这项工作得到了国家精神卫生研究所赠款（R00MH103531和R01MH116901至J.A.）、国家普通医学研究所（T32GM007635至S.R.）的博士前培训赠款和莱达·希尔·吉利亚姆高级研究奖学金（GT11021至S.R.）的支持。我们感谢凯文·伍尔弗雷博士在显微镜方面提供帮助，K·乌尔里希·拜尔博士使用他的显微镜，托马斯·塞多夫（斯坦福大学）为LRRTM2质粒。

1. 细胞培养和变性
<ol><li>使 HEK 细胞介质与 DMEM， 1x （杜尔贝科的鹰的介质的修改） 补充 4.5 克 / 升葡萄糖， L - 谷氨酰胺 + 硫酸钠和 10% Fbs 。无菌过滤器。</li>
	<li>预先确定合适的配体和受体进行聚合检测。 注:Neurexin3+SS4+/-及其已知的配体之一LRRTM2用于本研究。在 pcDNA3.1 中，cDNA 表示了兴趣的连体和受体。吉布森组件用于将纽雷辛3®插入pcDNA3.112。纽雷辛3+F/R:塔塔格塔格特格塔格塔格特加特克卡卡特加特卡特卡特克塔茨克塔克特克特克 加加格克茨加塔卡特克特加塔塔卡特加特加特加</li>
	<li>准备 HEK293T 细胞。<ol>
<li>将 HEK293T 细胞生长到一个 T-75 烧瓶中。</li>
		<li>一旦汇合，使用2 mL的试金石，并放置在37°C孵化器2分钟。将 6 mL 的 HEK 介质添加到烧瓶中以重新悬浮细胞，并将所有 8 mL 转移到 15 mL 圆锥管中。</li>
		<li>500 x g 的颗粒为 5 分钟，在 HEK 细胞介质中重新悬浮，共 8 mL。</li>
		<li>数细胞，在6井板的每口井中加入735，000个细胞。使用 HEK 细胞介质将每口井的最终体积调整为 2 mL。</li>
		<li>放置在37°C的孵化器中，让细胞在一夜之间生长，或直到它们达到50-60%的结合。</li>
	</ol></li>
	<li>使用磷酸钙方法13转染 HEK293T 细胞。<ol>
<li>与1微克荧光蛋白（pcDNA3.1-神经素3+WT SS4-和1微克的mCherry）共同感染3微克的蛋白质。</li>
		<li>与第 1.4.1 步一样， 转染井 2 。但与兴趣的突变蛋白（pcDNA3.1-纽瑞辛3®A687T SS4-）。</li>
		<li>与另外一种荧光蛋白的 1 微克 （pcDNA3.1 LRRTM2 和 GFP 的 1μg） 一起与 3 μg 的兴趣配体进行转染。</li>
		<li>转染好 4 和井 5 作为负控制: 井 4 与 1 μg 的 Gfp 和井 5 与 1μg 的 mcherry 。</li>
		<li>如果需要附加条件或控制（Neurexin®3WT/A687T SS4+），则准备另一个板材（如步骤 1.4.1-1.4.4）。 注:在表观荧光显微镜下进行24小时转位后24小时进行变性效率分析，并量化为表达其转染的荧光蛋白的细胞数量。更简化的方法将包括用双晶向量编码的 HEK 细胞的转染，以获得荧光蛋白和感兴趣的配体，并且强烈建议在共转体上方进行。在本研究中，阿尔法神经素为~4.3 kb，使用需要共同分流的双晶体系统观察到荧光强度低。</li>
	</ol></li>
	<li>转产后48小时，收获细胞进行聚集。<ol>
<li>用PBS清洗每口井两次。</li>
		<li>在 PBS 中加入 1 mL 的 10 mM EDTA，以轻轻地分离细胞对细胞的相互作用，并在 37 °C 下孵育板 5 分钟。 注:由于研究中粘附分子的潜在蛋白解，不建议使用 Trypsin 进行步骤 1 . 5 . 2 。此外，在 EDTA 添加后，协议在完成之前可能不会停止，因为细胞现在将暴露在环境条件下。</li>
		<li>轻轻敲击板分离细胞，并将每个细胞收获成单独的 15 mL 圆锥管。</li>
		<li>离心锥形管在500 x g 和室温5分钟。</li>
	</ol></li>
	<li>当细胞在造粒时，通过标记每个微中心管的顶部来准备6个孵化管。 注:GFP 和 mCherry 条件的每一次排列都应用于包含所有实验条件和适当的控制。例如:1. GFP/mCherry，2. 母马/LRRTM2-GFP 3。GFP/纽瑞辛3+WT SS4-梅里，4。GFP/纽瑞辛3+A687T SS4--切里，5.纽瑞辛3+WT SS4---梅里/勒特姆2-GFP，6.纽瑞辛3+A687T SS4--梅里/拉特姆2-GFP。制作额外的管子，以适应进一步的条件和控制。</li>
	<li>用 10 mM CaCl 2 和 10 mMMmgCl 2加热到 37 °C，在 500μL 的 HEK 介质中去除超纳坦和重新悬浮的细胞。 注:添加CaCl2 和MgCl2 允许粘附分子重新建立结合，并且只有在有关细胞相互作用伙伴需要二价粘附剂的情况下才需要粘合。</li>
	<li>使用血细胞计将每个 15 mL 圆锥管中的细胞计数，并将每个条件的 200，000 个细胞从步骤 1.6.1 计入适当的管中，总体积为 500 μL 的 1:1 混合物。 注:每种情况只需5分钟计算和引用金额。</li>
	<li>在室温下在慢管旋转器中孵育管。</li>
</ol>2. 图像采集
<ol><li>优化特定样品的显微镜采集参数。在此示例中，图像是在宽场显微镜上拍摄的。使用5倍空气目标（NA:0.15:WD:20000μm），以获得足够大的磁场进行分析。</li>
	<li>在第 1.8 步混合 HEK 细胞的两个条件后立即评估基线聚合。这些现在是"时间零"图像。<ol>
<li>每个样品混合物的移液器 40 μL 在 488 和 561 通道的荧光下通过带电显微镜滑动和图像。</li>
		<li>每次样品滴落在一个对焦平面上获取三个不同的视场。</li>
	</ol>
</li>
	<li>以"时间 60"图像在 60 分钟内获取最终图像。<ol>
<li>要在 60 分钟的潜伏期后获得混合物的"时间 60"图像，请从旋转管和移液器中将每个样品的每个情况的 40 μL 样本再取到带电滑梯上。图像为第 2.2.2 步。 注:细胞聚合应每15分钟检查一次，直到饱和。聚合的时间将取决于正在测试的蛋白质。</li>
	</ol>
</li>
</ol>3. 图像J/斐济分析
<ol><li>要使用斐济/ImageJ 量化聚合范围，请保存分析文件。<ol>
<li>将所提供的补充编码文件保存到计算机上的图像J宏文件夹中。</li>
		<li>安装所提供的聚合宏（插件、宏、安装，并选择"聚合分析.txt"文件）。</li>
	</ol>
</li>
	<li>确定阈值。<ol>
<li>将"时间零".tif文件加载到图像J中并拆分通道（图像|颜色|拆分通道）。 注:"时间零"图像用于确定整个实验的阈值和最小双关大小。</li>
		<li>屏蔽每个通道（插件|宏|AggregationAssay_MakeMask）。从映像窗口制作面具将出现。选框旁边确定阈值的图像和确定群集参数从直方图和点击确定。</li>
		<li>使用滑动栏确定图像的阈值，将数字记录到幻灯片栏的右侧，然后单击 "确定"。</li>
		<li>将显示 群集大小的直方图 。从适合实验的直方图中选择群集大小，在 最小群集大小 中键入此数字:框，然后单击 "确定"。此大小以下的群集将不进行分析。</li>
	</ol>
</li>
	<li>运行分析。<ol>
<li>在图像J中打开条件1的"时间60"图像，并像步骤3.2.1一样拆分通道。</li>
		<li>遮蔽每个通道（插件、宏、AggregationAssay_MakeMask）。使用步骤 3.2.3 和步骤 3.2.4 中确定的相同阈值和大小。取消选择 "确定图像阈值 "旁边的框， 从直方图中确定群集参数 ，然后手动键入大小和阈值到适当的字段中，然后单击 "确定"。</li>
		<li>计算聚合索引（插件|宏|AggregationAssay_CalculateOverlap）。选择要比较的蒙面通道，并编入生成的文件将保存的目录。</li>
		<li>每个"时间 60"图像在每种情况下重复步骤 3.3.1-3.3。 注:聚合索引定义为总重叠区域除以两个通道区域减去重叠区域乘以 100 的总和（聚合索引 = 重叠区域/[通道 1 的区域+ 通道 2 的区域 - 重叠区域] x 100）。此规范化是两个蒙面通道之间的"OR"操作，表示两个掩体中的总像素。</li>
	</ol>
</li>
</ol>

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</ol>

作者没有什么可透露的。

剖析细胞粘附过程中在跨细胞过程中发生的蛋白质-蛋白质相互作用，可以更好地了解基本细胞过程背后的分子机制，包括成熟和改造过程中突触的形成、功能和维护。细胞对细胞相互作用的意义超越了神经生物学，在信号传导、细胞迁移和组织发育等方面具有更广泛的作用。细胞粘附的畸变会破坏细胞过程，这是适当的细胞功能所必必须的，并且可能低于各种病因，如癌症、关...

突触粘附分子促进的突触相互作用是突触和神经网络生成的发展、组织、规范、维护和功能的基础。这些跨合成细胞粘附分子的识别正在迅速增加:因此，确定结合的伙伴并了解这些新的粘附分子是如何相互作用的，这一点至关重要。此外，基因组测序已经确定了许多这些粘附分子的突变，这些突变通常与多种神经发育、神经精神病和成瘾障碍1有关。编码突触细胞粘附分子的基因突变可能会有害地改变相互作用，并可能导致突触形成和或维持的病理生理变化。
存在多种测定，以定量评估蛋白质-蛋白质相互作用，如同热热量学，圆形二度，表面质子共振2， 虽然在性质上定量，他们有几个局限性。首先，它们需要重组蛋白，有时需要漫长而乏味的净化步骤。第二，它们需要先进的专门设备和技术专长。第三，它们可以高估结合的程度，因为它们允许自然系在体内膜上的蛋白质之间的cis和跨相互作用。在这里，我们提出了一个简单和相对快速的检测，专门测试跨交互。
为了规避与纯化蛋白检测相关的许多并发症，我们优化了基于细胞的蛋白质相互作用检测，该检测可以重述减少的异质细胞系统中的跨相互作用。这种检测以前曾以各种形式用于研究细胞相互作用。在此方法中，候选细胞粘附分子被转化为 HEK293T 细胞。在生理条件下，HEK293T细胞没有表现出自我聚合，因此它们成为这种检测的典范。然而，当表达受体和配体的单个 HEK 细胞群组合在一起时，受体和配体的结合会迫使 HEK 细胞聚集发生。此聚合仅通过跨交互进行调解，通常可在数十分钟内观察到。此方法无需蛋白质纯化步骤，该方法的效率依赖于表达认知粘连分子的 HEK 细胞群组合在一起，然后在几十分钟后成像的范式。此外，这种方法相对便宜，因为既不需要抗体，也不需要昂贵的设备。获取数据所需的唯一设备是标准荧光显微镜。这种基于细胞的检测的另一个优点是能够快速筛选疾病相关点突变对跨相互作用的影响。这可以通过用感兴趣的蛋白质突变变异的 cDNA 传染 HEK 细胞来执行。
在此协议中，我们举例说明，我们调查在诊断患有严重智力残疾和癫痫的患者中发现的 Neurexin3® （Neurexin®A687T）中的误区突变是否改变了与富含白细胞的重复性跨膜蛋白 2 （LRRTM2） 的跨性别相互作用。Neurexin3®是进化保存的先天性细胞粘附分子家族的成员，虽然最近的工作已经确定了突触3、4、5、6、7的多个角色，但我们对这种分子和神经素家族所有成员的突触理解仍然不完整。LRRTM2是一种激发后突触细胞粘附蛋白，参与突触形成和维护8，9，10。重要的是，LRRTM2 专门与缺乏拼接站点 4 替代异位素 （SS4-） 的神经素异形相互作用，但不与包含拼接站点 4 替代异位素 （SS4+） 的神经素异形交互。在Neurexin3®中发现的人类误区突变（A687T）位于一个未经研究的细胞外区域，该区域在进化保存后，在所有α神经素7之间保存。随着这两种分子之间的相互作用在8、9、11之间建立，我们提出了一个问题:Neurexin®SS4-LRRTM2的结合能力是否因A687T点突变而改变？这一分析表明，A687T点突变意外地增强了Neurexin3®与LRRTM2的聚合，表明点突变所在的细胞外区域在调解跨同步相互作用方面发挥作用。

<table><tbody><tr><td>1.5 mL disposable microtubes with snap caps</td><td>VWR</td><td>89000-028</td><td>Incubation of mixed population of HEK cells</td></tr><tr><td>1000 mL Rapid&amp;mdash;Flow Filter Unit, 0.2 um aPES membrane</td><td>Thermo Fisher</td><td>567-0020</td><td>Sterilization of HEK media</td></tr><tr><td>15 mL SpectraTube centrifuge tubes</td><td>Ward&amp;rsquo;s Science</td><td>470224-998</td><td>Harvesting HEK cells</td></tr><tr><td>6 well sterile tissue culture plates</td><td>VWR</td><td>100062-892</td><td>culturing HEK cells</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>223506-500G</td><td>Calcium phosphate transfection, HEK cell resuspension</td></tr><tr><td>Centrifuge- Sorvall Legend RT</td><td>Kendro Laboratory Products</td><td>75004377</td><td>Harvesting HEK cells</td></tr><tr><td>CO2 cell incubator</td><td>Thermo Scientific</td><td>HERACELL 150i</td><td>Incubation of HEK cells during growth</td></tr><tr><td>DMEM, 1x (Dulbecco&#039;s Modification of Eagle&#039;s Medium) with 4.5 g/L glucose, L-glutamine &amp;amp; sodium pyruvate</td><td>Corning</td><td>10-013-CV</td><td>HEK cell maintenance</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline PBS (1X)</td><td>Gibco</td><td>14190-144</td><td>Passaging/harvesting HEK cell</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma</td><td>ED-500G</td><td>Harvesting HEK cells</td></tr><tr><td>Falcon Vented culture flasks, 75cm&lt;sup&gt;2&lt;/sup&gt; growth area</td><td>Corning</td><td>9381M26</td><td>Culturing HEK cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>17L184</td><td>HEK cell maintenance</td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td></td><td>Model system</td></tr><tr><td>ImageJ</td><td>NIH</td><td>V: 2.0.0-rc-69/1.52p</td><td>Image analysis</td></tr><tr><td>Magnesium Chloride hexahydrate</td><td>Sigma</td><td>M9272-500G</td><td>HEK cell resuspension</td></tr><tr><td>Sodium phosphate dibasic anhydrous</td><td>Fisher BioReagents</td><td>BP332-500</td><td>Calcium phosphate transfection</td></tr><tr><td>Trypsin 0.25% (1X) Solution</td><td>GE Healthcare Life Sciences</td><td>SH30042.01</td><td>Passaging HEK cells</td></tr><tr><td>Tube rotator</td><td></td><td></td><td>Incubation of mixed population of HEK cells</td></tr><tr><td>UltraClear Microscope slides. White Frosted, Positive Charged</td><td>Denville Scientific Inc.</td><td>M1021</td><td>Image acquisition</td></tr><tr><td>Wide-field microscope</td><td>Zeiss</td><td>Axio Vert 200M</td><td>Image acquisition</td></tr></tbody></table>

measuring transcellular interactions through protein aggregation in a heterologous cell system

细胞接口的蛋白质相互作用决定着从组织发育和癌症进展到突触形成和维持等多种生物结果。许多这些基本相互作用发生在跨，通常是由表达膜锚定结合对的细胞之间的异亲或同亲相互作用引起的。阐明疾病相关突变如何破坏这些基本蛋白质相互作用，可以深入了解无数的细胞生物学领域。许多蛋白质-蛋白质相互作用分析通常不会区分cis和跨相互作用，这可能导致高估体内发生的结合程度，并涉及蛋白质和/或专门监测设备的劳动密集型纯化。在这里，我们提出了一个优化的简单协议，允许只观察和量化跨相互作用，而无需冗长的蛋白质纯化或专用设备。HEK细胞聚合检测涉及两个独立的HIK细胞群的混合，每个细胞都表达膜绑定同质配体。经过短暂的潜伏期后，对样品进行成像，并量化生成的聚合物。

A687T 突变增加神经素 3® SS4 - 绑定到 LRRTM27 为了研究两种已知突触蛋白的细胞间相互作用如何受到智力残疾和癫痫患者发现点突变的影响，我们使用了上述 HEK 细胞聚合检测（图 1）。细胞根据第1节进行转染，并根据协议第1节和第2节准备成像。细胞在基线上进行成像，没有像预期的那样观察到聚合（未显示）。在 60 分钟内获得的图像与协议第 3 节一?...

Watch this Scientific Journal Video about Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System at JoVE.com

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

在这里，我们提出了一个优化的协议，使用荧光显微镜快速和半定量测量异质细胞系统中跨体中的配体受体相互作用。

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

measuring-transcellular-interactions-through-protein-aggregation

Research

JoVE Journal

Neuroscience

3.5K 次观看.  University of Colorado Denver School of Medicine. 在这里，我们提出了一个优化的协议，使用荧光显微镜快速和半定量测量异质细胞系统中跨体中的配体受体相互作用。

视频: Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

科研

生物化学

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

生物工程

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

生物学

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估:兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

基于活细胞单分子成像的蛋白质相互作用定量

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

作者聚焦:活人细胞中蛋白质-凝聚物动力学的评估

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

生物分子缩合物中蛋白质相互作用动力学的单分子测量

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

摘要

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低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

珠聚集测定假定的细胞粘附分子的表征

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下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

在活细胞中蛋白质聚集的4D成像

遗传和生物化学方法

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

生物分子缩合物中蛋白质相互作用动力学的单分子测量

生物分子缩合物中蛋白质相互作用动力学的单分子测量