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Summary
Abstract
Introduction
Protocol
Results
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생체공학

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

Published: August 25, 2022
doi:
10.3791/64026
, ,
1Department of Biomedical Engineering,Yale University, 2Systems Biology Institute,Yale University, 3Department of Physics,Yale University

Summary

在这份手稿中，我们展示了将F-肌动蛋白细胞骨架封装到巨大的单层脂质囊泡（也称为脂质体）中的实验技术，以及在脂质体膜的内小叶处形成皮质仿生F-肌动蛋白层的方法。

Abstract

肌动蛋白细胞骨架是细胞中的主要机械机制，介导许多必需的物理细胞活动，包括细胞变形，分裂，迁移和粘附。然而，研究 体内 肌动蛋白网络的动力学和结构由于活细胞内的生化和遗传调控而变得复杂。为了建立一个缺乏细胞内生化调节的最小模型，肌动蛋白被封装在巨大的单层囊泡（GUV，也称为脂质体）中。仿生脂质体是细胞大小的，有助于定量了解细胞骨架网络的机械和动力学特性，为自下而上的合成生物学开辟了一条可行的途径。为了生成用于包封的脂质体，利用倒置乳化法（也称为乳化液转移法），这是将复杂溶液包封到脂质体中以制备各种细胞模拟系统的最成功的技术之一。使用这种方法，将目标蛋白质的混合物添加到内部缓冲液中，然后将其在含磷脂的矿物油溶液中乳化以形成单层脂质液滴。所需的脂质体是由穿过脂质/油水界面的单层脂质液滴产生的。该方法能够将浓缩的肌动蛋白聚合物包封到具有所需脂质组分的脂质体中，为仿生细胞骨架网络的 体外 重建铺平了道路。

Introduction

肌动蛋白细胞骨架通过协调分子水平的收缩性和力产生1，2，3在构建细胞的细胞内结构中起着重要作用。因此，它介导了许多基本的细胞活动，包括细胞变形4，5，分裂6，迁移7，8和粘附9。肌动蛋白网络的体外重建近年来获得了极大的关注10，11，12，13，14，15，16，17。重建的目标是建立一个细胞的最小模型，没有活细胞中存在的复杂生化调节。这为探测特定的细胞内活动提供了可控的环境，并有助于识别和分析肌动蛋白细胞骨架18，19的不同组分。此外，体外肌动蛋白网络在磷脂巨型单层囊泡（GUV，脂质体）内的包封提供了具有半渗透边界的受限但可变形的空间。它模仿细胞内肌动蛋白机制的生理和机械微环境9，20，21，22。

在制备脂质体的各种方法中，脂膜水合法（又称溶胀法）是最早的技术之一，23.干燥的脂质膜通过添加缓冲液进行水合，形成膜状气泡，最终变成囊泡24。为了产生具有较高产量的较大囊泡，从薄膜水合法推进的改进方法，称为电铸法25，施加交流电场以有效地促进水化过程26。这些基于水合的肌动蛋白包封方法的主要局限性在于它对高浓度蛋白质的包封效率低，并且仅与特定的脂质组合物24相容。相比之下，倒置乳液技术对脂质组分的限制较少，蛋白质浓度为20，27，28，29。在该方法中，将用于包封的蛋白质混合物加入到内部水缓冲液中，该缓冲液随后在含脂矿物油溶液中乳化，形成脂质单层液滴。然后，单层脂质液滴通过离心穿过另一个脂质/油水界面，形成双层脂质囊泡（脂质体）。该技术已被证明是肌动蛋白包封24，30的最成功的策略之一。另外，还有一些微流体装置方法，包括脉冲喷射31、32、瞬态膜喷射33和cDICE方法34。倒乳剂法与微流体法之间的相似之处在于所使用的脂质溶剂（油）和脂质/油 – 水界面的引入以形成脂质体的外小叶。相比之下，通过微流体方法产生脂质体需要设置微流体装置，并且伴随着在双层的两个小叶之间捕获的油，这需要额外的步骤来除油35。

在这份手稿中，我们使用倒置乳液技术制备包封聚合的F-肌动蛋白网络的脂质体，如之前22所示。首先将用于包封的蛋白质混合物置于具有非聚合条件的缓冲液中，以维持其球状（G）形式的肌动蛋白。整个过程在4°C下进行，以防止早期肌动蛋白聚合，随后通过允许样品升温至室温而触发。一旦在室温下，肌动蛋白聚合成其丝状（F）形式。可以将各种肌动蛋白结合蛋白添加到内部水缓冲液中以研究蛋白质的功能和性质，从而进一步深入了解其与肌动蛋白网络和膜表面的相互作用。该方法还可应用于包封感兴趣的各种蛋白质36和接近最终脂质体28，37大小的大型物体（微粒、自走式微游泳器等）。

Protocol

1. 缓冲液和蛋白质溶液的制备 通过混合0.1 mM氯化钙2，10 mM HEPES（pH 7.5），1mM DTT，0.5mM达布科，320mM蔗糖和0.2mM ATP，制备总体积为5mL的水性内部非聚合（INP）缓冲液。 通过在4°C下向INP缓冲液中加入蛋白质来制备蛋白质混合物（PM），浓度如下：11.2μM非荧光G-肌动蛋白，2.8μM荧光标记肌动蛋白和0.24μM Arp2/3（材料表）。为了形成F-肌动蛋白层，向PM中加?…

Representative Results

基于倒置乳液技术的脂质体的制备在 图1中以图形和示意性方式说明。 首先，制备由磷脂（EPC）和荧光脂质（DHPE）组成的空（裸）脂质体（直径约5-50μm）。作为对照实验，一种明亮的远红色荧光染料被封装在裸脂质体中。脂质单层是否在液滴的外周成功形成，可以通过观察在乳液中掺入荧光染料的单层脂质液滴来确定，如图2A所?…

Discussion

几个关键步骤决定了在制备过程中高产量脂质体的成功。为了将脂质膜完全溶解在油中，必须对样品进行超声处理，直到玻璃瓶底部的脂质膜完全消失。超声处理后，脂质油混合物必须在室温下在黑暗条件下储存过夜，以使脂质分子进一步分散29。混合物可以在4°C下储存长达一周。当使用含有目标蛋白质的单层脂质液滴制备FB / 油乳液时，必须通过玻璃注射器轻轻地来回泵送脂?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢向美国国立卫生研究院（NIH）提供 ARO MURI W911NF-1-0403 至 M.P.M.、美国国立卫生研究院 （NIH） R01 1R01GM126256、美国国立卫生研究院 （NIH） U54 CA209992、NIH RO1 GM126256、NIH U54 CA209992、密歇根大学/基因工程学院、SUBK00016255 和人类前沿科学计划 （HFSP） 拨款编号 RGY0073/2018 至 M.P.M.本材料中表达的任何意见，发现，结论或建议均为作者的观点，并不一定反映ARO，NIH或HFSP的观点。S.C.承认与V.亚达夫，C.穆雷桑和S.阿米里进行了富有成效的讨论。

Materials

1,2-dioleoyl-sn-glycero-3-{[n(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DOGS-NTA-Ni)  Avanti Polar Lipids Inc. 231615773 Nickel Lipid
1,4-Diazabicyclo[2.2.2]octane Sigma D27802-25G DABCO
Actin protein (>99% pure): rabbit skeletal muscle Cytoskeleton, Inc AKL99-D non-fluorescent G-actin
Actin protein (rhodamine): rabbit skeletal muscle Cytoskeleton, Inc AR05 fluorescently labeled actin
Adenosine 5′-triphosphate disodium salt hydrate Sigma A2383-10G ATP
Alexa Fluor 647 dye ThermoFisher fluorescent dye
Andor iQ3 Andor Technologies control and acquisition software for confocal microscope
Arp2/3 Protein Complex: Porcine Brain Cytoskeleton, Inc RP01P-A Arp 2/3
Calcium chloride dihydrate Sigma 10035048 CaCl2
Chamlide Chambers (4-well for 12 mm round coverslip) Quorum Technologies incubation chamber
Cofilin protein: human recombinant Cytoskeleton, Inc CF01-C cofilin
Confocal Microscope (63× oil-immersion objective) Andor Technologies LEICA DMi8
D-(+)-GLUCOSE BIOXTRA Sigma G7528 glucose
Dithiothreitol DOT Scientific  DSD11000-10 DTT
Gelsolin Protein: Homo Sapiens Recombinant Cytoskeleton, Inc HPG6 gelsolin
Hamilton 1750 Gastight Syringe, 500 µL, cemented needle, 22 G, 2" conical tip Cole-Parmer UX-07940-53 glass syringe
HEPES AmericanBio 7365-45-9
ImageJ/Fiji https://imagej.net/tutorials/
L-alpha-Phosphatidylcholine Avanti Polar Lipids Inc. 97281442 EPC
Magnesium chloride Sigma 7786303 MgCl2
Mineral oil, BioReagent, for molecular biology, light oil Sigma 8042475 mineral oil
N-WASP fragment WWA (aa400–501, VCA-His) VCA-His is purified using lab protocol. The protocol can be provided upon reasonable requests
Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (Oregon Green 488 DHPE) Thermo Fisher O12650 DHPE
Potassium chloride Sigma 7447407 KCl
Sucrose Sigma 57-50-1 sucrose
β-Casein from bovine milk Sigma C6905-250MG
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Tags

Actin CytoskeletonIn Vitro ReconstitutionGiant Unilamellar VesiclesLiposome YieldEncapsulation EfficiencyProtein EncapsulationMicroparticle EncapsulationSelf-propelling MicroswimmersNon-polymerization BufferPolymerization BufferEGGPCNickel Lipid

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Chen, S., Sun, Z. G., Murrell, M. P. In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles. J. Vis. Exp. (186), e64026, doi:10.3791/64026 (2022).
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