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Biochemistry

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

Published: February 21, 2019
doi:
10.3791/59111
,
1Biozentrum,University of Basel

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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。 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?…

Representative Results

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。 <p class="jove_c…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

NanobodyEndocytic UptakeRetrograde TransportTrans-Golgi NetworkCell Surface ReceptorsFluorescence MicroscopyElectron MicroscopyTyrosine Sulfation

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Buser, D. P., Spiess, M. Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells. J. Vis. Exp. (144), e59111, doi:10.3791/59111 (2019).
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