Name: the mechanics of (poro-)elastic contractile actomyosin networks as a model system of the cell cytoskeleton
Uploaded: 2023-03-10T12:45:00
Description: Watch this Scientific Journal Video about The Mechanics of Poro-Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton at JoVE.com

我们要感谢Dina Aranovich的蛋白质纯化和标记。G.L.感谢以色列科学，技术和空间部提供的Jabotinsky博士奖学金。A.B.G.感谢以色列科学基金会（赠款2101/20）和以色列国科学技术部（赠款3-17491）的财政支持。

1.玻璃表面处理及钝化：
注意：本节包括三个主要步骤（见 图1）：（i）清洁和亲水，（ii）硅烷化和（iii）表面钝化。
<ol><li>清洗和亲水<ol><li>使用食人鱼溶液清洁玻璃表面。 注意：食人鱼溶液是30%H2O 2和70%H2 SO4（材料表）的混合物。其主要目标是去除盖玻片表面的有机污染物，并暴露玻璃表面上的OH基...

<ol>
	<li>Alberts, B., 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> Genesis, Modulation, and Regeneration of Skeletal Muscle In Molecular Biology of the Cell. 4th edition. , (2002).</li><li>Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mechanics of Motor Proteins and the Cytoskeleton. , (2001).</li><li>Mogilner, A., Manhart, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+fluid+mechanics:+Coupling+cytoplasmic+flow+with+active+cytoskeletal+gel.">Intracellular fluid mechanics: Coupling cytoplasmic flow with active cytoskeletal gel.</a> Annual Review of Fluid Mechanics. 50 (1), 347-370 (2018).</li><li>Bausch, A. R., Kroy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bottom-up+approach+to+cell+mechanics.">A bottom-up approach to cell mechanics.</a> Nature Physics. 2 (4), 231-238 (2006).</li><li>Moeendarbary, 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=The+cytoplasm+of+living+cells+behaves+as+a+poroelastic+material.">The cytoplasm of living cells behaves as a poroelastic material.</a> Nature Materials. 12 (3), 253-261 (2013).</li><li>Charras, G. T., Mitchison, T. J., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+cell+hydraulics.">Animal cell hydraulics.</a> Journal of Cell Science. 122 (18), 3233-3241 (2009).</li><li>Charras, G. T., Yarrow, J. C., Horton, M. A., Mahadevan, L., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-equilibration+of+hydrostatic+pressure+in+blebbing+cells.">Non-equilibration of hydrostatic pressure in blebbing cells.</a> Nature. 435 (7040), 365-369 (2005).</li><li>Keren, K., Yam, P. T., Kinkhabwala, A., Mogilner, A., Theriot, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+fluid+flow+in+rapidly+moving+cells.">Intracellular fluid flow in rapidly moving cells.</a> Nature Cell Biology. 11 (10), 1219-1224 (2009).</li><li>Paluch, E., Piel, M., Prost, J., Bornens, M., Sykes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+actomyosin+breakage+triggers+shape+oscillations+in+cells+and+cell+fragments.">Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments.</a> Biophysical Journal. 89 (1), 724-733 (2005).</li><li>Liu, A. P., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+under+construction:+In+vitro+reconstitution+of+cellular+function.">Biology under construction: In vitro reconstitution of cellular function.</a> Nature Reviews Molecular Cell Biology. 10 (9), 644-650 (2009).</li><li>Siton-Mendelson, O., Bernheim-Groswasser, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+reconstitution+of+synthetic+cell+motility.">Toward the reconstitution of synthetic cell motility.</a> Cell Adhesion &amp; Migration. 10 (5), 461-474 (2016).</li><li>Bernheim-Groswasser, A., Wiesner, S., Golsteyn, R. M., Carlier, M. -. F., Sykes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+actin-based+motility+depend+on+surface+parameters.">The dynamics of actin-based motility depend on surface parameters.</a> Nature. 417 (6886), 308-311 (2002).</li><li>Bieling, P., 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=Force+feedback+controls+motor+activity+and+mechanical+properties+of+self-assembling+branched+actin+networks.">Force feedback controls motor activity and mechanical properties of self-assembling branched actin networks.</a> Cell. 164 (1-2), 115-127 (2016).</li><li>Carvalho, K., 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=Actin+polymerization+or+myosin+contraction:+two+ways+to+build+up+cortical+tension+for+symmetry+breaking.">Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 368 (1629), 20130005 (2013).</li><li>Dayel, M. 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=In+silico+reconstitution+of+actin-based+symmetry+breaking+and+motility.">In silico reconstitution of actin-based symmetry breaking and motility.</a> PLoS Biology. 7 (9), e1000201 (2009).</li><li>Manhart, A., 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=Quantitative+regulation+of+the+dynamic+steady+state+of+actin+networks.">Quantitative regulation of the dynamic steady state of actin networks.</a> eLife. 8, 42413 (2019).</li><li>Siton, O., 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=Cortactin+releases+the+brakes+in+actin-based+motility+by+enhancing+WASP-VCA+detachment+from+Arp2/3+branches.">Cortactin releases the brakes in actin-based motility by enhancing WASP-VCA detachment from Arp2/3 branches.</a> Current Biology. 21 (24), 2092-2097 (2011).</li><li>Pontani, L. -. 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=Reconstitution+of+an+actin+cortex+inside+a+liposome.">Reconstitution of an actin cortex inside a liposome.</a> Biophysical Journal. 96 (1), 192-198 (2009).</li><li>Ideses, Y., Sonn-Segev, A., Roichman, Y., Bernheim-Groswasser, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+II+does+it+all:+Assembly,+remodeling,+and+disassembly+of+actin+networks+are+governed+by+myosin+II+activity.">Myosin II does it all: Assembly, remodeling, and disassembly of actin networks are governed by myosin II activity.</a> Soft Matter. 9 (29), 7127-7137 (2013).</li><li>Backouche, F., Haviv, L., Groswasser, D., Bernheim-Groswasser, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+gels:+Dynamics+of+patterning+and+self-organization.">Active gels: Dynamics of patterning and self-organization.</a> Physical Biology. 3 (4), 264 (2006).</li><li>Alvarado, J., Sheinman, M., Sharma, A., MacKintosh, F. C., Koenderink, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+robustly+drive+active+gels+to+a+critically+connected+state.">Molecular motors robustly drive active gels to a critically connected state.</a> Nature Physics. 9 (9), 591-597 (2013).</li><li>Linsmeier, I., 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=Disordered+actomyosin+networks+are+sufficient+to+produce+cooperative+and+telescopic+contractility.">Disordered actomyosin networks are sufficient to produce cooperative and telescopic contractility.</a> Nature Communications. 7 (1), 12615 (2016).</li><li>Ideses, Y., 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=Spontaneous+buckling+of+contractile+poroelastic+actomyosin+sheets.">Spontaneous buckling of contractile poroelastic actomyosin sheets.</a> Nature Communications. 9 (1), 2461 (2018).</li><li>Spudich, J. A., Watt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+rabbit+skeletal+muscle+contraction:+I.+Biochemical+studies+of+the+interaction+of+the+tropomyosin-troponin+complex+with+actin+and+the+proteolytic+fragments+of+myosin.">The regulation of rabbit skeletal muscle contraction: I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin.</a> Journal of Biological Chemistry. 246 (15), 4866-4871 (1971).</li><li>Ono, S., 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=Identification+of+an+actin+binding+region+and+a+protein+kinase+C+phosphorylation+site+on+human+fascin.">Identification of an actin binding region and a protein kinase C phosphorylation site on human fascin.</a> Journal of Biological Chemistry. 272 (4), 2527-2533 (1997).</li><li>Margossian, S. S., Lowey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+myosin+and+its+subfragments+from+rabbit+skeletal+muscle.">Preparation of myosin and its subfragments from rabbit skeletal muscle.</a> In Methods in Enzymology. 85, 55-71 (1982).</li><li>Quinlan, M. E., Forkey, J. N., Goldman, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orientation+of+the+myosin+light+chain+region+by+single+molecule+total+internal+reflection+fluorescence+polarization+microscopy.">Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy.</a> Biophysical Journal. 89 (2), 1132-1142 (2005).</li><li>Cheng, N. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formula+for+the+viscosity+of+a+glycerol&#8722;water+mixture.">Formula for the viscosity of a glycerol&#8722;water mixture.</a> Industrial &amp; Engineering Chemistry Research. 47 (9), 3285-3288 (2008).</li><li>Marsh, D., Bartucci, R., Sportelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+membranes+with+grafted+polymers:+Physicochemical+aspects.">Lipid membranes with grafted polymers: Physicochemical aspects.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1615 (1-2), 33-59 (2003).</li><li>Strychalski, W., Guy, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pressure+dynamics+in+blebbing+cells.">Intracellular pressure dynamics in blebbing cells.</a> Biophysical Journal. 110 (5), 1168-1179 (2016).</li><li>Head, D. A., Levine, A. J., MacKintosh, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+regimes+of+elastic+response+and+deformation+modes+of+crosslinked+cytoskeletal+and+semiflexible+polymer+networks.">Distinct regimes of elastic response and deformation modes of crosslinked cytoskeletal and semiflexible polymer networks.</a> Physical Review E. 68 (6), 061907 (2003).</li><li>MacKintosh, F. C., Levine, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonequilibrium+mechanics+and+dynamics+of+motor-activated+gels.">Nonequilibrium mechanics and dynamics of motor-activated gels.</a> Physical Review Letters. 100 (1), 018104 (2008).</li><li>Melero, C., 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=Light-induced+molecular+adsorption+of+proteins+using+the+PRIMO+system+for+micro-patterning+to+study+cell+responses+to+extracellular+matrix+proteins.">Light-induced molecular adsorption of proteins using the PRIMO system for micro-patterning to study cell responses to extracellular matrix proteins.</a> Journal of Visualized Experiments. (152), e60092 (2019).</li><li>Bendix, P. M., 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+quantitative+analysis+of+contractility+in+active+cytoskeletal+protein+networks.">A quantitative analysis of contractility in active cytoskeletal protein networks.</a> Biophysical Journal. 94 (8), 3126-3136 (2008).</li></ol>

在这里，采用体外方法来表征多孔弹性肌动肌肽凝胶的力学，作为细胞骨架的模型系统，更一般地说，细胞质的模型系统，其已被证明表现为多孔弹性材料3,5。细胞骨架（细胞质）的流变学特征在于多孔弹性扩散常数，该常数决定了细胞内胞质液在施加机械应力后在细胞内重新分布所需的时间。已经提出细胞使用这种机制来调节它们的形状<sup cla...

活细胞具有独特的机械性能。除了被动地对施加的力做出反应的能力外，它们还能够主动产生响应外部刺激的力1。这些特征对于各种细胞过程（特别是在细胞运动期间）至关重要，主要归因于细胞骨架的机械和动态特性，尤其是肌动肌蛋白细胞骨架，它是极性肌动蛋白丝、肌球蛋白分子马达和辅助蛋白的活性凝胶。这些肌动蛋白网络表现出由肌球蛋白驱动的内在自组织和收缩特性，肌球蛋白运动蛋白交联肌动蛋白丝并在ATP水解2的推动下在网络中积极产生机械应力。
已经进行了大量的实验和理论研究来研究细胞骨架的材料性质3。普遍接受的观点是细胞骨架表现为粘弹性材料4。这意味着在短时间尺度上，细胞骨架表现为弹性材料，而在长时间尺度上，由于交联蛋白和肌球蛋白运动脱离（和重新附着），它表现为粘性流体，这允许网络动态周转。然而，在许多情况下，粘弹性模型无法描述实验结果，这更符合描述细胞骨架的图片，更一般地说，细胞质被描述为多孔弹性活性材料5,6。这些类型的材料有两个主要特征。（i）第一个主要特征是通过肌球蛋白马达驱动的收缩梯度产生穿透性细胞质（“溶剂”）流过凝胶孔，这是细胞起泡7，运动性8和细胞形状振荡9等过程的基础。这种胞质流的出现可以是局部的，用于起泡，也可以是全球性的，如细胞运动。在后一种情况下，细胞后部施加的收缩应力驱动细胞溶质液流向细胞前部，从而补充板状伪足组装所需的蛋白质库8。（ii）第二个主要特征是应力的松弛是扩散的，其特征在于有效扩散常数，这取决于凝胶弹性模量、凝胶孔隙率和溶剂粘度5。多孔弹性扩散常数决定了系统对施加应力的响应速度。扩散常数越高，应力再分布越快。反过来，这决定了细胞内胞质液在施加机械应力（无论是外部还是内部）后在细胞内重新分布所需的时间，例如肌球蛋白马达产生的主动收缩应力。因此，这些例子表明，细胞骨架和细胞质的力学是紧密耦合的，不能分开处理3。
由于细胞可以通过多种方式调节其机械性能，因此网络力学和流体流动动力学之间的相互作用仍然知之甚少。一种强大的替代方法是使用体外重组系统，该系统允许完全控制各种微观成分和系统参数，这使得这些模型系统最适合物理分析10,11。该方法已成功用于研究蛋白质组成和系统几何形状对基于肌动蛋白的运动的影响12,13,14,15,16,17,18，肌动肌蛋白网络的2D图案化19,20,21,22，以及多孔弹性肌动球蛋白凝胶的网络收缩性和流体流动动力学之间的相互作用，这是本文的重点23。
在本手稿中，基于Ideses等人的工作，讨论了可控尺寸和材料性质的收缩弹性肌动球蛋白网络的制备23。分析和量化了收缩凝胶和排出溶剂的动力学，通过这些动力学肌肉蛋白凝胶可以描述为多孔弹性活性材料。研究溶剂粘度对应力扩散率的影响进一步证实了这些网络的多孔弹性性质。提供了用于数据量化的各种缩放关系。最后，讨论了实验挑战、常见陷阱以及实验结果与细胞骨架的相关性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(3-Mercaptopropyl)trimethoxysilane</td>
			<td>Sigma-Aldrich Company</td>
			<td>175617</td>
			<td>Stored under Argon atmosphere at 4 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Bio-Lab ltd</td>
			<td>1070521</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-Fluor 488</td>
			<td>Invitrogene</td>
			<td>A10254</td>
			<td>Diluted with DMSO, stored under Argon atmosphere at -20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Alexa-Fluor 647</td>
			<td>Invitrogene</td>
			<td>A20347</td>
			<td>Diluted with DMSO, stored under Argon atmosphere at -20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma -Aldrich Company</td>
			<td>A3059</td>
			<td>Stored at 4 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma -Aldrich Company</td>
			<td>C9322</td>
			<td>The stock bottle is kept under dry atmosphere (silica gel) at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Mezel-glaser</td>
			<td>CG2222-1.5</td>
			<td>Kept in milliQ-water after the Piranha treatment and used within 3 weeks</td>
		</tr>
		<tr>
			<td>Creatine kinase</td>
			<td>Roche Life Science Products</td>
			<td>10736988001</td>
			<td>Prepared fresh in glycine buffer, kep on ice, and used within 3 days.&#160; The stock bottle is kept under dry atmosphere (silica gel) at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Creatine phosphate</td>
			<td>Roche Life Science Products</td>
			<td>10621714001</td>
			<td>When dissolved should be kept at -20 &#176;C and used within 3 months. The stock bottle is kept under Argon atmosphere and stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Roche Life Science Products</td>
			<td>10708984001</td>
			<td>When dissolved should be kept at -20 &#176;C and used within 3 months</td>
		</tr>
		<tr>
			<td>Dual view Simultaneous Imaging System&#160;</td>
			<td>Photometrics</td>
			<td>DV2-CUBE</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>MP Biomedicals</td>
			<td>195174</td>
			<td></td>
		</tr>
		<tr>
			<td>EM-CCD Camera</td>
			<td>Andor Technology Ltd</td>
			<td>DV 887</td>
			<td></td>
		</tr>
		<tr>
			<td>EM-CCD Camera</td>
			<td>Photometrics</td>
			<td>Evolve Delta</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Bio-Lab ltd</td>
			<td>525050300</td>
			<td></td>
		</tr>
		<tr>
			<td>Flourescence Lamp</td>
			<td>Rapp Optoelectronic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresbrite YG Microspheres</td>
			<td>Polysciences</td>
			<td>17151-10</td>
			<td>200 nm diameter</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>ICN Biomedicals Inc</td>
			<td>194024</td>
			<td>When dissolved should be kept at -20 &#176;C and used within 3 months.</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma-Aldrich Company</td>
			<td>G7141</td>
			<td>Kept in -20 &#176;C and used within 3 months. The stock powder is kept under Argon atmosphere and kept at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>ICN Biomedicals Inc</td>
			<td>800687</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>MP Biomedicals</td>
			<td>808822</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Sigma-Aldrich Company</td>
			<td>216763</td>
			<td>Stored at 4 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>EMD Millipore Corp.</td>
			<td>529552</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Bio-Lab ltd</td>
			<td>1368052100</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>EMD Millipore Corp.</td>
			<td>442615</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica Microsystems</td>
			<td>DMI3000</td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG-mal</td>
			<td>Nanocs</td>
			<td>PG1-ML-5k&#160;</td>
			<td>Mw = 5000 Da. Divided to small batches by weight. Stored under Argon atmosphere at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Nile red microspheres</td>
			<td>Spherotech</td>
			<td>FP-2056-2&#160;</td>
			<td>2300 nm diameter</td>
		</tr>
		<tr>
			<td>Objective (10x)</td>
			<td>Leica Germany</td>
			<td>HC PL AP0</td>
			<td>UPlanFL Numerical Aperture = 0.3</td>
		</tr>
		<tr>
			<td>Objective (2.5x)</td>
			<td>Leica Germany</td>
			<td>506304</td>
			<td>&#160;Plan-NEOFLUAR Numerical Aperture = 0.075</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>WTC Binder</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Amcor</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Buffer</td>
			<td>Sigma-Aldrich Company</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Shutter Driver</td>
			<td>Vincet Associates</td>
			<td>VMM D1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Merck</td>
			<td>1.01907.5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Elma</td>
			<td>Elmasonic P</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Carlo Erba reagents</td>
			<td>410301</td>
			<td></td>
		</tr>
		<tr>
			<td>DV2 Dual-Channel Simultaneous-Imaging System</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>MP Biomedicals</td>
			<td>819620</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-VIS Spectrophotometer</td>
			<td>Pharmacia</td>
			<td>Ultraspec 2100 pro</td>
			<td></td>
		</tr>
		<tr>
			<td>MICROMAN E</td>
			<td>Gilson</td>
			<td>FD10001</td>
			<td>1&#8211;10 uL</td>
		</tr>
		<tr>
			<td>MATLAB R2017b</td>
			<td>MathWorks</td>
			<td></td>
			<td>Data quantification&#160;</td>
		</tr>
		<tr>
			<td>MetaMorph&#160;</td>
			<td>Molecular devices</td>
			<td></td>
			<td>Control software of the optical imaging system; data quantification (particle tracking analysis, network mesh size)</td>
		</tr>
	</tbody>
</table>

the mechanics of (poro-)elastic contractile actomyosin networks as a model system of the cell cytoskeleton

细胞可以主动改变它们的形状并变得运动，这一特性取决于它们主动重组其内部结构的能力。这一特征归因于细胞骨架的机械和动态特性，特别是肌动肌蛋白细胞骨架，它是极性肌动蛋白丝、肌球蛋白马达和辅助蛋白的活性凝胶，具有内在收缩特性。通常接受的观点是细胞骨架表现为粘弹性材料。然而，该模型不能总是解释实验结果，这些结果更符合将细胞骨架描述为多孔弹性活性材料 - 嵌入细胞质的弹性网络的图片。肌球蛋白马达产生的收缩梯度驱动细胞质在凝胶孔中的流动，这推断细胞骨架和细胞质的力学是紧密耦合的。多孔弹性的一个主要特征是网络中应力的扩散松弛，其特征在于有效扩散常数取决于凝胶弹性模量、孔隙率和细胞质（溶剂）粘度。由于细胞有许多方法来调节其结构和材料特性，我们目前对细胞骨架力学和细胞质流动动力学如何耦合的理解仍然知之甚少。在这里，采用 体外 重建方法来表征多孔弹性肌动球蛋白凝胶的材料特性，作为细胞骨架的模型系统。凝胶收缩由肌球蛋白运动收缩性驱动，这导致渗透溶剂流动的出现。本文描述了如何制备这些凝胶并进行实验。我们还讨论了如何在局部和全球范围内测量和分析溶剂流动和凝胶收缩。给出了用于数据量化的各种缩放关系。最后，讨论了实验挑战和常见陷阱，包括它们与细胞骨架力学的相关性。

每个实验使用两个玻璃盖玻片。玻璃盖玻片经过清洁并用PEG聚合物钝化。钝化对于防止溶解的蛋白质在早期实验阶段粘附在玻璃表面上以及最大限度地减少收缩网络与玻璃壁的相互作用至关重要。未能实现良好的钝化会导致低效收缩，在极端情况下，甚至会抑制肌动蛋白网络的形成。
图1描述了表面处理程序的三个主要步骤。这些步骤包括以下内容：（i...

Watch this Scientific Journal Video about The Mechanics of Poro-Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton at JoVE.com

The Mechanics of Poro-Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

本工作采用 体外 重构方法研究动粒肌肽凝胶在受控条件下的多孔弹性。量化了肌动球蛋白凝胶和包埋溶剂的动力学，通过这些动力学证明了网络多孔弹性。我们还讨论了实验挑战，常见陷阱以及与细胞骨架力学的相关性。

（poro-）弹性收缩肌动球蛋白网络作为细胞骨架模型系统的机制

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This ...

我们已经开发了一种体外方法来表征多孔弹性肌动肌肽凝胶的力学，作为细胞骨架的模型系统，更一般地说，细胞质已被证明表现为多孔弹性活性材料。该方法提供了肌球蛋白收缩性如何促进流体流动的出现以及网络和细胞质速度如何在时间和空间上相关的见解。这种简单的方法使我们能够提取动态演变系统的机械性能，而无需使用任何无法使用AFIM等传统工具的复杂设备或技术。该方法可以深入了解多孔弹性活性材料的流变学，而不管活性凝胶和包埋流体的特异性如何。首先，将 10 到 12 个 1.5 号玻璃盖玻片放入自制的聚四氟乙烯支架中，并在 400 毫升烧杯中制备食人鱼溶液。在化学罩中的冰上制备溶液。将支架转移到装满食人鱼的烧杯中。三个小时后，将支架转移到装有新鲜双蒸水的干净烧杯中，从盖玻片上洗涤多余的食人鱼，然后将烧杯转移到 80 赫兹的超声浴中，并在 25 摄氏度下全功率 10 分钟。用新鲜的双蒸水再重复此步骤两次。对于盐碱化，将支架转移到装有300毫升纯甲醇的干净烧杯中，然后放入超声浴中30分钟。然后将支架转移到硅烷溶液中。用封口膜密封烧杯，并将其保存在冰箱中，温度为四摄氏度过夜。第二天，将支架转移到装有 300 毫升纯甲醇的干净烧杯中 15 秒。干燥支架底部以去除多余的甲醇并将其转移到干净的烧杯中。然后将烧杯转移到烤箱中，在 120 摄氏度下将玻璃盖玻片干燥五分钟。为了实现表面钝化，每个实验取两个盖玻片，并将它们放置在涂有最初用乙醇清洁的封口膜层的培养皿中。将每个盖玻片与一毫升甲氧基聚乙二醇马来酰亚胺在 1X PBS 中在 22 摄氏度下孵育一小时。在孵育过程结束时，倾斜培养皿以取出并钉住。用五毫升双蒸水冲洗每个盖玻片，并用氮气流干燥。由于盖玻片在钝化后必须保持湿润，因此立即将一毫升10毫摩尔Tris放在聚乙二醇化表面上，并在两小时内使用盖玻片。为了形成大的肌球蛋白聚集体，用10毫摩尔Tris稀释肌球蛋白储备溶液，以达到25毫摩尔氯化钾的最终浓度。将稀释的肌球蛋白溶液在室温下保持10分钟，然后将其转移到冰上直至使用。电机完全启动长达两个小时。为了跟踪溶剂流过凝胶孔，制备钝化荧光珠，并通过将1微升珠子与5微摩尔G-肌动蛋白孵育20分钟来减少磁珠与肌动蛋白网络之间的相互作用。通过离心去除多余的G-肌动蛋白。用每毫升10毫克的牛血清白蛋白重复此步骤，以阻止剩余的未包被表面，并在实验中以1至10， 000的最终稀释度使用珠子。要制备肌动肌肽溶液，解冻储存在零下80摄氏度的蛋白质等分试样并将其放在冰上。然后制备溶液，达到终浓度为10毫摩尔Tris，5微摩尔G-肌动蛋白，280纳摩尔GST筋膜和1.67纳摩尔肌球蛋白II.取聚乙二醇钝化盖玻片中的一种。将涂有油脂的副成型垫片放在其顶部，然后将其放入样品架中。将1.1微升肌动肌肽溶液加入支架安装的盖玻片中，并将第二个盖玻片放在顶部。拧紧支架以密封样品。将样品架放在显微镜上并开始采集。提前准备显微镜以减少初始采集时间。要使用倒置荧光显微镜对样品进行成像，请使用2.5倍物镜的低放大倍率可视化整个凝胶区域，并随时间跟踪其宏观收缩。使用10倍物镜表征网络孔隙度和结构，并跟踪网络自组织和随时间的收缩。对于同时进行双色成像，以488纳米激发肌动蛋白，在561纳米激发肌球蛋白II马达，并使用双发射装置在EMCCD相机上同时记录图像。使用相同的成像系统同时对肌动蛋白凝胶和荧光肌球蛋白II马达进行成像。对于同时双色成像，在488纳米处激发肌动蛋白，在561纳米处激发2， 300纳米磁珠。并使用双发射设备在EMCCD相机上同时记录图像。使用相同的成像系统同时对肌动蛋白凝胶和荧光珠进行成像。第一天涉及证明肌动球蛋白网络表现为弹性材料。此处描述了肌动球蛋白网络的示意图。荧光显微镜图像表明，肌动蛋白丝自发成核并聚合成各向同性的互连网络，该网络随时间变粗并最终在宏观上收缩。网络吸引在凝胶外围启动并向内传播到凝胶块中。凝胶中心用C标记，肌动蛋白丝束在网络收缩期间保持笔直。此处显示了 T316 秒和 T327 秒处等高线长度与端到端距离之间的比率分布。第二个目的是证明向外的溶剂流动是由肌球蛋白收缩力产生的。此处显示了收缩中间阶段低放大倍率下的凝胶图像。这些圆圈标记了四个珠子的位置。箭头标记珠子运动的全局方向。这里描绘了九颗选定珠子的轨迹。这里描述了在收缩的高级阶段解析网络孔隙率和溶剂在凝胶孔中的移动。第三个目标是证明应力松弛的特征在于有效的多孔弹性扩散常数。此处显示了收缩行动球蛋白凝胶在低放大倍率下从混合时间到稳态的俯视图荧光图像。肌动蛋白网络表现为多孔弹性活性材料。在尝试此过程时，请快速准确地工作，并牢记表面保存的重要性。

Research

JoVE Journal

Bioengineering

The Mechanics of (Poro-)Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

科研

生物工程

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

癌症细胞球体机器人生产与药物测试的双水相系统

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

用人工晶状体担架模拟透镜体的力学性能

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...