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Bioengineering

巨型单层囊泡内重组细胞骨架的快速封装

Published: November 10, 2021
doi:
10.3791/63332
, , , ,
1Department of Mechanical Engineering,University of Michigan, Ann Arbor, 2John A. Paulson School of Engineering and Applied Sciences,Harvard University, 3Department of Cellular and Molecular Biophysics,Max Planck Institute of Biochemistry, 4Department of Biomedical Engineering,University of Michigan, Ann Arbor, 5Department of Biophysics,University of Michigan, Ann Arbor, 6Cellular and Molecular Biology Program,University of Michigan, Ann Arbor

Summary

本文介绍了一种简单的方法，用于快速生产具有包封细胞骨架蛋白的巨型单层囊泡。该方法被证明可用于自下而上的细胞骨架结构在约束和细胞骨架 – 膜相互作用中的重建。

Abstract

巨型单层囊泡（GUV）经常被用作生物膜的模型，因此是 研究体外膜相关细胞过程的绝佳工具。近年来，GUV中的封装已被证明是细胞生物学和相关领域重建实验的有用方法。它更好地模拟活细胞内的限制条件，而不是传统的生化重建。在GUV内部封装的方法通常不容易实施，并且不同实验室的成功率可能会有很大差异。一种已被证明可以成功封装更复杂的蛋白质系统的技术称为连续液滴界面交叉封装（cDICE）。这里提出了一种基于cDICE的方法，用于在GUV中快速包封细胞骨架蛋白，具有高包封效率。在该方法中，首先，通过在脂质/油混合物中乳化感兴趣的蛋白质溶液来产生脂质 – 单层液滴。在被添加到旋转的3D打印室后，这些脂质单层液滴随后在腔室内的水/油界面处通过第二个脂质单层，形成含有蛋白质系统的GUV。该方法简化了GUV内封装的整体过程并加快了该过程，从而使我们能够限制和观察脂质双层囊泡内网络组装的动态演变。该平台对于研究禁闭中细胞骨架 – 膜相互作用的力学非常方便。

Introduction

脂质双层隔室用作模型合成细胞，用于研究封闭的有机反应和基于膜的过程，或用作药物递送应用中的载体模块1，2。具有纯化成分的自下而上的生物学需要最少的实验系统来探索生物分子（例如蛋白质和脂质）之间的性质和相互作用3，4。然而，随着该领域的进步，对更复杂的实验系统的需求增加，这些系统可以更好地模仿生物细胞中的条件。GUV中的包封是一种实用的方法，可以通过提供可变形和选择性渗透的脂质双层和有限的反应空间来提供一些这些细胞样特性。特别是，细胞骨架系统的 体外 重建，作为合成细胞的模型，可以从膜室5中的包封中受益。许多细胞骨架蛋白与细胞膜结合并相互作用。由于大多数细胞骨架组装形成跨越整个细胞的结构，因此它们的形状自然由细胞大小的限制6决定。

使用不同的方法来产生GUV，例如溶胀7，8，小囊泡融合9，10，乳液转移11，12，脉冲喷射13，以及其它微流体方法14，15。尽管这些方法仍在使用，但每种方法都有其局限性。因此，非常需要一种具有高GUV封装率的稳健而直接的方法。尽管诸如自发溶胀和电膨胀之类的技术被广泛用于GUV的形成，但这些方法主要与特定的脂质组合物16，低盐浓度缓冲液17，较小的封装胶分子尺寸18相容，并且需要大量封装胶。将多个小囊泡融合到GUV中本质上是不利于能量的，因此需要在带电脂质组合物9 和/或外部融合诱导剂（例如肽19或其他化学物质）中具有特异性。另一方面，乳液转移和微流体方法可能需要在双层形成后通过表面活性剂和溶剂去除来稳定液滴，分别为18，20。微流体技术（如脉冲喷射）中实验设置和装置的复杂性带来了额外的挑战21。cDICE是一种基于乳液的方法，其衍生自类似的支配乳液转移的原理22，23。水溶液（外溶液）和脂质油混合物在旋转圆柱形室（cDICE室）中通过离心力分层，形成脂质饱和界面。将脂质单层水滴穿梭到旋转的cDICE室中导致双层的拉链，因为液滴穿过脂质饱和界面进入外层水溶液22，24。cDICE方法是一种用于GUV封装的强大技术。使用所提出的改进方法，不仅可以实现cDICE典型的高囊泡产量，并且具有显着缩短的封装时间（几秒钟），而且允许观察时间依赖性过程（例如，肌动蛋白细胞骨架网络形成）的GUV生成时间显着降低。该方案从开始到GUV收集和成像大约需要15-20分钟。在这里，GUV的产生是使用修饰的cDICE方法包封肌动蛋白和肌动蛋白结合蛋白（ABP）来描述的。然而，所提出的技术适用于包封广泛的生物反应和膜相互作用，从生物聚合物的组装到无细胞蛋白质表达再到基于膜融合的货物转移。

Protocol

1. 油脂混合物的制备 注意：该步骤需要在通风橱中按照处理氯仿的所有安全指南进行。 在15毫升玻璃小瓶中取0.5毫升氯仿。将88μL的25mg / mL二油酰基磷酸胆碱（DOPC），9.3μL的50mg / mL胆固醇和5μL的1mg / mL二分子酰基 – 磷酸乙醇胺 – 赖胺罗丹明B（罗丹明PE）（参见 材料表）加入15ml玻璃小瓶中。注意：硅油/矿物油中DOPC和胆固醇的最终摩尔分数?…

Representative Results

为了证明使用当前方案成功生成细胞骨架GUV，重建了GUV中的fascin-actin束结构。Fascin是肌动蛋白丝的短交联剂，其形成坚硬的平行排列的肌动蛋白束，并从 大肠杆菌 中纯化为谷胱甘肽-S-转移酶（GST）融合蛋白26。首先重构5 μM肌动蛋白，包括肌动蛋白聚合缓冲液中的0.53 μM ATTO488肌动蛋白和7.5%的密度梯度培养基。在以2.5μM的浓度加入筋膜并包封法氏菌素 – 肌动蛋白混合物时?…

Discussion

已经探索了产生GUV的不同方法来创建合成细胞然而，程序的复杂性，获得封装的时间延长，密封剂的脂质类型和分子组成的限制，需要非生理化学物质来促进封装，低GUV产率以及封装效率的不一致一直困扰着该领域的研究人员。考虑到可以在自下而上的合成生物学中开展的广泛潜在研究，一种无缝的高通量GUV封装方法，与不同的脂质组成兼容，并且可以封装任何分子，无论大小如何，都可能激发?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

APL感谢洪堡研究奖学金对有经验的研究人员以及国家科学基金会（1939310和1817909）和美国国立卫生研究院（R01 EB030031）的支持。

Materials

18:1 Liss Rhod PE lipid in chloroform Avanti Polar Lipids 810150C
96 Well Optical Btm Pit PolymerBase ThermoFisher Scientific 165305
Actin from rabbit skeletal muscle Cytoskeleton AKL99-A
ATTO 488-actin from rabbit skeletal muscle Hypermol 8153-01
Axygen microtubes (200 µL) Fisher Scientific 14-222-262 for handling ABPs
Black resin Formlabs RS-F2-GPBK-04
Cholesterol (powder) Avanti Polar Lipids 700100P
Choloroform Sigma Aldrich 67-66-3
Clear resin Formlabs RS-F2-GPCL-04
CSU-X1 Confocal Scanner Unit YOKOGAWA CSU-X1
Density gradient medium (Optiprep) Sigma-Aldrich D1556
DOPC lipid in chloroform Avanti Polar Lipids 850375C
Fascin homemade N/A
F-buffer homemade N/A
Fisherbrand microtubes (1.5 mL) Fisher Scientific 05-408-129
FS02 Sonicator Fischer Scientific FS20
G-buffer homemade N/A
Glucose Sigma-Aldrich 158968
iXon X3 camera Andor DU-897E-CS0
Mineral oil Acros Organics 8042-47-5
Olympus IX81 Inverted Microscope Olympus IX21
Olympus PlanApo N 60x Oil Microscope Objective Olumpus 1-U2B933
Silicone oil Sigma-Aldrich 317667
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Tags

Giant Unilamellar VesiclesCytoskeleton ReconstitutionIn Vitro ReconstitutionSynthetic Cell ResearchCell-free Transcription-translationMinimal ProtocellsOil-lipid MixtureLipid-oil DispersionActin PolymerizationActin Binding Proteins
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Bashirzadeh, Y., Wubshet, N., Litschel, T., Schwille, P., Liu, A. P. Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles. J. Vis. Exp. (177), e63332, doi:10.3791/63332 (2021).
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