JoVE Journal > Biochemistry
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Abstract
Introduction
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Biochemistry

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

Published: May 05, 2022
doi:
10.3791/63891
, , , ,
1Université Paris Cité, CNRS,Institut Jacques Monod

Summary

我们提出了简单的肌动蛋白丝微流体测定方案，结合荧光显微镜，使人们能够实时准确地监测单个肌动蛋白丝，同时按顺序将它们暴露于不同的蛋白质溶液中。

Abstract

为了破译调节肌动蛋白细丝组装和拆卸的复杂分子机制，在良好控制的条件下监测单个反应是一项重要资产。为此，在过去的20年中出现了实时单丝实验，主要使用全内反射荧光（TIRF）显微镜，并提供了大量关键结果。2011年，为了进一步扩大这些实验的可能性并避免反复出现有问题的伪影，我们在这些测定中引入了简单的微流体。本研究详细介绍了我们的基本方案，其中单个肌动蛋白细丝由一端锚定在钝化的盖玻片表面上，与流动对齐，并且可以连续暴露于不同的蛋白质溶液中。我们还介绍了特定应用的协议，并解释了如何施加受控的机械力，这要归功于流动溶液的粘性阻力。我们强调了这些实验的技术警告，并简要介绍了基于该技术的可能发展。这些方案和解释，以及当今易于使用的微流体设备的可用性，应该允许非专业人员在其实验室中实施该测定。

Introduction

肌动蛋白长丝和肌动蛋白丝网络的组装和拆卸由几种生化反应控制，并取决于机械环境。为了深入了解这些复杂的机制，能够观察单个细丝上的单个反应（足够大的数量）是非常宝贵的。在过去的几十年中，实时观察动态肌动蛋白丝，主要使用全内反射荧光（TIRF）显微镜，已成为一项关键技术，并提供了令人印象深刻的结果列表，这些结果无法通过本体溶液生化测定1获得。

为了实现这一点，需要将荧光标记的肌动蛋白丝保持在显微镜盖玻片表面附近，同时将它们暴露于肌动蛋白结合蛋白（ABP）的溶液中，这些溶液也可以进行荧光标记。这样做提供了一种在良好控制的生化条件下监测单个细丝上发生的事件的方法，从而量化反应速率。但是，应考虑一些具体的限制。人为地保持细丝靠近表面，通常由于多个锚定点或使用拥挤剂如甲基纤维素，可以改变它们的行为（例如，导致其聚合和解聚的暂停2）。跟踪每根灯丝的轮廓可能具有挑战性，特别是如果新的灯丝或灯丝碎片随着时间的推移在视野中积聚。反应发生在有限的体积中，其中肌动蛋白单体和ABP的浓度会随时间变化，可能难以获得准确的速率常数。最后，更新或改变ABP的溶液很难在不到30秒的时间内实现，并且通常会导致样品中的蛋白质含量不均匀。

10多年前，受到已经完成的单个脱氧核糖核酸（DNA）链3的启发，我们引入了一种基于微流体的新技术来观察和操纵单个肌动蛋白丝4。它允许人们规避上述经典单丝技术的局限性。在这些微流体测定中，肌动蛋白细丝是从吸附在盖玻片上的光谱蛋白 – 肌动蛋白种子中生长的。因此，细丝仅由一端锚定在微流体室的底部，并在表面上方波动而不粘附。细丝与进料溶液的流动保持一致，从而简化了对其轮廓长度的监控，并将它们保持在可以使用TIRF的盖玻片上方的浅区域。不同的溶液同时流入腔室而不混合，并且细丝可以依次快速地暴露在它们中。

在这里，我们提出了一系列基本方案，以在实验室中建立单肌动蛋白丝微流体测定。盖玻片和微流体室可以提前（在半天内）制备，实验本身可以在不到一天的时间内完成，其中可以测试几种生化条件。

Protocol

1. 微流控室制备 选择具有多个腔室图案的SU-8主模具。典型的腔室是十字形的，有三个入口和一个出口，高20μm，宽800μm（图1）。这种母模可以从外部公司购买或在学术实验室制造（例如，Gicquel，Y.等人5）。 将胶带放在模具边缘周围。 将约50厘米长，19毫米宽的标准透明办公胶带（见 材料表）放在长凳上，粘?…

Representative Results

对于上述所有实验，荧光标记的肌动蛋白丝应清晰可见，具有良好的对比度，表明表面的背景荧光较低（图4，请参阅 补充文件1 以解决常见问题）。肌动蛋白细丝也不应粘附在表面上：当显性流速较低时，当观察它们活着时，肌动蛋白丝的横向波动应该是可感知的，并允许人们清楚地确定它们仅由其末端之一锚定。同样，当使用TIRF成像时，它们的垂直波动应该…

Discussion

与标准的单丝方法相比，肌动蛋白细丝沿其长度通过多个点锚定在表面上或通过甲基纤维素等拥挤剂保持靠近表面，微流体具有许多优点。由于与表面的相互作用很小，因此避免了在伸长和解聚过程中这些相互作用可能引起的人为停顿。灯丝按流动对齐，彼此平行，便于监控和测量长度。细丝周围的溶液不断更新，使它们暴露于恒定的蛋白质浓度。能够在细丝暴露的不同蛋白质溶液之间快速（<1?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢拉杜克斯和拉多姆。梅格实验室使用他们的紫外线清洁设备，以及J.休文和0。du Roure接受的关于在硅晶圆上准备模具的初步培训，并提供有关微流体的提示。我们感谢欧洲研究理事会拨款StG-679116（授予AJ）和国家研究基金的资助，以资助肌肉酸和顺应蛋白（授予G.R.-L.）。

Materials

β-Casein Merck C6905 Used at 8 mg/mL
Biopsy punch (with plunger) Ted Pella 15115-2 ID 0.75 mm, OD 1.07 mm
Biotin-BSA Merck A8549 Used at 1 mg/mL
BSA Merck A8022 Used at 50 mg/mL
Coverslip Mini-Rack
Teflon holder		 Invitrogen C14784 for 8 coverslips
Coverslips 22x40mm
Thickness #1.5		 Menzel Gläser 631-1370
DABCO Merck D27802 component in f-buffer
DTT Euromedex EU0006-D component in f-buffer
Ester NHS Alexa Fluor 488 Invitrogen A20000 Fluorophore for actin labeling on Lys328.
EZ-Link Sulfo-NHS-Biotin Thermo Scientific 21338 To biotinylate actin on Lys328
Hellmanex III Hellma 9-307-011-4-507 Glass cleaning detergent
ImageJ NIH N/A open source software
Laboport KNF 811kn.18 vacuum pump (ultimate vacuum: 240 mbar)
Magic invisible tape Scotch 7100024666 standard transparent office tape
Micrewtube Simport T341-6T 2 mL microfluidic reservoir tubes
Microfluidic device Part 1: Flow Unit S Fluigent FLU-S-D-PCKB Flowmeter
Microfluidic device Part 2: Fluiwell-4C-2 mL Fluigent 14002001PCK Reservoir holder
Microfluidic device Part 3: MFCS-EZ Fluigent EZ-11000001
EZ-00345001		 Pressure controller
Model 42 – UVO-Cleaner Jelight Inc. 42-220 Ultraviolet cleaner
N6-(6-Aminohexyl)-ATP-ATTO-488 Jena Bioscience NU-805-488 ATP-ATTO used to label actin
neutravidin Thermo Scientific 31000
PLL-PEG SuSoS PLL(20)-g[3.5]- PEG(2) Use at 1 mg/mL in PBS.
Polydimethylsiloxane (PDMS) Sylgard 184 Silicon Elastomer Dow Corning 1673921 Contains PDMS base and curing agent
Polyetheretherketone (PEEK) tubing Merck Z226661 “Blue” : I.D. = 0.25 mm
Safety blow gun Coilhose Pneumatics 700-S filtered air
Silicon tubing VWR 228-0701P connect PEEK to coupler
Stainless steel catheter coupler Prime Bioscience SC22/15 Inserted into PDMS inlets and outlet to connect to PEEK tubing
Thermoplastic film Sigma Aldrich PM996 Standard "parafilm"
Ultrapure ethanol VWR 64-17-5
Ultrasonic cleaning bath VWR USC200TH To accomodate 1 L beakers
Vacuum dessicator SP Bel-Art F42022-0000 to degas the PDMS or solutions
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Tags

Keywords: MicrofluidicsFluorescence MicroscopyActin FilamentsActin BundlesAssembly DynamicsActin-binding ProteinsPDMS ChamberF-bufferPressure ControlAir Bubbles
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Wioland, H., Ghasemi, F., Chikireddy, J., Romet-Lemonne, G., Jégou, A. Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles. J. Vis. Exp. (183), e63891, doi:10.3791/63891 (2022).
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