Name: imaging integrin tension and cellular force at submicron resolution with an integrative tension sensor
Uploaded: 2019-04-25T15:00:00
Description: Watch this Scientific Journal Video about Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor at JoVE.com

这项工作得到了爱荷华州立大学和国家普通医学研究所 (R35GM128747) 提供的启动基金的支持。

这里描述的所有方法都得到了爱荷华州立大学动物护理和使用机构委员会 (IACUC, 8-16-8331-i) 的批准。
1. 集成张力传感器的合成
<ol><li>自定义和订购 ssDNAs (请参阅材料表)。 注: ssDNA 序列如下所示。上链是/5Thiomc6-dgg AGG acg cg GCC/3BHQ_2/。较低的股如下所示。 12 pn its:5 cycc CCG CGT CCT CCT c/3 bi/3 23 PN IT:5 cycc CCG CGT CCC/BIODT/CCC 33 PN IT:5 cycc CCG CTG Cg/biodt/cct CCC 43 pn its:5 ...

<ol>
	<li>Price, L. S., Leng, J., Schwartz, M. A., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Rac+and+Cdc42+by+Integrins+Mediates+Cell+Spreading.">Activation of Rac and Cdc42 by Integrins Mediates Cell Spreading.</a> Molecular Biology of the Cell. 9 (7), 1863-1871 (1998).</li><li>Cavalcanti-Adam, E. 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=Cell+Spreading+and+Focal+Adhesion+Dynamics+Are+Regulated+by+Spacing+of+Integrin+Ligands.">Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands.</a> Biophysical Journal. 92 (8), 2964-2974 (2007).</li><li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+in+cell+migration.">Integrins in cell migration.</a> Cold Spring Harbor Perspectives in Biology. 3 (9), a005074 (2011).</li><li>Hood, J. D., Cheresh, 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=Role+of+integrins+in+cell+invasion+and+migration.">Role of integrins in cell invasion and migration.</a> Nature Reviews Cancer. 2 (2), 91-100 (2002).</li><li>Giancotti, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+signaling:+specificity+and+control+of+cell+survival+and+cell+cycle+progression.">Integrin signaling: specificity and control of cell survival and cell cycle progression.</a> Current Opinion in Cell Biology. 9 (5), 691-700 (1997).</li><li>Illario, 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=Integrin-Dependent+Cell+Growth+and+Survival+Are+Mediated+by+Different+Signals+in+Thyroid+Cells.">Integrin-Dependent Cell Growth and Survival Are Mediated by Different Signals in Thyroid Cells.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 88 (1), 260-269 (2003).</li><li>Aoudjit, F., Vuori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+Signaling+in+Cancer+Cell+Survival+and+Chemoresistance.">Integrin Signaling in Cancer Cell Survival and Chemoresistance.</a> Chemotherapy Research and Practice. 2012, 1-16 (2012).</li><li>Hou, 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=Distinct+effects+of+&#946;1+integrin+on+cell+proliferation+and+cellular+signaling+in+MDA-MB-231+breast+cancer+cells.">Distinct effects of &#946;1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells.</a> Scientific Reports. 6, 18430 (2016).</li><li>Shankar, G., Davison, I., Helfrich, M. H., Mason, W. T., Horton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+receptor-mediated+mobilisation+of+intranuclear+calcium+in+rat+osteoclasts.">Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts.</a> Journal of Cell Science. 105 (Pt 1) (1), 61-68 (1993).</li><li>Moreno-Layseca, P., Streuli, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+pathways+linking+integrins+with+cell+cycle+progression.">Signalling pathways linking integrins with cell cycle progression.</a> Matrix Biology. 34, 144-153 (2014).</li><li>G&#243;mez-Lamarca, M. J., Cobreros-Reguera, L., Ib&#225;&#241;ez-Jim&#233;nez, B., Palacios, I. M., Mart&#237;n-Bermudo, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+regulate+epithelial+cell+differentiation+by+modulating+Notch+activity.">Integrins regulate epithelial cell differentiation by modulating Notch activity.</a> Journal of Cell Science. 127 (Pt 1), 4667-4678 (2014).</li><li>Wang, H., Luo, X., Leighton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+and+Integrins+in+Embryonic+Stem+Cell+Differentiation.">Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation.</a> Biochemistry Insights. 8 (Suppl 1), 15-21 (2015).</li><li>Schwarz, U. S., Soin&#233;, J. 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=Traction+force+microscopy+on+soft+elastic+substrates:+A+guide+to+recent+computational+advances.">Traction force microscopy on soft elastic substrates: A guide to recent computational advances.</a> Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1853 (11), 3095-3104 (2015).</li><li>Xie, T., Hawkins, J., Sun, Y., Ritti&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+Force+Measurement+Using+Deformable+Microposts.">Traction Force Measurement Using Deformable Microposts.</a> Fibrosis. Methods and Protocols. , 235-244 (2017).</li><li>Radmacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+Mechanics+of+Cellular+Processes+by+Atomic+Force+Microscopy.">Studying the Mechanics of Cellular Processes by Atomic Force Microscopy.</a> Methods in Cell Biology. 83, 347-372 (2007).</li><li>Charras, G. T., Lehenkari, P. P., Horton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+can+be+used+to+mechanically+stimulate+osteoblasts+and+evaluate+cellular+strain+distributions.">Atomic force microscopy can be used to mechanically stimulate osteoblasts and evaluate cellular strain distributions.</a> Ultramicroscopy. 86 (1-2), 85-95 (2001).</li><li>Miller, J. 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=Bioactive+hydrogels+made+from+step-growth+derived+PEG-peptide+macromers.">Bioactive hydrogels made from step-growth derived PEG-peptide macromers.</a> Biomaterials. 31 (13), 3736-3743 (2010).</li><li>Legant, W. R., Miller, J. S., Blakely, B. L., Cohen, D. M., Genin, G. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mechanical+tractions+exerted+by+cells+within+three-dimensional+matrices.">Measurement of mechanical tractions exerted by cells within three-dimensional matrices.</a> Nature Methods. 7 (12), 969 (2010).</li><li>Brenner, M. 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=Spider+Silk+Peptide+Is+a+Compact,+Linear+Nanospring+Ideal+for+Intracellular+Tension+Sensing.">Spider Silk Peptide Is a Compact, Linear Nanospring Ideal for Intracellular Tension Sensing.</a> Nano Letters. 16 (3), 2096-2102 (2016).</li><li>Zhang, Y., Ge, C., Zhu, C., Salaita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-based+digital+tension+probes+reveal+integrin+forces+during+early+cell+adhesion.">DNA-based digital tension probes reveal integrin forces during early cell adhesion.</a> Nature Communications. 5, 5167 (2014).</li><li>Liu, 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=DNA-based+nanoparticle+tension+sensors+reveal+that+T-cell+receptors+transmit+defined+pN+forces+to+their+antigens+for+enhanced+fidelity.">DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (20), 5610-5615 (2016).</li><li>Wang, X., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+Single+Molecular+Forces+Required+to+Activate+Integrin+and+Notch+Signaling.">Defining Single Molecular Forces Required to Activate Integrin and Notch Signaling.</a> Science. 340 (6135), (2013).</li><li>Blakely, B. 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=A+DNA-based+molecular+probe+for+optically+reporting+cellular+traction+forces.">A DNA-based molecular probe for optically reporting cellular traction forces.</a> Nature Methods. 11 (12), 1229-1232 (2014).</li><li>Wang, Y., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+outside+focal+adhesions+transmit+tensions+during+stable+cell+adhesion.">Integrins outside focal adhesions transmit tensions during stable cell adhesion.</a> Scientific Reports. 6 (1), 36959 (2016).</li><li>Wang, 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=Force-activatable+biosensor+enables+single+platelet+force+mapping+directly+by+fluorescence+imaging.">Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.</a> Biosensors and Bioelectronics. 100, 192-200 (2018).</li><li>Zhao, Y., Wang, Y., Sarkar, A., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratocytes+Generate+High+Integrin+Tension+at+the+Trailing+Edge+to+Mediate+Rear+De-adhesion+during+Rapid+Cell+Migration.">Keratocytes Generate High Integrin Tension at the Trailing Edge to Mediate Rear De-adhesion during Rapid Cell Migration.</a> iScience. 9, 502-512 (2018).</li><li>Crisalli, P., Kool, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Path+Quenchers:+Efficient+Quenching+of+Common+Fluorophores.">Multi-Path Quenchers: Efficient Quenching of Common Fluorophores.</a> Bioconjugate Chemistry. 22 (11), 2345-2354 (2011).</li><li>Mondal, G., Barui, S., Chaudhuri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+the+cyclic-RGDfK+ligand+and+&#945;v&#946;3+integrin+receptor.">The relationship between the cyclic-RGDfK ligand and &#945;v&#946;3 integrin receptor.</a> Biomaterials. 34 (26), 6249-6260 (2013).</li><li>Wang, X., 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=Integrin+Molecular+Tension+within+Motile+Focal+Adhesions.">Integrin Molecular Tension within Motile Focal Adhesions.</a> Biophysical Journal. 109 (11), 2259-2267 (2015).</li><li>Euteneuer, U., Schliwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent,+directional+motility+of+cells+and+cytoplasmic+fragments+in+the+absence+of+microtubules.">Persistent, directional motility of cells and cytoplasmic fragments in the absence of microtubules.</a> Nature. 310 (5972), 58-61 (1984).</li><li>Kucik, D. F., Elson, E. L., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+does+not+produce+membrane+flow.">Cell migration does not produce membrane flow.</a> The Journal of Cell Biology. 111 (4), 1617-1622 (1990).</li><li>Mueller, 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=Load+Adaptation+of+Lamellipodial+Actin+Networks.">Load Adaptation of Lamellipodial Actin Networks.</a> Cell. , (2017).</li><li>Sarkar, A., Zhao, Y., Wang, Y., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-activatable+coating+enables+high-resolution+cellular+force+imaging+directly+on+regular+cell+culture+surfaces.">Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.</a> Physical Biology. 15 (6), 065002 (2018).</li><li>Mosayebi, M., Louis, A. A., Doye, J. P. K., Ouldridge, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-Induced+Rupture+of+a+DNA+Duplex:+From+Fundamentals+to+Force+Sensors.">Force-Induced Rupture of a DNA Duplex: From Fundamentals to Force Sensors.</a> ACS Nano. 9 (12), 11993-12003 (2015).</li><li>Bockelmann, U., Essevaz-Roulet, B., Heslot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Stick-Slip+Motion+Revealed+by+Opening+DNA+with+Piconewton+Forces.">Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces.</a> Physical Review Letters. 79 (22), 4489-4492 (1997).</li><li>Krautbauer, R., Rief, M., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unzipping+DNA+oligomers.">Unzipping DNA oligomers.</a> Nano Letters. 3 (4), 493-496 (2003).</li><li>de Gennes, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+pull+out+force+on+DNA+hybrids.">Maximum pull out force on DNA hybrids.</a> Comptes Rendus de l&#8217;Acad&#233;mie des Sciences - Series IV - Physics. 2 (10), 1505-1508 (2001).</li><li>Hatch, K., Danilowicz, C., Coljee, V., Prentiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+that+the+shear+force+required+to+separate+short+double-stranded+DNA+does+not+increase+significantly+with+sequence+length+for+sequences+longer+than+25+base+pairs.">Demonstration that the shear force required to separate short double-stranded DNA does not increase significantly with sequence length for sequences longer than 25 base pairs.</a> Physical Review E. 78 (1), 011920 (2008).</li></ol>

ITS 是一种高度可访问但功能强大的细胞力映射技术, 在合成和应用方面都是有效的。在所有材料准备好的情况下, ITS 可以在1天内合成。在实验过程中, 在细胞电镀之前只需要三个步骤的表面涂层。最近, 我们进一步简化了涂层程序, 将 ITS 与牛血清白蛋白直接连接, 从而使 ITS 能够直接物理吸附到玻璃或聚苯乙烯表面 33.ITS 将细胞力信号的荧光强度提升到可比的细胞结构成像水平。?...

细胞依靠整合素来粘附和施加细胞外基质的细胞力。整合素介导的细胞粘附和力传递是细胞扩散1,2、迁移3,4 和生存5, 6,7的关键。从长期来看, 整合素生物力学信号也会影响细胞增殖8、9、10 和分化11、12.研究人员已经开发出了各种方法来测量和映射细胞矩阵界面上的整合蛋白传递的细胞力。这些方法是基于弹性底层13, 阵列的微柱 14, 或原子力显微镜 (afm)15,16。弹性底层和微柱方法依靠基板的变形来报告细胞应力, 在空间分辨率和力敏感性方面存在局限性。AFM 具有较高的力灵敏度, 但不能同时检测多个点的力, 因此很难绘制整合物传递的细胞力图。
近年来, 在分子水平上研究细胞力的几种技术已经发展起来。开发了一套基于聚乙二醇17、18、蜘蛛丝肽 19和 dna20、21、22 、23的分子张力传感器。可视化和监测分子蛋白传递的张力。在这些技术中, dna 首先被用作张力量系系 (tgt) 的合成材料, tgt 是一种可破坏的链接器, 可调节活细胞22,24中整合素张力的上限。后来, DNA 和荧光共振转移技术结合起来, 首先由陈氏23组和萨拉伊塔第20组创建了基于发夹 dna 的荧光张力传感器。基于发夹 dna 的张力传感器实时报告整合蛋白张力, 已成功地应用于一系列细胞功能21的研究。随后, 王的实验室将 TGT 与氟淬火器对结合起来, 报告整合素张力。这个传感器被命名为 its25,26。ITS 基于双链 DNA (dsDNA), 具有更广泛的动态范围 (10-60 pN) 的整合素张力校准。与基于发夹 dna 的传感器不同的是, ITS 不实时地报告蜂窝力, 而是将所有历史整合事件记录为细胞力的足迹;这种信号积累过程提高了细胞力成像的灵敏度, 即使使用低端荧光显微镜也可以对细胞力进行成像。ITS 的合成相对更方便, 因为它是通过杂交两个单链 Dna (ssDNA) 而产生的。
ITS 是一个18基配对 dsDNA 与生物素、荧光蛋白、淬火剂 (黑洞淬火 2 [BHQ2])27和环状精氨酰糖基环氨酸 (rgd) 肽28作为整合素肽配体 (图 1)结合而成。较低的链与荧光体结合 (Cy3 在本手稿中使用, 而其他染料, 如 Cy5 或亚历克莎系列, 也已被证明是可行的, 在我们的实验室) 和生物素标签, its 被固定在基板上的生物素-阿维丁键。上链与 rgd 肽和黑洞淬火结合, 淬火 cy3 约98% 淬火效率 26,27。根据本文提出的协议, ITS 在基板上的涂层密度约为 1100/μm 2.这是我们先前通过遵循相同的涂层协议29在中和功能化基板上包覆的 18 bp 生物基化 dsdna 的密度。当细胞粘附在涂有 ITS 的基板上时, 整合蛋白通过 RGD 将 its 结合起来, 并将张力传递到 ITS。ITS 有一个特定的张力耐受性 (ttol), 它被定义为在 dsDNA 内机械分离 Its dsDNA 的张力阈值。ITS 被整合蛋白张力断裂导致淬火器与随后发出荧光的染料分离。因此, 将不可见的整合素张力转化为荧光信号, 并通过荧光成像映射细胞力。
为了证明 its 的应用, 我们在这里使用鱼类角化细胞, 这是一种广泛使用的细胞迁移模型,用于细胞迁移研究 30,31, 32, chok1 细胞, 一种常用的非运动细胞系, 和 nih 3t3 成纤维细胞。还对整合素张力和细胞结构进行了成像。

<table>
	<tbody>
		<tr>
			<td>BSA-biotin</td>
			<td>Sigma-Aldrich</td>
			<td>A8549</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>434301</td>
			<td></td>
		</tr>
		<tr>
			<td>upper strand DNA</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td>Customer designed. DNA sequence is shown in PROTOCOL section</td>
		</tr>
		<tr>
			<td>lower strand DNA</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td>Customer designed. DNA sequences are shown in PROTOCOL section.</td>
		</tr>
		<tr>
			<td>sulfo-SMCC</td>
			<td>Thermo Fisher Scientific</td>
			<td>A39268</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclic peptide RGD with an amine group</td>
			<td>Peptides International</td>
			<td>PCI-3696-PI</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>ATCC</td>
			<td>&#8206;62996227</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ATCC</td>
			<td>302020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma-Aldrich</td>
			<td>C4706</td>
			<td></td>
		</tr>
		<tr>
			<td>200 uL petri dish</td>
			<td>Cellvis</td>
			<td>D29-14-1.5-N</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td>N/A</td>
			<td>spectrometer</td>
		</tr>
		<tr>
			<td>SE410 Tall Air-Cooled Vertical Protein Electrophoresis Unit</td>
			<td>Hoefer</td>
			<td>SE410-15-1.5</td>
			<td>Device for electroporesis</td>
		</tr>
		<tr>
			<td>CHO-K1 cell line</td>
			<td>ATCC</td>
			<td>CCL-61</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH/3T3 cell line</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Vinculin Antibody</td>
			<td>EMD Millipore</td>
			<td>90227</td>
			<td>Primary antibody for vinculin immunostaining</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A28175</td>
			<td>Secondary antibody for vinculin immunostaining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A22287</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>microscope</td>
		</tr>
	</tbody>
</table>

imaging integrin tension and cellular force at submicron resolution with an integrative tension sensor

整合素配体键传递的分子张力是整合素通路中的基本机械信号, 在许多细胞功能和行为中发挥着重要作用。为了对高力灵敏度和空间分辨率的图像整合子张力进行校准和图像整合, 我们开发了一种基于 dna 的集成张力传感器 (ITS)。如果维持分子张力, ITS 被激活以荧光, 从而在分子水平上将力转化为荧光信号。ITS 激活的张力阈值可在 10-60 pN 范围内进行调谐, 很好地覆盖了细胞内整合蛋白张力的动态范围。在用 ITS 接枝的基板上, 用荧光显示粘附细胞的整合素张力, 并以亚微米分辨率进行成像。ITS 还与活细胞和固定细胞的细胞结构成像兼容。ITS 已成功地应用于血小板收缩和细胞迁移的研究。本文详细介绍了智能交通系统的合成和应用在整合物传递细胞力研究中的应用。

利用 ITS 捕获了鱼类角质细胞的整合素张力图。它表明, 角质细胞在两个力轨迹上迁移并产生整合素张力 (图 2 a)。力图的分辨率被校准为 0.4μm (图 2B)。高整合素张力集中在后缘 (图 3 a)。ITS 还显示了不同细胞的不同特定模式。非运动细胞 NIH-3T3 形成了与快速迁移的角质形成细胞的特定整合蛋白张力模式 (<st...

Watch this Scientific Journal Video about Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor at JoVE.com

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

整合素张力在各种细胞功能中起着重要作用。利用集成张力传感器, 用 picoNewton (pN) 灵敏度校准整合素张力, 并以亚微米分辨率进行成像。

集成张力传感器在亚微米分辨率下的成像整合和细胞力

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...

这种基于DNA的综合张力传感器（ITS）可将力信号在局部上转换为荧光，从而通过荧光显微镜直接成像细胞力。ITS 具有高空间分辨率和高灵敏度。激活阈值是可调的，允许在不同级别选择性地显示集成张力。在收到感兴趣的定制单链DNA（或ssDNA）将RGD肽加入硫化sSSDNA淬火剂后，首先在400微升水中加入100微升0.5摩尔EDTA，加入约450微升新鲜准备的TCEP溶液中。然后，在PBS中20微升的1毫摩尔硫醇DNA淬火器中加入10微升的TCEP-EDTA溶液，在室温下孵育30分钟。要将RGD胺与磺胺-SMCC结合，将40微升新鲜准备的23毫摩尔磺胺-SMCC加入100微升新鲜准备好的11毫摩尔RGD胺溶液，在室温下孵育20分钟。要将RGD胺SMCC与硫化sSDNA淬火器结合，将硫化sSSDNA淬火剂溶液的整个体积与RGD胺SMCC溶液的整个体积混合，在室温下进行一小时的孵育。在孵化结束时，将溶液转移到四摄氏度的储存过夜。第二天早上，将三摩尔氯化钠与硫醇结合DNA溶液和零下20摄氏度、冷藏、100%乙醇混合在1比10-25的比例下，将反应在零下20摄氏度左右，约30分钟。当DNA沉淀时，通过离心收集DNA，并在PBS中重新预支颗粒，以测量光谱仪上的DNA浓度。对于综合张力传感器（或 ITS）合成，以 1.1 比 1 摩尔比混合上链和下链 DNA 溶液，并在 4 摄氏度下孵化反应，然后将混合物的等分在零下 20 摄氏度进行长期储存。对于 ITS 的固定，在 4 摄氏度下涂上 200 微升 0.1 毫克每毫升 0.1 毫克生物素化牛血清白蛋白（BSA-生物素）的玻璃底培养皿，30分钟。当 BSA-生物素在玻璃表面物理吸附时，每次洗涤用 200 微升新鲜 PBS 洗三次，然后用 200 微升 50 微克每毫升 avidin 蛋白质孵育 30 分钟，温度为 4 摄氏度。在孵化结束时，如证明，用PBS洗三次，并添加200微升0.1微摩尔 ITS，在4摄氏度下再孵育30分钟。在孵育结束时，用PBS洗三次盘子，最后一次洗在盘子里直到细胞电镀。要启动鱼角细胞培养，请使用平尖的钳子从鱼身上拔出一块秤，然后轻轻地将水平压在未修改的玻璃底培养皿的井上，与玻璃质接触。等待约 30 秒，让鱼量表粘附在鱼的玻璃表面，然后用一毫米完整的 Iscove 的修改 Dulbecco 介质覆盖鱼型水表。然后，在室温下在黑暗潮湿的盒子里孵育6至48小时。要将角核细胞板到 ITS 表面，请用 1X 细胞分离溶液替换角蛋白培养皿中的介质，在室温下孵育三分钟。在孵育结束时，用新鲜的完整介质替换分离溶液，用温和的移液分离细胞。然后，将细胞调整到适当的浓度进行电镀，并在成像前在室温下将其播种到 ITS 涂层盘中 30 分钟。对于活细胞中的准实时整数张力成像，将细胞转移到荧光显微镜的阶段，并选择40或100倍的目标。在成像软件中，将曝光时间设置为 1 秒，帧间隔为 20 秒，以获取 ITS 图像的视频。然后，使用适当的图像分析软件程序从当前帧中减去 ITS 视频的前一帧，以计算最新帧间隔中新产生的 ITS 信号。新生成的 ITS 信号表示最新帧间隔中产生的整数张力，从而以准实时方式报告蜂窝力。在这里，在角细胞迁移过程中，集成张力映射演示了在两个力轨上诱导的整流蛋白张力。然后，力图的分辨率可校准为0.4微米。高整数张力集中在莫蒂鱼角细胞的后缘。在非分子细胞中，形成了一种与快速迁移角细胞中观察到的非常不同的特异性张力模式。可以共同成像鱼角细胞的聚焦粘附和整数张力，以评估细胞内整数张力与细胞结构之间的关系。使用 ITS 时，细胞粘附力以分辨率和与细胞结构成像相媲美的灵敏度变得可见。这项技术为研究细胞中的力-结构相互作用铺平了道路。

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SCIENTISTS IN ACTION

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

生物分子检测的干涉反射成像传感器（IRIS）

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Three-dimensional Optical-resolution Photoacoustic Microscopy

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

在激光诱导轴突病变张力释放测量评估轴突在Piconewton和毫秒级的分辨率对基材的附着力

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...