携带相对较弱的单纯疱疹病毒 - 胸腺嘧啶激酶（HSV-TK）启动子，这确保了低表达水平，是获得特定结果的最佳选择。然而，在结果欠佳的情况下，使用基于CMV的载体或优化用于转染的质粒量可能是有益的。
所提出的方法虽然功能强大且方便，但存在一些局限性。首先，该方法不允许得出所鉴定的复合物是否对应于二聚体或高阶低聚物的结论。此外，无法监测相互作用蛋白质的亚细胞定位。然而，在扩展生物发光成像的基本方案时，这是可能的，尽管与基于荧光的方法相比，这些图像的空间分辨率会显着降低。
所提出的方法非常快速和有效（测量需要几分钟，数据来自数千个细胞）。数据处理和解释也相对简单。很少或根本不需要对基本协议进行优化。唯一需要的特定设备是光度计。分裂荧光素酶互补测定和BiFC的工作原理相同，即从其非功能性片段中重建功能蛋白。与基于荧光的方法相比，基于生物发光的方法的一般优势已在引言中列出。与BiFC相比，拆分荧光素酶补体测定的一个具体优点是前者是完全可逆的15。在BiFC中，一旦荧光蛋白被重组，它就不会解离回相应的非荧光片段17。相比之下，NanoLuc亚基的组装是可逆的，这为研究PPI的动力学创造了一个独特的机会。最后，所提出方法的特殊灵敏度允许假设这种方法应该适用于难以转染的细胞系。
这里提出的方案允许确定两种高尔基体驻留的III型膜蛋白是否相互作用。如前所述，该方法的基本设置可以与生物发光成像相结合，以确认目标PPI的亚细胞定位。这种分裂荧光素酶互补测定的可逆性允许实时研究PPI的动力学。来自呋喃嗪的发光信号持续约2小时。然而，用于分裂荧光素酶互补测定的底物也可用，以确保所得信号的持续时间大大延长（数小时至数天）。这种方法允许识别触发或阻止感兴趣的PPI的因素。其应用的一些最新例子包括对G蛋白与G蛋白偶联受体之间相互作用的研究18，蛋白质构象变化19，蛋白质泛素化20，细胞表面受体内化21以及鉴定调节PPI的因子22，23。因此，这种方法似乎是一种多功能工具，具有很高的潜力，甚至可以实现极具挑战性的实验目标。

本手稿描述了一种分步方案，以使用分裂荧光素酶互补测定的最新变体检查瞬时转染的人细胞中高尔基体驻留的III型膜蛋白之间是否存在异源相互作用。该程序已经针对核苷酸糖转运蛋白（NST）进行了最广泛的测试，但我们也能够获得其他高尔基体驻留的III型膜蛋白的阳性结果，其N和/或C-末端面向细胞质。
我们的研究小组探索了NST在大分子糖基化中的作用。NST是高尔基体和/或ER驻留的III型膜蛋白，其N端和C端朝向器官膜的细胞质侧1。NST被认为携带核苷酸激活的糖穿过细胞器膜，为其底物提供糖基转移酶。NST形成二聚体和/或更高的低聚物2，3，4，5，6，7，8，9，10。此外，还报道了不同NST之间的异源相互作用6，11。NST也被证明与功能相关的糖基化酶形成复合物12，13，14。我们寻求一种替代目前使用的技术，基于荧光寿命成像（FLIM）的FRET方法，用于研究NST和功能相关的高尔基体驻留蛋白的相互作用，因此我们决定测试分裂荧光素酶互补测定。它使我们能够确定NST和功能相关的糖基化酶之间的新相互作用9。
拆分荧光素酶补体测定的最新修改，NanoBiT，用于此处介绍的方案15。它依赖于从两个片段中重建荧光素酶（例如，NanoLuc） - 大的，称为大BiT或LgBiT，一种17.6 kDa蛋白，以及小的，仅由11个氨基酸组成，称为小BiT或SmBiT。感兴趣的两种蛋白质与互补片段融合，并在人类细胞系中瞬时表达。如果两种融合蛋白相互作用，则在添加细胞可渗透底物时原位产生发光。这两个片段已经过优化，因此它们与最小的亲和力结合，除非它们通过它们融合到的目标蛋白质之间的相互作用而聚集在一起。
一般来说，基于生物发光的方法比基于荧光的方法具有一些优势。生物发光信号具有更高的信噪比，因为与荧光素酶衍生的信号相比，背景发光可以忽略不计16。相比之下，基于荧光的方法通常受到由自发荧光现象引起的相对较高的背景的影响。此外，生物发光对分析细胞的危害小于荧光，因为在前一种情况下，不需要激发样品。由于这些原因，在体内研究PPI的生物发光方法优于常用的荧光方法，如Förster共振能量转移（FRET）或双分子荧光互补（BiFC）。
我们的方案依赖于将感兴趣的蛋白质组合获得的发光参考为对照组合获得的发光。后者包括一种被测试的蛋白质，它与较大的片段和对照蛋白（例如HaloTag）融合，与较小的片段融合。后者是一种细菌来源的蛋白质，预计不会与任何哺乳动物蛋白质相互作用。使用这种蛋白质作为对照会限制要分析的高尔基体驻留蛋白质对的拓扑结构。由于在哺乳动物细胞中，这种蛋白质是在细胞质中合成的，因此感兴趣的两种蛋白质都应该至少有一个细胞质尾巴。
这种方法对于PPI的初始筛选特别有用。当感兴趣的融合蛋白以不足以应用其他方法的水平表达时，它可能成为首选方法。同样，如果感兴趣的蛋白质以高水平表达，则分裂荧光素酶互补测定可能是最佳选择，但这会对其亚细胞定位产生不利影响或已知会迫使非特异性相互作用。由于较小的片段只有11个氨基酸，因此当使用较大的标签是不可能的时，可以应用分裂的荧光素酶互补测定。最后，它可以用来进一步确认使用其他技术获得的数据，如这里介绍的情况。

<table><tbody><tr><td>0.25% trypsin-EDTA solution</td><td></td><td></td><td></td></tr><tr><td>Adherent mammalian cell line</td><td></td><td></td><td></td></tr><tr><td>BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid</td><td>Corning</td><td>356651</td><td></td></tr><tr><td>Cell culture centrifuge</td><td></td><td></td><td></td></tr><tr><td>Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptamycin)</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments</td><td></td><td></td><td></td></tr><tr><td>FuGENE HD Transfection Reagent</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>GloMax Discover Microplate Reader (or a different luminescence microplate reader)</td><td>Promega</td><td>GM3000</td><td></td></tr><tr><td>Growth medium dedicated to the cell line used</td><td></td><td></td><td></td></tr><tr><td>Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification)</td><td></td><td></td><td></td></tr><tr><td>NanoBiT MCS Starter System</td><td>Promega</td><td>N2014</td><td>This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair.</td></tr><tr><td>Nano-Glo Live Cell Assay System</td><td>Promega</td><td>N2011</td><td>This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer.</td></tr><tr><td>Opti-MEM I Reduced Serum Medium, no phenol red</td><td>Gibco</td><td>11058021</td><td></td></tr><tr><td>Oribital shaker</td><td></td><td></td><td></td></tr><tr><td>Software for data analysis (e.g. GraphPad Prism)</td><td></td><td></td><td></td></tr><tr><td>Thermocycler</td><td></td><td></td><td></td></tr></tbody></table>

demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay

该协议的目标是探索分裂荧光素酶互补的最新变体的适用性，以证明由核苷酸糖转运蛋白（NST）形成的异源复合物。这些ER和高尔基体驻留的多跨膜蛋白携带细胞质合成的核苷酸糖穿过细胞器膜，以提供介导糖基化及其底物的酶。NST以二聚体和/或更高的低聚物形式存在。不同 NST 之间的异源相互作用也有报道。为了验证该技术是否适合研究NST异色化现象，我们针对两种高尔基体驻留的NSTs的组合对其进行了测试，这两种NSTs先前已被证明可以通过其他几种方式关联。荧光素酶补体测定似乎特别适合研究高尔基体驻留膜蛋白之间的相互作用，因为它不需要高表达水平，这通常会引发蛋白质错位并增加假阳性的风险。

为了在这种方法中获得最可靠的数据，应测试所有可能的组合（参见 图1）。同时，应包括阳性和阴性对照。阳性对照应由已知相互作用的两种蛋白质组成，其中一种与较大的片段融合，另一种与较小的片段融合。理想情况下，阴性对照应由同样标记的两种非相互作用的III型膜蛋白组成。然而，建立这种对照可能具有挑战性，因为应使用几种替代方法彻底确认两种对照蛋白缺乏相互作用。因此，如果蛋白质的N-和/或C-末端（其相互作用有待确定）像这里介绍的情况一样细胞质暴露，则可以使用预计不会与任何人类蛋白质（例如HaloTag）相互作用的非人源胞质蛋白作为阴性对照。我们强烈建议通过共表达任何一种目标蛋白质的未标记变体并结合相应质粒的滴定，对使用这种类型的对照获得的结果进行额外的验证。如果两种蛋白质特异性相互作用，则其中任何一种缺乏荧光素酶片段的额外拷贝应导致发光的特定降低。
相对发光单元（RLU）值通常为正负组合，如表1所示。两种NST，SLC35A2和SLC35A3的结果被证明通过共免疫沉淀和FLIM-FRET6以及原位接近连接测定11相关联。SLC35A2和SLC35A3都是高尔基体驻留的III型膜蛋白，其N端和C端朝向细胞质（图1A）。因此，有八种可能的标记选项，并且所得的融合蛋白也可以以八种可能的方式设置在一起（图1B）。与所有测试和对照组合相对应的平均RLU值如图2A所示。阳性对照也包括在内。要将所选结果视为交互作用的指示，最初的要求是，为感兴趣的组合获得的RLU值在统计上显着高于为相应对照组合获得的RLU值。在图2A中可以看到三种这样的组合（LgBiT-SLC35A2 + SLC35A3-SmBiT，SmBiT-SLC35A2 + LgBiT-SLC35A3和SLC35A2-SmBiT + LgBiT-SLC35A3）。结果分析的下一步涉及通过将相应的RLU值除以为相应控件获得的RLU值来获得感兴趣组合的比率值。这种分析的结果如图2B所示。根据制造商的建议，10 到 1000 之间的比率高度指示了特定的相互作用。有两种组合符合这些标准（SmBiT-SLC35A2 + LgBiT-SLC35A3 和 SLC35A2-SmBiT + LgBiT-SLC35A3），并支持 SLC35A2 和 SLC35A3 相互作用的想法。然而，其他六种组合为阴性的事实并不意味着各自的融合蛋白之间根本没有相互作用，而是表明标记策略在这些情况下不是最佳的。此示例显示了测试所有可能的组合的重要性。
图2中提供的数据还通过HA标记的SLC35A2或SLC35A3与导致最高相对发光的组合（即SLC35A2-LgBiT + SmBiT-SLC35A3）的共表达得到证实。使用不同量的编码HA标记的NST变体的质粒进行共转染。这导致RLU值的剂量依赖性降低，具有统计学意义（图3）。通过HA标记的NST变体同时共表达SLC35A2-LgBiT和SmBiT-SLC35A3之间相互作用的特异性和剂量依赖性破坏如表2所示。
与本文介绍的方法相关的最常见问题是转染效率低。为了验证这是否是次优结果的原因，请在所有实验中包括阳性对照。我们用于获得此处提供的数据的阳性对照由两种相互作用的融合蛋白组成:与较小片段融合的cAPM依赖性蛋白激酶催化亚基α（PRKACA）和与较大片段融合的cAPM依赖性蛋白激酶II-α型调节亚基（PRKAR2A）。在我们手中，相应的RLU值达到了~6 x 105。这个数字的显着下降（2-3个数量级）可能表明所进行转染的效率欠佳。在这种情况下，我们建议检查培养细胞的健康状况，并确保接种适当数量的细胞进行转染。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61669/61669fig1v3.jpg"> 图1:膜拓扑结构和标记的示意图。 （A）SLC35A2和SLC35A3蛋白的膜拓扑结构。（B）使用分裂的荧光素酶互补测定片段标记SLC35A2和SLC35A3蛋白的可能性，并将所得的融合蛋白组合用于测定。 <a href="https://www.jove.com/files/ftp_upload/61669/61669fig1v3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61669/61669fig2v3.jpg"> 图2:在HEK293T细胞中对所选蛋白质组合和相应的阴性对照（处理数据）进行的分裂荧光素酶互补测定的结果。 （A）为所选蛋白质组合和相应的阴性对照获得的RLU值。负面，HaloTag标记有SmBiT;PRKAR2A，cAPM依赖性蛋白激酶II-α型调节亚基;PRKACA，cAPM依赖性蛋白激酶催化亚基α。使用单因素方差分析进行多重比较，并以三个技术重复的平均±标准差（SD）表示。 p < 0.1 *， p < 0.05 **， p < 0.01 ***.（B）通过将测试组合获得的平均发光（RLU样品）除以相应阴性对照获得的平均发光（RLU CONTROL）来计算的折叠变化。被视为指示交互作用的阈值设置为 10。 <a href="https://www.jove.com/files/ftp_upload/61669/61669fig2v3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61669/61669fig3v3.jpg"> 图3:通过同时共表达HA标记的NST变体，SLC35A2-LgBiT和SmBiT-SLC35A3之间相互作用的特异性和剂量依赖性破坏。HEK293T细胞转染编码SLC35A2-LgBiT和SmBiT-SLC35A3的质粒，此外，用空的pSelect质粒（模拟）或编码HA标记的SLC35A2（左图）和SLC35A3（右图）的pSelect质粒量增加。使用单因素方差分析进行多重比较，并以三个技术重复的平均±标准差（SD）表示。 p < 0.05 **， p < 0.001 ****。 <a href="https://www.jove.com/files/ftp_upload/61669/61669fig3v3large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
表1:在HEK293T细胞中对所选蛋白质组合和相应的阴性对照进行的测定结果（原始数据）。 显示为所选蛋白质组合和相应的阴性对照获得的未处理的RLU值。负面，HaloTag标记有SmBiT;PRKAR2A，cAPM依赖性蛋白激酶II-α型调节亚基;PRKACA，cAPM依赖性蛋白激酶催化亚基α，TR，技术复制。 <a href="https://cloudflare.jove.com/files/ftp_upload/61669/JoVE_table_1.xlsx" target="_blank">请点击此处下载此表格。</a>
表2:通过同时共表达HA标记的NST变体（原始数据），SLC35A2-LgBiT和SmBiT-SLC35A3之间相互作用的特异性和剂量依赖性破坏。 图中显示了为所选蛋白质组合获得的未处理的RLU值。模拟，表达SLC35A2-LgBiT和SmBiT-SLC35A3的细胞与空pSelect载体共同转染;HA-SLC35A2，表达SLC35A2-LgBiT和SmBiT-SLC35A3的细胞与编码SLC35A2的pSelect质粒的指示量共同转染，编码SLC35A2在N末端标记HA表位;HA-SLC35A3，表达SLC35A2-LgBiT和SmBiT-SLC35A3的细胞与编码SLC35A3的pSelect质粒的指示量共同转染，编码SLC35A3在N末端标记HA表位;TR，技术复制。 <a href="https://cloudflare.jove.com/files/ftp_upload/61669/JoVE_table_2.xlsx" target="_blank">请点击此处下载此表格。</a>

Watch this Scientific Journal Video about Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay at JoVE.com

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

这里提出的方案旨在使用分裂荧光素酶互补测定的最新变体，证明高尔基体驻留的III型膜蛋白与活哺乳动物细胞中细胞质暴露的N-和/或C-末端之间异源相互作用的发生。

使用分裂荧光素酶互补测定法展示高尔基体驻留III型膜蛋白形成的异源复合物

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

demonstration-heterologous-complexes-formed-golgi-resident-type-iii

Universidad San Sebastian

Research

JoVE Journal

Biology

2.2K 次观看.  University of Wroclaw. 这里提出的方案旨在使用分裂荧光素酶互补测定的最新变体，证明高尔基体驻留的III型膜蛋白与活哺乳动物细胞中细胞质暴露的N-和/或C-末端之间异源相互作用的发生。

视频: Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

科研

癌症研究

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

生物化学

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

生物学

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

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 activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

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 ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

发育生物学

<meta charset="utf8"></head><body>一种用于高通量筛选的测量 ER-线粒体接触的简单检测

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

<meta charset="utf8"></head><body>作者聚焦：ER-线粒体接触的调节和失调 — 对神经退行性疾病发病机制的影响

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

SNARE蛋白介导的膜融合酶的细胞融合含量的分析

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

使用分裂荧光素酶互补测定法展示高尔基体驻留III型膜蛋白形成的异源复合物

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此视频中的章节

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

原位近接结扎法检测蛋白异构体的 Heterodimerization

研究与基于荧光素酶的记者基因检测膜生源

通过测量活细胞中的 eIF4E-eIF4G 交互来监控 eIF4F 装配

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

SNARE蛋白介导的膜融合酶的细胞融合含量的分析