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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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、关节炎、成瘾、自闭症和精神分裂症1，15，16。在这里，我们提供了一个优化的详细协议，涉及HIK细胞聚合，允许测试细胞粘附相互作用的跨。
使用此 HEK 细胞聚合协议，我们可以剖析聚合中的差异，以接近先天性蛋白与其后突触配体之间的结合能力。因此，我们可以提出这样的问题:两种基本突触蛋白之间的相互作用是否受到点突变的影响？更具体地说，Neurexin3®与LRRTM2的跨相互作用是否受到A687T的影响？这里研究的A687T误区位于Neurexin®7细胞外域一个以前未研究的链接区域。结果表明，A687T突变增强了含有Neurexin3®A687T 的细胞与含有LRRTM2（图2C）的细胞的聚集。这一发现意义重大，因为它以前曾显示，LRRTM仅通过名为LNS617的Neurexin域与神经素结合，并进一步表明LNS6上游的序列可以对Neurexin3®SS4-和LRRTM27之间的跨相互作用产生独立影响。
图 2中的结果显示此检测作为所有负控制（GFP/mCherry） 的特异性:纽雷辛3+WT/A687T SS4---切里/GFP或LRRTM-GFP/mCherry）在60分钟后没有可观察到的聚合。本研究中使用的关键控制，Neurexin3+WT/A687T SS4+ - mCherry/LRRTM-GFP，在60分钟后也没有表现出聚集，因为LRRTM2已知只与缺乏纽雷辛（SS4-）异形的SS4结合。这一控制表明，膜绑定分子的简单过度表达不足以迫使这个系统聚集，这进一步说明了这种检测的特殊性。
这里描述的细胞粘附检测已被广泛用于测试跨合成细胞粘附分子8，18，19的相互作用:然而，它可以用来测试膜系绳蛋白的粘合细胞对细胞相互作用。此协议经过一段时间的演变，通过更改三个实验参数，从原始已发布的协议和协议的后续变体中进行了优化。其一，细胞的孵化温度发生了变化。原始协议要求在 4 °C 下以步骤 1.9 孵化细胞。 这种低温可以起到环境压力的作用，降低细胞存活率，导致细胞死亡和裂解。在细胞裂解过程中，细胞将基因组DNA和细胞碎片分泌到介质中，导致细胞在成像过程中聚集在溶液中导致误报。第二，每种情况的细胞数增加到每人口200 000个，目标大小改为5倍:这使得研究人员能够对每个视场进行更多的交互，从而增加每个n内的统计能力。第三，聚合指数的衡量方式不同。先前的聚合指数受到实验者选择集群大小的限制，导致排除"次要"聚类。相比之下，现在为"时间零"的单个细胞大小设定阈值，聚合现在被计算为重叠区域除以两个通道区域减去重叠区域的总和，在"时间 60"下乘以 100，从而允许加入较小的正簇，使检测对其他蛋白相关对更敏感（图 2B，C）。
可能需要故障排除和优化，具体取决于测试的相互作用蛋白质。虽然最终图像采集之前的潜伏期会因测试的蛋白质而异，但在"时间零"处不观察到聚合至关重要。如果聚合在零点时看到，它可能意味着细胞一开始是不健康的。这可能是由于几个因素，包括突然暴露在37°C以下的温度或缓慢的实验过程。重要的是，第1.7步的悬停介质应在37°C，这将使细胞逐渐达到室温，而不会突然严酷的温度下降。在整个实验中实现最佳细胞健康的一种方法是确保步骤 1.7 到 1.9 在 25 分钟内完成，两者之间没有中断。建议在细胞收获细胞1.5步之前设置并准备使用显微镜:这可确保在第 2.2 步中立即拍摄真正的"时间零"图像。
如果在60分钟的潜伏期后没有观察到聚合，则可能导致这种粘附力的缺乏。首先，所涉蛋白质可能不是结合伙伴，或可能从事这种检测无法检测到的低亲和力相互作用。在这种情况下，可能需要更灵敏的检测。 其次，当单独在 HEK 细胞中表达时，感兴趣的蛋白质可能无法本地化到膜上。在这种情况下，通过使用生化技术（表面生物素化）确认蛋白质是局部化的，或直接用荧光标记标记感兴趣的蛋白质，并以 40 倍或更高的放大倍数成像 HEK293T 细胞来评估表面表达。但是，在确认膜定位后，我们建议使用未标记的变种进行这种 HEK 细胞聚合检测，以便更准确地评估原生蛋白质的结合能力。第三，在此协议中，我们允许Neurexin3+表达48小时:但是，蛋白质表达时间可能因测试的蛋白质而异。第四，如果有关蛋白质依赖于二价 cations，则有必要在第 1.7 步中用 MgCl2 和 CaCl2 来补充介质，例如神经素和 LRRTM211。如果未知交互伙伴是否需要二价粘附，则将 EDTA 添加到一个条件中。EDTA 应将天然存在于 DMEM 中的重新矿化 cations 切碎，以确保无钙和无镁溶液。如果在添加EDTA后观察到粘附，则有关蛋白质不需要二价的粘附剂。
有许多方法可以测试蛋白质相互作用:然而，大多数不是专门测试只发生从细胞到细胞的跨相互作用，需要乏味的蛋白质纯化步骤。虽然 HEK 细胞聚合检测不是直接的亲和力测量方法，不应用于取代更定量的方法来恢复分离常数，但据我们所知，这种检测代表了以半定量和相对有效的方式测试真正跨交互的方法。此外，由于这种检测相对容易，HEK细胞聚合检测可以与这些更定量的方法一起使用，以获得对发生的相互作用的更广泛和更完整的描述。

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 border="0" cellpadding="0" cellspacing="0" style="width:788px;" width="787">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:339px;">1.5 mL disposable microtubes with snap caps</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">89000-028</td>
			<td style="width:167px;">Incubation of mixed population of HEK cells</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:339px;">1000 mL Rapid&#8212;Flow Filter Unit, 0.2 um aPES membrane</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:161px;">567-0020</td>
			<td style="width:167px;">Sterilization of HEK media</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:339px;">15 mL SpectraTube centrifuge tubes</td>
			<td style="width:121px;">Ward&#8217;s Science</td>
			<td style="width:161px;">470224-998</td>
			<td style="width:167px;">Harvesting HEK cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:339px;">6 well sterile tissue culture plates</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">100062-892</td>
			<td style="width:167px;">culturing HEK cells</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:339px;">Calcium Chloride</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">223506-500G</td>
			<td style="width:167px;">Calcium phosphate transfection, HEK cell resuspension</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:45px;width:339px;">Centrifuge- Sorvall Legend RT</td>
			<td style="width:121px;">Kendro Laboratory Products</td>
			<td align="right" style="width:161px;">75004377</td>
			<td style="width:167px;">Harvesting HEK cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:339px;">CO2 cell incubator</td>
			<td style="width:121px;">Thermo Scientific</td>
			<td style="width:161px;">HERACELL 150i</td>
			<td style="width:167px;">Incubation of HEK cells during growth</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:339px;">DMEM, 1x (Dulbecco&#39;s Modification of Eagle&#39;s Medium) with 4.5 g/L glucose, L-glutamine &#38; sodium pyruvate</td>
			<td style="width:121px;">Corning</td>
			<td style="width:161px;">10-013-CV</td>
			<td style="width:167px;">HEK cell maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">Dulbecco&#8217;s Phosphate Buffered Saline PBS (1X)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">14190-144</td>
			<td style="width:167px;">Passaging/harvesting HEK cell</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:339px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">ED-500G</td>
			<td style="width:167px;">Harvesting HEK cells</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:339px;">Falcon Vented culture flasks, 75cm2 growth area</td>
			<td style="width:121px;">Corning</td>
			<td style="width:161px;">9381M26</td>
			<td style="width:167px;">Culturing HEK cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">Fetal Bovine Serum</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">17L184</td>
			<td style="width:167px;">HEK cell maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">HEK293T cells</td>
			<td style="width:121px;">ATCC</td>
			<td style="width:161px;"></td>
			<td style="width:167px;">Model system</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:339px;">ImageJ</td>
			<td style="width:121px;">NIH</td>
			<td style="width:161px;">V: 2.0.0-rc-69/1.52p</td>
			<td style="width:167px;">Image analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">Magnesium Chloride hexahydrate</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">M9272-500G</td>
			<td style="width:167px;">HEK cell resuspension</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:339px;">Sodium phosphate dibasic anhydrous</td>
			<td style="width:121px;">Fisher BioReagents</td>
			<td style="width:161px;">BP332-500</td>
			<td style="width:167px;">Calcium phosphate transfection</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:339px;">Trypsin 0.25% (1X) Solution</td>
			<td style="width:121px;">GE Healthcare Life Sciences</td>
			<td style="width:161px;">SH30042.01</td>
			<td style="width:167px;">Passaging HEK cells</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:339px;">Tube rotator</td>
			<td style="width:121px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">Incubation of mixed population of HEK cells</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:339px;">UltraClear Microscope slides. White Frosted, Positive Charged</td>
			<td style="width:121px;">Denville Scientific Inc.</td>
			<td style="width:161px;">M1021</td>
			<td style="width:167px;">Image acquisition</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:339px;">Wide-field microscope</td>
			<td style="width:121px;">Zeiss</td>
			<td style="width:161px;">Axio Vert 200M</td>
			<td style="width:167px;">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 节一样进行分析。为了尽量减少选择偏差，条件被随机化，使实验者失明。出于类似的原因，每个图像的整个视场都被选为投资回报率。
细胞没有表达任何突触配体（GFP/mCherry）的情况显示，在60分钟的潜伏期后，聚集量最小（图2）。同样，在两个细胞群中只有一个表达突触配体（mCherry/LRRTM2-GFP或GFP/神经素3+T/A687T SS4--mCherry）的条件下，在60分钟的潜伏期（图2）之后，其聚集量极小。不出所料，包含两个具有不兼容粘附分子的细胞群的条件（Neurexin3+WT/A687T SS4+-mCherry/LRRTM2-GFP）在60分钟内没有聚集（图2B）。 这些条件作为关键的负控制，因为 LRRTM2 已知只与缺乏神经素异形 （SS4-） 的 SS4 结合，并突出了此聚合检测的特殊性。
与兼容粘附分子8，9，10（纽雷辛3+WT SS4--mCherry/LRRTM2-GFP）的条件在60分钟的潜伏期（图2C）后显示出显著的聚集。聚合可视化为黄色，并存在于细胞之间的重叠（图 1和图 2）中。令人惊讶的是，Neurexin3®A687T SS4-与LRRTM2（纽雷辛+A687T SS4--mCherry/LRRTM2-GFP）共同孵育的状况与其野生型对应物相比表现出更多的聚合（Neurexin3+WT SS4-mCherry/LRRTM2-GFP;正对照）。这表明，Neurexin3®中的A687T点突变增强了纽雷辛3®SS4-到LRRTM2的结合能力。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61237/61237fig1.jpg"> 图 1.聚合检测的工作流程。（A） HEK293T细胞使用蛋白质-1/mCherry或蛋白质-2/GFP进行转染。（B） 神经素3®的表达需要48小时（C）细胞使用10m EDTA收获，然后颗粒化（D）。（E） 细胞以1:1的比例混合，并重新悬浮在含有10 mM CaCl2和MgCl2的500μL的HIK介质中。（F） 细胞在室温下孵育，直到聚合发生，并在"时间60"获得最终图像。聚合可视化为在视野中可见的黄色双关语数量。当细胞没有表达正确的受体配体对，因此被视为单独的红色或绿色双关语时，不会观察到聚合。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61237/61237fig1large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

补充编码文件。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61237/Supplemental_Coding_Files.zip" target="_blank">请点击这里下载这些文件。</a>

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.4K 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

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein G&alpha;olf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

测定在异种细胞表面的化学感应受体的表达

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

科研

神经科学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Peering into the Dynamics of Social Interactions: Measuring Play Fighting in Rats

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. The frequency of play fighting can be measured by counting the number of playful nape attacks occurring per unit time. However, playful defense, as it can only occur in response to attack, is necessarily a contingent measure that is best measured as a percentage (#attacks defended/total # attacks X 100%). How a particular attack is defended against can involve one of several tactics, and these are contingent on defense having taken place; consequently, the type of defense is also best expressed contingently as a percentage. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.

窥视到社会相互作用的动力学测量打打架的影响

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...