Name: using microfluidics and fluorescence microscopy to study the assembly dynamics of single actin filaments and bundles
Uploaded: 2022-05-05T15:00:00
Description: Watch this Scientific Journal Video about Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles at JoVE.com

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.）。

1. 微流控室制备
<ol><li>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）。</li>
	<li>将胶带放在模具边缘周围。<ol>
<li>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?...

<ol>
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(134), e57133 (2018).</li><li>Chandradoss, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> Journal of Visualized Experiments: JoVE. (86), e50549 (2014).</li><li>Schaedel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+self-repair+in+response+to+mechanical+stress.">Microtubules self-repair in response to mechanical stress.</a> Nature Materials. 14 (11), 1156-1163 (2015).</li><li>Zimmermann, D., Morganthaler, A. N., Kovar, D. R., Suarez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+biochemical+characterization+of+cytokinesis+actin-binding+proteins.">In vitro biochemical characterization of cytokinesis actin-binding proteins.</a> Methods in Molecular Biology. 1369, 151-179 (2016).</li><li>Funk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profilin+and+formin+constitute+a+pacemaker+system+for+robust+actin+filament+growth.">Profilin and formin constitute a pacemaker system for robust actin filament growth.</a> eLife. 8, 50963 (2019).</li><li>Pandit, N. 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F., Romet-Lemonne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formin+mDia1+senses+and+generates+mechanical+forces+on+actin+filaments.">Formin mDia1 senses and generates mechanical forces on actin filaments.</a> Nature Communications. 4, 1883 (2013).</li><li>Breitsprecher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rocket+launcher+mechanism+of+collaborative+actin+assembly+defined+by+single-molecule+imaging.">Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging.</a> Science. 336 (6085), 1164-1168 (2012).</li><li>Courtemanche, N., Lee, J. Y., Pollard, T. D., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+modulates+actin+filament+polymerization+mediated+by+formin+and+profilin.">Tension modulates actin filament polymerization mediated by formin and profilin.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (24), 9752-9757 (2013).</li><li>Niedermayer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+depolymerization+of+actin+filaments+is+caused+by+photo-induced+dimerization+of+actin+protomers.">Intermittent depolymerization of actin filaments is caused by photo-induced dimerization of actin protomers.</a> Proceedings of the National Academy of Sciences. 109 (27), 10769-10774 (2012).</li><li>Gateva, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropomyosin+isoforms+specify+functionally+distinct+actin+filament+populations+in+vitro.">Tropomyosin isoforms specify functionally distinct actin filament populations in vitro.</a> Current Biology: CB. 27 (5), 705-713 (2017).</li><li>Aratyn, Y. S., Schaus, T. E., Taylor, E. W., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+dynamic+behavior+of+fascin+in+filopodia.">Intrinsic dynamic behavior of fascin in filopodia.</a> Molecular Biology of the Cell. 18 (10), 3928-3940 (2007).</li><li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate+constants+for+the+reactions+of+ATP-+and+ADP-actin+with+the+ends+of+actin+filaments.">Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments.</a> The Journal of Cell Biology. 103, 2747-2754 (1986).</li><li>Wioland, H., Jegou, A., Romet-Lemonne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torsional+stress+generated+by+ADF/cofilin+on+cross-linked+actin+filaments+boosts+their+severing.">Torsional stress generated by ADF/cofilin on cross-linked actin filaments boosts their severing.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (7), 2595-2602 (2019).</li><li>Colombo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+family+of+fluorescent+nucleotide+analogues+to+investigate+actin+dynamics+and+energetics.">A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics.</a> Nature Communications. 12 (1), 548 (2021).</li><li>Spudich, J. 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Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin.</a> The Journal of Biological Chemistry. 246 (15), 4866-4871 (1971).</li><li>Romet-Lemonne, G., Guichard, B., J&#233;gou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microfluidics+single+filament+assay+to+study+formin+control+of+actin+assembly.">Using microfluidics single filament assay to study formin control of actin assembly.</a> Methods in Molecular Biology. 1805, 75-92 (2018).</li><li>Gieselmann, R., Kwiatkowski, D. J., Janmey, P. A., Witke, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+biochemical+characteristics+of+the+two+human+profilin+isoforms.">Distinct biochemical characteristics of the two human profilin isoforms.</a> European Journal of Biochemistry. 229 (3), 621-628 (1995).</li><li>Lin, D. C., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization+induced+by+a+motility-related+high-affinity+cytochalasin+binding+complex+from+human+erythrocyte+membrane.">Actin polymerization induced by a motility-related high-affinity cytochalasin binding complex from human erythrocyte membrane.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (5), 2345-2349 (1979).</li><li>Casella, J. F., Maack, D. J., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+initial+characterization+of+a+protein+from+skeletal+muscle+that+caps+the+barbed+ends+of+actin+filaments.">Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments.</a> The Journal of Biological Chemistry. 261 (23), 10915-10921 (1986).</li><li>Kremneva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin-2+controls+actin+filament+length+in+muscle+sarcomeres.">Cofilin-2 controls actin filament length in muscle sarcomeres.</a> Developmental Cell. 31 (2), 215-226 (2014).</li><li>Le Clainche, C., Carlier, M. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-based+motility+assay.">Actin-based motility assay.</a> Current Protocols in Cell Biology. , 1-20 (2004).</li><li>Vignjevic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+filopodia-like+bundles+in+vitro+from+a+dendritic+network.">Formation of filopodia-like bundles in vitro from a dendritic network.</a> The Journal of Cell Biology. 160 (6), 951-962 (2003).</li><li>Duellberg, C., Cade, N. I., Holmes, D., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+size+of+the+EB+cap+determines+instantaneous+microtubule+stability.">The size of the EB cap determines instantaneous microtubule stability.</a> eLife. 5, 13470 (2016).</li><li>Duellberg, C., Cade, N. I., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+aging+probed+by+microfluidics-assisted+tubulin+washout.">Microtubule aging probed by microfluidics-assisted tubulin washout.</a> Molecular Biology of the Cell. 27 (22), 3563-3573 (2016).</li><li>Suzuki, E. 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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-Casein</td>
			<td>Merck</td>
			<td>C6905</td>
			<td>Used at 8 mg/mL</td>
		</tr>
		<tr>
			<td>Biopsy punch (with plunger)</td>
			<td>Ted Pella</td>
			<td>15115-2</td>
			<td>ID 0.75 mm, OD 1.07 mm</td>
		</tr>
		<tr>
			<td>Biotin-BSA</td>
			<td>Merck</td>
			<td>A8549</td>
			<td>Used at 1 mg/mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Merck</td>
			<td>A8022</td>
			<td>Used at 50 mg/mL</td>
		</tr>
		<tr>
			<td>Coverslip Mini-Rack 
			Teflon holder</td>
			<td>Invitrogen</td>
			<td>C14784</td>
			<td>for 8 coverslips</td>
		</tr>
		<tr>
			<td>Coverslips 22x40mm 
			Thickness #1.5</td>
			<td>Menzel Gl&#228;ser</td>
			<td>631-1370</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Merck</td>
			<td>D27802</td>
			<td>component in f-buffer</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Euromedex</td>
			<td>EU0006-D</td>
			<td>component in f-buffer</td>
		</tr>
		<tr>
			<td>Ester NHS Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A20000</td>
			<td>Fluorophore for actin labeling on Lys328.</td>
		</tr>
		<tr>
			<td>EZ-Link Sulfo-NHS-Biotin</td>
			<td>Thermo Scientific</td>
			<td>21338</td>
			<td>To biotinylate actin on Lys328</td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Hellma</td>
			<td>9-307-011-4-507</td>
			<td>Glass cleaning detergent</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>N/A</td>
			<td>open source software</td>
		</tr>
		<tr>
			<td>Laboport</td>
			<td>KNF</td>
			<td>811kn.18</td>
			<td>vacuum pump (ultimate vacuum: 240 mbar)</td>
		</tr>
		<tr>
			<td>Magic invisible tape</td>
			<td>Scotch</td>
			<td>7100024666</td>
			<td>standard transparent office tape</td>
		</tr>
		<tr>
			<td>Micrewtube</td>
			<td>Simport</td>
			<td>T341-6T</td>
			<td>2 mL microfluidic reservoir tubes</td>
		</tr>
		<tr>
			<td>Microfluidic device Part 1: Flow Unit S</td>
			<td>Fluigent</td>
			<td>FLU-S-D-PCKB</td>
			<td>Flowmeter</td>
		</tr>
		<tr>
			<td>Microfluidic device Part 2: Fluiwell-4C-2 mL</td>
			<td>Fluigent</td>
			<td>14002001PCK</td>
			<td>Reservoir holder</td>
		</tr>
		<tr>
			<td>Microfluidic device Part 3: MFCS-EZ</td>
			<td>Fluigent</td>
			<td>EZ-11000001 
			EZ-00345001</td>
			<td>Pressure controller</td>
		</tr>
		<tr>
			<td>Model 42 - UVO-Cleaner</td>
			<td>Jelight Inc.</td>
			<td>42-220</td>
			<td>Ultraviolet cleaner</td>
		</tr>
		<tr>
			<td>N6-(6-Aminohexyl)-ATP-ATTO-488</td>
			<td>Jena Bioscience</td>
			<td>NU-805-488</td>
			<td>ATP-ATTO used to label actin</td>
		</tr>
		<tr>
			<td>neutravidin</td>
			<td>Thermo Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>PLL-PEG</td>
			<td>SuSoS</td>
			<td>PLL(20)-g[3.5]- PEG(2)</td>
			<td>Use at 1 mg/mL in PBS.</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) Sylgard 184 Silicon Elastomer</td>
			<td>Dow Corning</td>
			<td>1673921</td>
			<td>Contains PDMS base and curing agent</td>
		</tr>
		<tr>
			<td>Polyetheretherketone (PEEK) tubing</td>
			<td>Merck</td>
			<td>Z226661</td>
			<td>&#8220;Blue&#8221; : I.D. = 0.25 mm</td>
		</tr>
		<tr>
			<td>Safety blow gun</td>
			<td>Coilhose Pneumatics</td>
			<td>700-S</td>
			<td>filtered air</td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>VWR</td>
			<td>228-0701P</td>
			<td>connect PEEK to coupler</td>
		</tr>
		<tr>
			<td>Stainless steel catheter coupler</td>
			<td>Prime Bioscience</td>
			<td>SC22/15</td>
			<td>Inserted into PDMS inlets and outlet to connect to PEEK tubing</td>
		</tr>
		<tr>
			<td>Thermoplastic film</td>
			<td>Sigma Aldrich</td>
			<td>PM996</td>
			<td>Standard &#34;parafilm&#34;</td>
		</tr>
		<tr>
			<td>Ultrapure ethanol</td>
			<td>VWR</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaning bath</td>
			<td>VWR</td>
			<td>USC200TH</td>
			<td>To accomodate 1 L beakers</td>
		</tr>
		<tr>
			<td>Vacuum dessicator</td>
			<td>SP Bel-Art</td>
			<td>F42022-0000</td>
			<td>to degas the PDMS or solutions</td>
		</tr>
	</tbody>
</table>

using microfluidics and fluorescence microscopy to study the assembly dynamics of single actin filaments and bundles

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。

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...

Watch this Scientific Journal Video about Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles at JoVE.com

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

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。

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

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

我们将荧光显微镜与微流体相结合，研究肌动蛋白结合蛋白如何调节肌动蛋白丝的组装和拆卸。通过微流体，我们能够同时量化数十个单个肌动蛋白丝上发生的反应，同时精确控制生化和机械条件。请记住，溶液首先平行注射，直到它们到达腔室，然后通过改变压力设置将细丝依次暴露于每个溶液中。要开始制备聚二甲基硅氧烷或PDMS，腔室组装，将热板预热至100摄氏度。在深层紫外线清洁剂中，将清洁培养皿中的一个清洁的PDMS室和一个玻璃盖玻片暴露在紫外线下三到五分钟。完成后，将PDMS腔室放在盖玻片上，使直接暴露在紫外线下的两个表面接触。要去除困在 PDMS 盖玻片接口处的气泡，请用手指轻轻按压腔室表面。用力按压角落和侧面，确保腔室的天花板不会与玻璃表面接触。将玻璃底朝向热板的腔室置于100摄氏度下五分钟。然后立即使用该室，或将其存放在干净的培养皿中一周。要冲洗入口和出口管，请用300微升F缓冲液填充一个2毫升的储液管，将其拧在微流体支架上，并将压力设置为300毫巴。让五到八滴F缓冲液被浪费，然后将压力设置为零并重复每个通道的过程。接下来，设置4至50毫巴的储液管出口压力，一旦液滴从油管尖端出来，将油管连接到PDMS室的出口。当液体充满腔室并从所有入口流出时，将出口压力设置为20毫巴。之后，将储液管的压力设置为 1 至 50 毫巴。为避免引入气泡，请确保在将管道连接到入口之前，有液滴从管道和 PDMS 入口中流出。将入口压力设置为 30 毫巴。如图所示连接入口 2 和 3 后，将所有入口的压力设置为 20 毫巴，将出口压力设置为零毫巴。确保入口处的流速大致相等。更换储液罐时，将所有入口压力设置为 12 毫巴，将出口压力设置为 5 毫巴。为了快速注入溶液，请将相应入口的压力设置为150毫巴，并将其他入口中的压力调节到100毫巴左右，使得入口处的流速为每分钟500纳升。要更换溶液，请在新的储液管中填充200至300微升的溶液，并将压力设置为变化设置。然后，拧下入口的储液管。一旦在管尖上形成一个小液滴，用新鲜的溶液拧入新管，将压力设置更改为高流量。根据微流体配置和腔室几何形状，等待三到五分钟，让溶液填充管子并到达腔室。该过程之后可以测量荧光随时间的增加。对于表面官能化，在F-缓冲液中将溶液3至200微升50皮摩尔光谱蛋白肌动蛋白种子，并以高流量三将溶液注入两分钟。对于表面钝化，在F缓冲液中用300微升的5%BSA更换管三，然后在高流量三处注入五分钟，然后在中流三处注入五分钟，在通道1和2中减压7至8毫巴，以获得每分钟100纳升的逆流。整个腔室表面将被BSA钝化。将试管三改为F缓冲液，在高流量三下冲洗通道五分钟。之后，如手稿中所述，分别用一个微摩尔肌动蛋白profilin，0.15微摩尔肌动蛋白和F缓冲液更换一至三个管，并使用高流量全部预设注入新鲜制备的溶液三至四分钟。打开显微镜并将相机曝光时间调整为100至200毫秒，激发激光功率至10至20%，全内反射荧光或TIRF深度为200至300纳米，以使用60X物镜捕获图像。接下来，将压力设置为中流一，持续10分钟，以每20秒一帧的速率记录长丝聚合，然后以中流二速记录15分钟，用于长丝老化。在落射荧光模式下，每五秒钟一帧，一到两帧后，切换到中流三，以记录以每秒约10个亚基解聚的细丝。为了重置实验，通过将荧光标记的灯丝以最大功率连续暴露在激光下两分钟来打破所有荧光标记的灯丝。通过更换溶液一，二或三并以高流量注入三到四分钟来测试不同的条件。这里给出了基本聚合/解聚实验的结果。由此产生的缩模照片用于量化聚合和解聚速率。当生长的肌动蛋白细丝暴露于荧光标记的科菲林溶液中时，科菲林簇成核并朝向尖头和带刺的末端生长。当单个细丝暴露于迷彩时，它们会形成看起来更亮的束，并且不完全与流动对齐。在更换储液管时，避免在系统中引入气泡非常重要，并且注意不要让储油管变空。该技术对于在数十根单丝上施加拉力，查看formin和cofilin机械灵敏度以及ARP2 /3去支散至关重要。

Research

JoVE Journal

Biochemistry

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

科研

生物化学

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

使用扩张显微镜对波多细胞蛋白尼腓林、阿克汀和波多辛的成像

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...