Name: measuring transcellular interactions through protein aggregation in a heterologous cell system
Uploaded: 2020-05-22T14:45:00
Description: Watch this Scientific Journal Video about Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System at JoVE.com

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...

<ol>
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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。

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			<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>
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		<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>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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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 ...

Research

JoVE Journal

Neuroscience

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

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

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

本地化的蛋白质三维神经干细胞培养：一个混合的可视化方法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

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

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

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

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

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 Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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

A Caenorhabditis elegans Model System for Amylopathy Study

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

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

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 ...