Name: analysis of endocytic uptake and retrograde transport to the trans-golgi network using functionalized nanobodies in cultured cells
Uploaded: 2019-02-21T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells at JoVE.com

这项工作得到了瑞士国家科学基金会31003a-162643 赠款的支持。我们感谢 nicole beuret 和 biozentrum 成像核心设施 (imcf) 的支持。

1. 功能化纳米化的细菌转化
请注意:该协议针对功能化抗 gfp 纳米波模的表达、纯化和分析进行了优化, 如前面所述.与其他蛋白质分子的衍生化可能需要修改这个标准协议。
<ol><li>解冻化学主管细菌 (~ 100μl), 适合于蛋白质表达 (例如, 罗塞塔bl21 (de3) 细胞), 将它们放在冰上。 请注意:根据标准实验室程序制备具有化学能力的?...

纳米机器人代表了一类新兴的蛋白质粘合剂支架, 与传统抗体相比具有许多优点: 它们体积小、稳定、单体化, 可选择高亲和力和缺乏二硫键33,35,44,45. 它们被用于许多应用, 例如在发育生物学的细胞培养系统和生物体中,46、47、48...

蛋白质和脂质从细胞表面逆行到不同的细胞内隔间, 对于维持膜内的稳态以平衡分泌和回收前级运输机械的成分至关重要 1,2. 通过环素依赖性或-独立性依赖性的内化后, 蛋白质和脂质货物首先填充早期内质体, 从那里它们沿着溶酶体系统进一步重定向, 回收到质膜,或针对跨 golgi 网络 (tgn)。从内质体和细胞表面或细胞表面到 tgn 的回收是一些前品位跨膜货物受体的功能循环的一部分, 例如依赖阳离子和与阳离子无关的曼鼻-6-磷酸盐受体 (cdmpr 和 cimpr)新合成的从 tgn 到晚期内生体和溶酶体 3、4、5、旋毛蛋白和索拉6、7和 Wntless (wls) 的溶酶体水解酶将 wnt 配体输送到细胞表面8,9,10,11. 回收回 tgn 的其他蛋白质是 tgn46 及其相关的异形12、13、14、snares (可溶性n-乙基马来酰亚胺敏感的融合因子附着受体)15,16,17、淀粉样体前体蛋白 (app)18,19, 进行性强直 (ank) 蛋白20, 金属转运体如 atp7a/b 或 dmt121、22和跨膜加工酶, 包括羧肽酶 d, 呋喃或 bce123,24,25。除了这些内源性蛋白质外, 细菌和植物毒素 (如滋贺和霍乱毒素、黄素和阿林) 还劫持逆行运输机械, 使其到达 er, 以便逆行进入细胞溶胶26、27, 28,29。
为了直接分析逆行交通, 我们以前开发了一个基于纳米生物的工具包, 用于标记和跟踪从细胞表面到细胞内隔间的货物蛋白质.纳米生物是一种新的蛋白质粘合剂家族, 它们来源于同源重链的抗体 (hcabs), 自然存在于骆驼和软骨鱼类中 31,32。它们构成了 hcabs 的可变重链域 (vhh), 与传统抗体 (例如 igg) 相比具有许多优点: 它们是单体、小 (~ 15 kda)、高度溶解性、无二硫键、可细菌表达, 并可选择高亲和力结合33,34,35,36。为了使我们的纳米机器人工具用途广泛, 我们使用功能化的抗 gfp 纳米机器人在表面标记和跟踪标记为 gfp 的蛋白质在他们的外皮膜内。通过纳米棒与 mcherry、抗坏血酸过氧化物酶 2 (apex2)或酪氨酸硫化 (ts) 序列的功能化, 可以通过固定和活细胞成像分析真正跨膜货物蛋白的逆行迁移, 通过电子显微镜, 或生物化学。由于酪氨酸磺酸硫化介导的酪氨酸蛋白磺酸盐转移酶 (tpst1 和 tpst2) 是一种后置修饰, 仅限于跨 golgi/tgn, 我们可以直接研究感兴趣的蛋白质从细胞表面到这个的迁移和动力学。细胞内的 golgi隔间38,39,40。
在这篇方法的文章中, 我们描述了功能化纳米棒模具 (vh-2xts,-apex2,-mcherry 和衍生物) 的易用性, 适用于许多应用, 以分析哺乳动物细胞中的逆行运输30。我们主要研究了 ts 位修饰纳米无菌分析细胞内交通从细胞表面到硫化室的方法。

<table >
	
		
		
		
		
	
	<tbody>
		<tr >
			<td >Anti-GFP antibody</td>
			<td >Sigma-Aldrich</td>
			<td >118144600001</td>
			<td >Product is distributed by Sigma-Aldrich, but manufactured by Roche</td>
		</tr>
		<tr >
			<td >Anti-His6 antibody</td>
			<td >Bethyl Laboratories</td>
			<td >A190-114A</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-actin antibody</td>
			<td >EMD Millipore</td>
			<td >MAB1501</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-rabbit HRP</td>
			<td >Sigma-Aldrich</td>
			<td >A-0545</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-mouse HRP</td>
			<td >Sigma-Aldrich</td>
			<td >A-0168</td>
			<td ></td>
		</tr>
		<tr >
			<td >4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td >Sigma-Aldrich</td>
			<td >D9542</td>
			<td >dissolved in 1 x PBS/1%BSA</td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide (DMSO)</td>
			<td >Applichem</td>
			<td >A3672</td>
			<td ></td>
		</tr>
		<tr >
			<td >D-biotin</td>
			<td >Sigma-Aldrich</td>
			<td >B4501</td>
			<td >dissolved in sterile 500 mM NaH2PO4 or DMSO</td>
		</tr>
		<tr >
			<td >5-aminolevuilnic acid (dALA) hydrochloride</td>
			<td >Sigma-Aldrich</td>
			<td >A3785</td>
			<td >dissolved in sterile water</td>
		</tr>
		<tr >
			<td >DNase I</td>
			<td >Applichem</td>
			<td >A3778</td>
			<td >dissolved in sterile water</td>
		</tr>
		<tr >
			<td >Lysozyme</td>
			<td >Sigma-Aldrich</td>
			<td >18037059001</td>
			<td >Product is distributed by Sigma-Aldrich, but manufactured by Roche</td>
		</tr>
		<tr >
			<td >Brefeldin A (BFA)</td>
			<td >Sigma-Aldrich</td>
			<td >B5936</td>
			<td ></td>
		</tr>
		<tr >
			<td >Puromycin</td>
			<td >Invivogen</td>
			<td >ant-pr-1</td>
			<td ></td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin</td>
			<td >Bioconcept</td>
			<td >4-01F00-H</td>
			<td ></td>
		</tr>
		<tr >
			<td >L-glutamine</td>
			<td >Applichem</td>
			<td >A3704</td>
			<td ></td>
		</tr>
		<tr >
			<td >Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td >Sigma-Aldrich</td>
			<td >D5796</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fetal calf serum (FCS)</td>
			<td >Biowest</td>
			<td >S181B-500</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sulfur-35 as sodium sulfate</td>
			<td >Hartmann Analytics</td>
			<td >ARS0105</td>
			<td >Product contains 5 mCi</td>
		</tr>
		<tr >
			<td >Earle&#39;s balanced salts</td>
			<td >Sigma-Aldrich</td>
			<td >E6267</td>
			<td ></td>
		</tr>
		<tr >
			<td >MEM amino acids (50 x) solution</td>
			<td >Sigma-Aldrich</td>
			<td >M5550</td>
			<td ></td>
		</tr>
		<tr >
			<td >MEM vitamin solution (100 x)</td>
			<td >Sigma-Aldrich</td>
			<td >M6895</td>
			<td ></td>
		</tr>
		<tr >
			<td >cOmplete, Mini Protease inhibitor cocktail</td>
			<td >Sigma-Aldrich</td>
			<td >11836153001</td>
			<td >Product is distributed by Sigma-Aldrich, but manufactured by Roche</td>
		</tr>
		<tr >
			<td >Isopropyl-&#946;-D-thiogalactopyranosid (IPTG)</td>
			<td >Applichem</td>
			<td >A1008</td>
			<td >dissolved in sterile water, stock is 1 M</td>
		</tr>
		<tr >
			<td >Carbenicillin disodium salt</td>
			<td >Applichem</td>
			<td >A1491</td>
			<td >dissolved in sterile water, stock is 100 mg/mL</td>
		</tr>
		<tr >
			<td >Kanamycin sulfate</td>
			<td >Applichem</td>
			<td >A1493</td>
			<td >dissolved in sterile water, stock is 100 mg/mL</td>
		</tr>
		<tr >
			<td >Coomassie-R (Brilliant Blue)</td>
			<td >Sigma-Aldrich</td>
			<td >B-0149</td>
			<td ></td>
		</tr>
		<tr >
			<td >Paraformaldehyde (PFA)</td>
			<td >Applichem</td>
			<td >A3813</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bovine serum albumin (BSA)</td>
			<td >Sigma-Aldrich</td>
			<td >A2153</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fluoromount-G</td>
			<td >Southern Biotech</td>
			<td >0100-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ni Sepharose High Performance</td>
			<td >GE Healthcare</td>
			<td >17-5268-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >His GraviTrap columns</td>
			<td >GE Healthcare</td>
			<td >GE11-0033-99</td>
			<td ></td>
		</tr>
		<tr >
			<td >His buffer kit</td>
			<td >GE Healthcare</td>
			<td >GE11-0034-00</td>
			<td ></td>
		</tr>
		<tr >
			<td >Disposable PD10 desalting columns</td>
			<td >GE Healthcare</td>
			<td >GE17-0851-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini-Protean TGX gels, 4-20%, 15-well</td>
			<td >Bio-Rad</td>
			<td >456-1096</td>
			<td ></td>
		</tr>
		<tr >
			<td >Dulbecco&#8217;s phosphate buffered saline (DPBS) w/o Ca2+/Mg2+</td>
			<td >Sigma-Aldrich</td>
			<td >D8537</td>
			<td ></td>
		</tr>
		<tr >
			<td >35-mm dishes</td>
			<td >Falcon</td>
			<td >353001</td>
			<td ></td>
		</tr>
		<tr >
			<td >6-well plates</td>
			<td >TPP</td>
			<td >92406</td>
			<td ></td>
		</tr>
		<tr >
			<td >Glass coverslips (No. 1.5H)</td>
			<td >VWR</td>
			<td >631-0153</td>
			<td ></td>
		</tr>
		<tr >
			<td >Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td >Applichem</td>
			<td >A0999.0025</td>
			<td >dissolved in 40% DMSO 60% isopropanol, stock in 500 mM</td>
		</tr>
		<tr >
			<td >Tryptone</td>
			<td >Applichem</td>
			<td >A1553</td>
			<td ></td>
		</tr>
		<tr >
			<td >Yeast extract</td>
			<td >Applichem</td>
			<td >A1552</td>
			<td></td>
		</tr>
		<tr >
			<td >Magnesium chloride hexahydrate</td>
			<td >Merck Millipore</td>
			<td >105833</td>
			<td>dissolved in sterile water, stock is 1 M</td>
		</tr>
		<tr >
			<td >Calcium chloride dihydrate</td>
			<td >Merck Millipore</td>
			<td >102382</td>
			<td>dissolved in sterile water, stock is 1 M</td>
		</tr>
		<tr >
			<td >Sodium chloride</td>
			<td >Merck Millipore</td>
			<td >106404</td>
			<td>dissolved in sterile water, stock is 5 M</td>
		</tr>
	</tbody>
</table>

analysis of endocytic uptake and retrograde transport to the trans-golgi network using functionalized nanobodies in cultured cells

蛋白质和膜从细胞表面输送到 golgi 和更远的地方对于稳态、细胞器身份和生理是必不可少的。为了研究逆行蛋白的流量, 我们最近开发了一个基于纳米生物的多功能工具包, 通过固定和活细胞成像、电子显微镜或生化方法来分析从细胞表面到 golgi 复合体的迁移。我们设计了功能化的抗绿色荧光蛋白 (gfp) 纳米棒--小的、单体的、高亲和力的蛋白质粘合剂--它可以应用于细胞外 gfp 分子表达感兴趣的膜蛋白的细胞系。衍生纳米棒绑定到 gfp 记者是专门内化和运输搭载沿记者的排序路线。用荧光显微镜和活体成像技术对纳米无孔进行了功能化, 以跟踪荧光的逆行迁移, 并利用抗坏血酸过氧化物酶 2 (apex2) 研究了报告-纳米-机器人复合物电子的超微结构定位显微镜, 并与酪氨酸硫化 (ts) 的主题, 以评估动力学跨 golgi 网络 (tgn) 的到来。在这篇方法性文章中, 我们概述了细菌表达和纯化功能化纳米棒的一般程序。我们说明了我们的工具的强大用途, 使用 mcherry-l 和 ts 修饰纳米生物来分析内循环吸收和货物蛋白的 tgn 到达。

为了研究逆行蛋白运输到各种细胞内目的地, 我们最近建立了一个抗 gfp 纳米生物为基础的工具, 以标记和跟踪重组融合蛋白从细胞表面30。在这里, 我们展示了这种衍生纳米棒的细菌生产, 并演示了它们在荧光显微镜和免疫印迹法研究内循环吸收方面的应用, 以及它们在硫化分析研究 tgn 到达方面的应用。后一种适用是本议定书的方法重点。
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Watch this Scientific Journal Video about Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells at JoVE.com

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

培养细胞功能化纳米化对跨 golgi 网络的内分泌吸收和逆行迁移分析

蛋白质从细胞表面逆行运输到 golgi 是维持膜稳态所必需的。在这里, 我们描述了一种方法, 生物化学分析细胞地对golgi 的重组蛋白运输使用功能化纳米棒在海拉细胞。

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

这种方法使细菌表达、功能化纳米体的生产和应用能够分析从细胞表面到细胞间隔间的逆行蛋白传输。特别是跨戈尔吉网络。这种技术的主要优点是使用硫化。高度具体、分门别类和敏感的翻译后修改，以评估记者纳米体复杂到达DTN隔间。该方法与其他衍生纳米体相结合，用于生命细胞成像和电子显微镜，也可用于研究疾病相关细胞表面受体的内皮过程。我们的功能化纳米体系列已被设计成用于研究培养细胞的逆行运输。然而，这些工具也可以应用于多细胞生物体。要开始这个程序，从板中挑选一个含有感兴趣的载体的细菌菌落，并接种含有20毫升LB介质的烧瓶，并辅以抗生素。37 岁孵育过夜

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SCIENTISTS IN ACTION

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

科研

生物化学

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

密度梯度超速离心研究哺乳动物细胞内吞循环

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

JUMPn：蛋白质共表达聚类和网络分析在蛋白质组学中的简化应用

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

使用200 KV透射电子显微镜常规收集高分辨率冷冻电镜数据集

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...