Name: analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads
Uploaded: 2017-03-08T15:00:00
Description: Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

CG是由来自法新社法国国家（ANR-13-JSV1-0008），来自欧盟第七框架计划（居里夫人职业集成n˚8304162），由根据欧盟的地平线欧洲研究委员会（ERC）资助项目2020年研究和创新计划（ERC启动格兰特n˚639300）。

1.配体偶联磁珠注意：使用超顺磁性甲苯磺酰基活化的珠用直径2.8微米，进行配体缀合（原液浓度10 8珠/毫升，30毫克珠粒/毫升）。以下协议是基于约2×10 5个细胞，这对应于在一个60mm组织培养板上生长至80％汇合MRC-5细胞的样品。相应地，如果使用不同大小或细胞的板在不同汇流调整的珠粒和试剂的体积。使用顺序的超顺磁珠的量具有每单元2珠。因此，需要用?...

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此处所描述的方法构成，以施加张力到细胞表面粘附受体，并允许其随后的纯化一个简单的方法。然而，一些步骤是进行高效粘附纯化的和潜在的优化可以做取决于目标粘附受体的关键。我们目前的用户可能会遇到下面的潜在问题。 我们使用直径2.8微米的磁性珠粒但较大的小珠可用于直径如4.5微米。然而，珠子直径应限制在2-5微米自吞噬可能在30-60分钟温育发生更快速和更大...

在后生动物，机械张力通过细胞过程如增殖，分化和存活1,2无数的调节指导组织发育和内环境稳定。机械张力可以从细胞外基质产生，或可通过贴壁细胞，其样品通过拉动到细胞外基质，并通过张力敏感分子探测其刚性的肌动球蛋白收缩机械它们的胞外环境中产生的。为了应对紧张局势，机械敏感粘附蛋白发生触发复杂的信号级联反应的构象变化。反过来，这些信号传导途径编排一个mechanoresponse包含增殖，分化和存活是调节细胞行为到细胞外环境。这样的方法可以在短期时间周期进行结算（数秒至数分钟）快速反馈到机甲的环<html>通过修改机械敏感结构notransduction。例如，基于整联粘连加强在通过的Rho GTP酶-介导的细胞骨架重构3，4，5响应于张力。与此同时，其他的信号通路超过几小时或几天来控制的遗传程序，最终影响细胞的命运6激活。然而，许多研究强调矩阵刚度对细胞确定性和疾病发展1,2的效果，粘附介导的机械传导的精确分子机制仍然不清楚。 各种方法已被开发来研究细胞的行为的细胞产生的力或外力，包括流动系统，荧光共振能量转移（FRET）-tension传感器7的影响，小姑娘=“外部参照”&gt; 8，柔性基板9，磁镊子，光学镊子10和原子力显微镜（AFM）11。在这里，我们使用超顺磁珠响应于施加到特定粘附受体的张力来表征机械传导途径提出的协议。超顺磁珠是放置在一个磁场时可逆地磁化的颗粒。一旦涂覆有对特定受体的配体，这些珠粒提供了有力的工具来研究细胞外力的应用程序的影响。此方法已被若干研究3，5，12验证- 17和呈现的优点在很大程度上促进在贴壁细胞的生化分析。使用类似的胶原包被的磁珠，随后通过生物化学分析，早期工作报告中的增加蛋白酪氨酸磷酸化和RhoA激活响应于张力5，18，19。下面描述的方法也被用于与纤连蛋白（FN）包被的珠从施加到整3张力下游表征信号通路。在这项研究中，Guilluy等。表明张力通过两个鸟嘌呤核苷酸交换因子（全环基金），LARG和全球环境基金-H1，以整合的粘附物招募激活的RhoA。因为，在其他的研究已经表明，GEF-H1被用不同的方法20，21，表现出这里描述的方法的稳健性招募到粘附物响应于细胞产生的张力。其结果是，活化的RhoA被证明促进粘附加固，通过细胞骨架重构。该系统也被用来探索张力施加吨O单元/细胞粘附受体。力到涂有诱导纽蛋白招募增加同样地整合伴生附着配合物12 E-钙粘蛋白的胞外域磁珠应用。柯林斯和他的同事观察到紧张的应用PECAM-1促进整合和RhoA的活性13。采用磁珠另一种实验方法是紧张适用于孤立的核研究。使用涂覆有对核包膜蛋白nesprin-1抗体珠，核膜复合物进行纯化，以显示它们响应于机械张力22动态地调节。这些结果支持在机械传导途径的研究该方法的有力度。此外，虽然流或牵引力系统刺激一般的细胞过程，磁珠特别通过使用受体配体靶向细胞粘附受体<sup系列=“外部参照”&gt; 3或反对细胞表面受体13，15单克隆抗体。 这种方法的另一个优点是粘附复合物通过一个简单的配体的亲和纯化过程的隔离。这是众所周知的，加入的涂覆配体珠的细胞结合粘附受体和诱导几个粘附蛋白23的募集。力的涂覆配体磁珠进一步应用开启这些粘附配合成介导的各种张力依赖性信号通路4,24大分子平台。细胞裂解，随后通过使用磁铁珠浓度允许的附着平台的隔离。用于纯化粘附物的其它方法已经在贴壁细胞使用。他们结合化学交联，以节省蛋白质 - 蛋白质相互作用和细胞裂解步骤由洗涤剂和剪切流动或超声处理20，21，25，26，27，28。最后一步是含有粘合性复合物所得到的腹侧质膜的集合。不像这些方法，磁珠允许通过选择性靶向粘附受体的特定家族细胞粘附复合物的更大的纯化水平。磁珠已经用于纯化粘附络合物附着到涂覆配体的微珠29,30非贴壁细胞。下面模拟生物环境中描述的方法，其中施加短持续时间（数秒至数分钟）力。因此，它提供了一个有力的工具，调查两个纯化粘附复合物的分子组成和下游机械敏感-信号通路。 在这里，我们提出了一个详细的实验协议利用磁珠的张力适用于粘接面的蛋白质。永久钕磁铁被放置在培养皿表面的顶部。磁铁的磁极面被放置在6mm的高度，使得在单个2.8微米磁珠上的力是恒定的（约30-40 PN）31。张力刺激的持续时间是由根据兴趣和其时间尺度活化的分子上的操作者确定。将细胞最后裂解，粘附络合物通过珠粒分离利用磁铁纯化和生物化学分析被处理。这个协议包括的涂覆配体超顺磁珠的制备，和张力通过磁体的应用，随后通过生物化学分析。此外，我们还提供应用于基于整联-ADH数据表明张力的代表性样品esions诱导粘附重塑和改变蛋白质的酪氨酸磷酸化。

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the upper face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">DX88-N52</td>
			<td style="width:375px;">grade N52 dimension: 1 1/2&#34; dia. x 1/2&#34; thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the lower face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">D84PC-BLK</td>
			<td>grade N42 dimension: 1/2&#34; dia. x 1/4&#34; thick Black Plastic Coated&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dynabeads M280 Tosylactivated</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">14203</td>
			<td>superparamagnetic beads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DynaMag-2 Magnet</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fibronectin&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">F1141-5MG</td>
			<td>Fibronectin from bovine plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Poly-D-Lysine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">P7280-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Apo-Transferrin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">T1428-50MG</td>
			<td>Bovine Apo-Transferrin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">A7906-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">DMEM high glucose, GlutaMAX supplement, pyruvate&#160;</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:103px;">31966-021</td>
			<td>DMEM+GlutaMAX-I 500 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">60*15 mm culture dish</td>
			<td style="width:115px;">Falcon</td>
			<td style="width:103px;">353004</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

机械敏感细胞表面粘附物使细胞感测与其周围环境的机械性能。最近的研究已经确定了在固定区域既力传感分子和调节谱系特异性基因表达和驱动表型输出力依赖性转录因子。然而，信令网络转换机械张力成生化途径仍然难以捉摸。探索时施加到细胞表面受体的机械张力接合的信号传导途径，可以使用超顺磁性微珠。在这里，我们提出了一个协议利用磁珠力量应用到细胞表面粘附蛋白。使用这种方法，可以通过安装在涂覆配体珠粘附复合物的磁隔离，调查通过各种生物化学方法不仅力依赖性细胞质信号传导途径，而且粘附重塑。这个协议包括配体 - 共聚的制备ated超顺磁珠，和应用程序中定义的拉力接着生物化学分析。此外，我们提供的数据表明应用于基于整合素的粘附张力的代表性样品触发粘附重塑和改变蛋白质的酪氨酸磷酸化。

该技术的原理在图1a中示出。以下的配体缀合，磁珠与细胞孵育20分钟，然后一个永久磁铁用于施加约30-40 PN的拉伸力对各种量的时间。 图1b示出了2.8微米势必MRC5细胞粘附受体的FN包被的磁珠。 细胞裂解后的超顺磁珠洗涤步骤是关键的，并确定纯化的程度。建议三次洗涤最低。 GADPH免疫印迹与长时间曝光可以测试粘附复合物的纯度（图2a）是</stro...

Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

细胞表面粘连是在机械传导的中心，因为它们传输机械张力，并开始参与组织稳态和发展的信号通路。这里，我们提出了解剖被响应于张力激活生化途径，使用包被配体 - 磁珠和施力到粘附受体的协议。

分析细胞表面粘附重塑回应机械张力使用磁珠

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Research

JoVE Journal

Biology

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

Quantification of ÃƒÅ½Ã‚Â³H2AX Foci in Response to Ionising Radiation

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

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基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

生化分析的分析ATP依赖的染色质重塑酶活性的影响

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

Measuring Glutathione-induced Feeding Response in Hydra

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Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

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对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...