Name: in situ monitoring of transiently formed molecular chaperone assemblies in bacteria, yeast, and human cells
Uploaded: 2019-09-02T15:00:00
Description: Watch this Scientific Journal Video about In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells at JoVE.com

NBN由莫纳什大学医学护理与健康科学学院提供特别招聘补助金，维多利亚州政府和澳大利亚政府提供资金。我们感谢伯恩德·布考（德国海德堡大学ZMBH）和哈姆·坎金加（荷兰格罗宁根大学细胞与系统生物医学科学系）对试剂霍尔格·洛伦茨（ZMBH）的宝贵支持和共享德国海德堡大学成像设施，支持共聚焦显微镜和图像处理，克莱尔·赫斯特（澳大利亚莫纳什大学ARMI）对手稿进行批判性阅读。

1. 赫拉细胞制备
<ol><li>准备以下材料： PBS （137 mM NaCl， 2.7 mM KCl， 10 mM Na2HPO4， 1.8 mM KH2PO4），pH 7.4;DMEM，辅以 10% FCS 和 1% 笔链;4%甲醛在PBS中;0.5% Triton-X100 在 PBS 中;TBS-T （150 mM NaCl， 20 mM Tris， 0.05% 补间）， pH 7.4;0.0001% 无菌多L-莱辛溶液;10 well 诊断幻灯片;潮湿的室内;纸巾;和科普林滑动染色罐。 注：为确保最佳的固定效率，在每次实验前应新鲜制备...

<ol>
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基于共免疫沉淀和共同定位的方法一直被用作描述蛋白质组装的长期方法。检测瞬时形成的特定伴郎复合物是这种传统方法的一大挑战，因此，以前的发现主要限于定性解释。基于细胞莱沙的共免疫沉淀技术通常需要交联以稳定蛋白质-蛋白质的相互作用。细胞莱沙会增加破坏瞬时形成的伴生复合物的风险，而交联可能引入非原生相互作用，特别是由大多数伴生者固有的"粘性"驱动。当使用共免疫?...

由于我们对细胞相互作用的不完全了解，大量的基因组信息仍然无法解释。传统的蛋白质-蛋白质相互作用检测方法，如蛋白质共免疫沉淀与/没有化学交联和蛋白质共定位，虽然广泛使用，造成一系列缺点。一些主要缺点包括相互作用的量化不佳，以及可能引入非本机绑定事件。相比之下，新兴的基于接近的技术为捕获细胞中的蛋白质相互作用提供了另一种强有力的方法。接近结扎测定（PLA）1，现在作为专有试剂盒提供，利用抗体根据相互作用亚单位的接近度专门靶向蛋白质复合物。
PLA是由在靶向蛋白复合物表面形成一个脚手架，由具有小DNA标记（PLA探针）的初级和二级抗体组成（图1，步骤1-3）。接下来，由DNA标记的接近性决定，一个圆形DNA分子通过与连接器寡核苷酸杂交产生（图1，步骤4）。圆形DNA的形成通过DNA结扎步骤完成。新形成的圆形DNA片作为后续滚环扩增（RCA）型聚合酶链反应（PCR）的模板，由其中一个结合的寡核苷酸标记。这通过抗体支架产生单链连体DNA分子附着在蛋白质复合物上（图1，步骤6）。串联DNA分子是使用荧光标记的寡核苷酸进行可视化的，这种核苷酸杂交到分散在扩增DNA上的多个独特序列（图1，步骤7）2。生成的PLA信号，显示为荧光点（图1，步骤7），对应于细胞中靶向蛋白质复合物的位置。因此，该测定可以检测高空间精度的蛋白质复合物。该技术并不仅限于捕获蛋白质相互作用，但也可用于检测高灵敏度1，2的蛋白质上的单个分子或蛋白质修饰。
Hsp70 通过参与一系列内务管理和与压力相关的功能，形成了一个高度通用的护套系统，对于维持细胞蛋白平衡至关重要。Hsp70伴郎系统的家政活动包括脱新蛋白折叠、细胞膜中蛋白质易位、蛋白质复合物的组装和拆解、蛋白质活性的调节以及连接不同的蛋白质折叠/质量控制机器3.同样的伴松系统还重新折叠错折叠/展开的蛋白质，防止蛋白质聚集，促进蛋白质分解，并与细胞蛋白酶合作，降解最终错折叠/损坏的蛋白质，以促进细胞修复后蛋白毒性应力4，5。为了实现这种功能多样性，Hsp70 伴侣依赖于 JDP 家族的合作伙伴和核苷酸交换因子 （NEF），这些因子可微调 Hsp70 的基于 ATP 的基板结合和释放3的依合体控制， 6.此外，JDP共同护带器在选择这种多功能护带系统的基板方面发挥着至关重要的作用。这个家族的成员根据他们与原型JDP（大肠杆菌DnaJ）的结构同源性，被细分为三类（A、B和C）。 A 类 JKP 包含一个 N 端 J 域，该域与 Hsp70、甘氨酸-苯丙氨酸富集区、由锌手指状区域 （ZFLR） 和两个 β管域组成的基板结合区域以及一个 C-终端二分化域。具有 N 端 J 域和甘氨酸-苯丙氨酸富区但缺少 ZFLR 的 JSP 属于 B 类。通常，这两个类的成员都参与伴校函数。属于C类的会员，其中只包含共享J域4的JdP，招募Hsp70执行各种非伴奏功能。Hsp70 系统作为可互换基板识别"适配器"的重要作用体现在在进化过程中家庭成员的扩展。例如，人类有超过42个不同的JDP成员4。这些JJD功能作为单体，同构体和/或同质/异质寡聚物4，5。最近，通过A类之间的瞬态复杂形成进行功能合作（例如，H.智人DNAJA2;S. cerevisae Ydj1） 和 B 类（例如，H.智人DNAJB1;据报道，S.cerevisiae Sis1）真核JGP促进在体外7、8对非晶蛋白聚集物的有效识别。这些混合类JDP复合物大概在聚合蛋白的表面组装，以促进Hsp70-和Hsp70_Hsp100基蛋白的分解7，8，9，10.支持这些在真核细胞中暂时形成的混合类JDP复合物存在的关键证据被PLA8提供。
PLA越来越多地用于评估元动物的蛋白质相互作用，主要是哺乳动物细胞。在这里，我们报告这项技术的成功扩展，以监测真核和原核单细胞生物中暂时形成的伴生复合物，如萌芽酵母S.cerevisae和大肠杆菌。重要的是，这种扩展凸显了PLA在检测和分析感染人类和动物细胞的微生物方面的潜在用途。

<table>
	<tbody>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Merck</td>
			<td>103999</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>32201</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DNAJA2 antibody</td>
			<td>Abcam</td>
			<td>ab157216</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DNAJB1 Antibody</td>
			<td>Enzo Life Sciences</td>
			<td>ADI-SPA-450</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DnaK antibody</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mCherry antibody</td>
			<td>Abcam</td>
			<td>ab125096</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sis1 Antibody</td>
			<td>Cosmo Bio Corp</td>
			<td>COP-080051</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Ydj1 antibody</td>
			<td>StressMarq Biosciences</td>
			<td>&#160;SMC-150,</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-YFP antibody</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin slide-staining jar</td>
			<td>Sigma-Aldrich</td>
			<td>S5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Diagnostic slides</td>
			<td>Marienfeld</td>
			<td>1216530</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo-Fischer</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ Detection Reagents Orange</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92007</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ Mounting Medium + DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ PLA Probe Anti-Mouse MINUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ PLA Probe Anti-Rabbit PLUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td></td>
		</tr>
		<tr>
			<td>DuoLink In Situ Wash Buffers, Fluorescence</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82049</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Thermo-Fischer</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>62971</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>32213</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo-Fischer</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P47-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S7547</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Merck</td>
			<td>108643</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo-Fischer</td>
			<td>25300096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolase 100T / / Lyticase</td>
			<td>United States Biological</td>
			<td>Z1004</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ monitoring of transiently formed molecular chaperone assemblies in bacteria, yeast, and human cells

J-domain蛋白（JdPs）是真核细胞中最大、最多样化的共体细胞家族。最近的发现表明，JDP家族的特定成员可以在真核生物中形成瞬态异质复合物，以微调70 kDa热休克蛋白（Hsp70）基于配体蛋白的基质选择。JDP复合物针对急性/慢性应激诱导的聚合蛋白，并可能通过在蛋白质聚合体表面招募多个Hsp70s来帮助组装分离的气体。这些物理相互作用的JdPs形成的蛋白质质量控制（PQC）网络在体内基本上没有特征。在这里，我们描述了一种基于显微镜的原位蛋白相互作用测定，名为接近结扎测定（PLA），它能够在真核细胞的不同细胞隔间中强有力地捕获这些瞬时形成的伴结复合物。我们的工作将PLA的用法从人类细胞扩展到酵母（糖母细胞）和细菌（大肠杆菌），从而成为监测两者中瞬态形成的蛋白质组体的动力学的重要工具。原核细胞和真核细胞。

我们以前使用纯化蛋白质的体外研究表明，人类A类和B类JDPs的子集形成瞬态混合类JDP复合物，以有效地靶向广泛的聚合蛋白，并可能促进基于Hsp70的组装蛋白质异化7。我们利用PLA来确定混血类（A+B）JDP复合物是否发生在人类宫颈癌细胞（HeLa）中。人类JDPsDNAJA2（A类）和DNAJB1（B类）被以高度特异性原抗体和二级PLA探针（图2A-C）为目标。红色荧光...

Watch this Scientific Journal Video about In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells at JoVE.com

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

Cognate J-domain 蛋白质与 Hsp70 伴护器合作，协助处理从蛋白质折叠到降解的各种生物过程。在这里，我们描述了一个原位接近结扎测定，它允许监测这些瞬时形成的伴菌机在细菌，酵母和人类细胞。

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 ...

此方法捕获细菌和酵母等生物体以及不同细胞类型的瞬态蛋白质相互作用。在这里，我们监测暂时形成的特定伴郎复合物，涉及细胞蛋白转移。该技术的主要优点是能够生成关于原核细胞和真核细胞中蛋白质组件的动态和亚细胞定位的信息。在细菌和酵母等单细胞生物体中，这种技术的适应使得研究人员能够用它作为研究与微生物感染有关的疾病的有力工具。这种技术有可能用于分析几乎所有生物体的蛋白质相互作用，只需改变协议样品制备阶段的条件即可。选择在免疫组织化学、免疫荧光、ELISA 或免疫沉淀中特异性和测试的优质抗体非常重要。首先使用聚 L-Lysine 预处理 10 个井诊断幻灯片。将 100 微升无菌和过滤 0.0001%聚-L-莱辛添加到每一个井中，并在无菌层水流罩中孵育幻灯片 30 分钟。然后，用50微升无菌水清洗每一个井，以去除多余的聚L-莱辛。根据细胞类型，每井加入约15，000个细胞，并在37摄氏度的无菌湿润培养箱中生长细胞，将5%的二氧化碳培养成60至80%的汇合。24小时后，将纸巾放在每井边缘，取出生长介质，用50微升PBS洗三次细胞。然后，将50微升新鲜准备好的4%甲醛在PBS中添加到每井中，并在室温下孵化10分钟，来修复细胞。固定后，将幻灯片与 PBS 一起淹没在滑动染色罐中，将幻灯片冲洗三次。要渗透附着细胞的膜，将幻灯片淹没在PBS中0.5%Triton-X100的100毫升，并在室温下孵育细胞10分钟。然后用 TBS-T 洗三次幻灯片。最后一次洗涤后，用纸巾取出多余的缓冲液。根据手稿指示生长和稀释S.cerevisiae细胞，然后将它们转移到50毫升的离心管中，以665倍g的离心进行造粒，三分钟。取出上一除素，用五毫升新鲜介质重新暂停细胞。然后加入550微升37%甲醛的最终浓度为4%，并在室温下孵育培养物15分钟以修复细胞。孵育后，将细胞粒粒，取出上经剂，将颗粒重新在一毫升新鲜准备的4%甲醛洗涤缓冲液中。在室温下孵育细胞45分钟。同时，通过在每个井中加入100微升0.0001%聚-L-Lysine溶液，并在室温下将幻灯片孵育30分钟，来准备诊断幻灯片。然后用超纯水洗去多余的聚L-莱辛，让滑梯风干。孵育步骤后，用一毫升的洗涤缓冲液通过离心洗涤细胞两次。取出上流液，在洗涤缓冲液中重新填充细胞。上次洗涤后，将细胞切开，然后取出上清液，将细胞重新在含有1.2摩尔松醇的洗涤缓冲液中重新暂停。要消化细胞壁，再次对细胞进行颗粒化，并在250微升新鲜准备好的Lyticase溶液中重新给细胞重新下水。在30摄氏度下用温和的震动孵育细胞15分钟。消化步骤后，在洗涤缓冲液中清洗细胞三次，并在含有1.2摩尔索比托的250微升洗涤缓冲液中重新填充细胞。在每个聚L-Lysine涂层的井中加入20微升再悬浮细胞，使固定和渗透细胞附着30分钟。用50微升洗涤缓冲液洗三次，以去除非粘附细胞，并继续进行接近结扎测定。启动接近结扎测定，或PLA协议，在每一个井中加入一滴阻塞缓冲液，并在37摄氏度下在潮湿室中孵化滑梯30分钟。将一张纸巾放在每一个井的边缘，将细胞洗涤两次，在100毫升的洗涤缓冲液中清洗细胞。取出初级抗体溶液，在洗涤缓冲液中洗涤两次，洗涤后，将四十微升的二次抗体溶液添加到每井中，并在潮湿室的37摄氏度下孵育幻灯片60分钟。然后取出溶液，在洗涤缓冲液中洗涤两次，然后将含有连接器寡核苷酸和DNA结扎的40微升加入每个井，并在37摄氏度的潮湿室中孵育30分钟的幻灯片。孵育后，去除结扎混合物，用洗涤缓冲液A.加入40微升DNA扩增混合物，其中含有DNA聚合酶和荧光标记的寡核苷酸，到每井中孵育100分钟，在37摄氏度的潮湿室中孵育。去除放大混合物，在 100 毫升洗涤缓冲液 B 中清洗幻灯片 10 分钟。然后用100毫升的洗涤缓冲液B洗涤滑梯，在超纯水中稀释1至100。每孔添加 20 微升含 DAPI 安装介质。用盖滑盖住滑轨的井，用指甲油密封。该协议已成功用于监测不同 J 域蛋白和 Hsp70 伴郎和 J 域蛋白之间形成的瞬态伴郎组件，这些蛋白在 HeLa、S.cerevisiae 和大肠杆菌细胞中形成。人类 A 类和 B 类 J 域蛋白以高度特异性原抗体为目标。每个红色荧光双子表示在 HeLa 细胞中两种不同的 J 域蛋白之间形成的蛋白质复合物。类似的混合类J-域蛋白复合物也观察到在单细胞真核生物S.cerevisiae，或面包师的酵母。由于细胞尺寸小，从PLA生成的一些荧光蓬蒂更难作为个体的细胞解决。在大肠杆菌细胞中未观察到 A 类和 B 类 J 域蛋白之间的复杂形成，这与使用纯化蛋白质进行的生化研究一致。但其他涉及J域蛋白和Hsp70伴郎的陪护组件是使用PLA协议捕获的。在原本完整的PLA反应中，缺乏对相互作用的J域蛋白或伴用的一种初级抗体的技术控制表明细胞中很少或没有荧光蓬蒂形成，表明缺乏假阳性信号放大。要使用接近结扎测定获得可靠的结果，请事先验证所用抗体是否足够质量。此外，采取预防措施，避免含有细胞壁的细胞过度消化。

Research

JoVE Journal

Biology

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

科研

生物学

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...