Name: generating a fractal microstructure of laminin-111 to signal to cells
Uploaded: 2020-09-28T13:00:00.000+00:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

这项工作由劳伦斯利弗莫尔国家实验室LDRD 18-ERD-062（C.R.）资助。感谢 John Muschler 赠送的 DgKO 和 DgKI 细胞。感谢加州大学伯克利分校电子显微镜实验室的工作人员在电子显微镜样品制备和数据收集方面提供的建议和帮助。

1. 解冻和等分试样层粘连蛋白-111
注意:纯化的层粘连蛋白-111 可在市面上买到（通常从作为基质出售的 EHS 基质中纯化），但并非所有来源都能提供完整、未降解的蛋白质。
<ol><li>通过获取 Ln1 和运行尺寸排阻色谱26 或聚丙烯酰胺凝胶电泳26 确认 Ln1 未降解。变性 Ln1 应具有 337 kDa、197 kDa 和 177 kDa 的条带，而天然 Ln1 应具有 711 kDa 的单条带。</li>
	<li>在冰箱中将 Ln1 在冰上解冻过夜，并使用低蛋白结合尖端和无菌技术等分到低蛋白结合微量离心管中（通常以 100 μg/管等分试样进行培养覆盖，或 10 μg 用于包被）27。冷冻等分试样并储存在-80°C。</li>
</ol>2. 使用低pH缓冲液进行层粘连蛋白聚合

<ol><li>制备polyLM聚合缓冲液:称取1 mM CaCl2 和20 mM乙酸钠在超纯水中混合。然后使用HCl或乙酸将pH调节至4.0。通过0.2μm过滤器进行无菌过滤，并储存在4°C。</li>
	<li>制备对照聚合缓冲液:混合 1 mM CalCl2 和 20 mM Tris，并根据需要使用 10 N HCl 或 12 N NaOH 将 pH 调节至 7.0。通过0.2μm过滤器进行无菌过滤，并储存在4°C。 注意:协议可以在此处暂停。</li>
	<li>在冰箱中解冻所需数量的 Ln-1 等分试样过夜。</li>
	<li>使用无菌细胞培养技术，将适当的缓冲液（步骤2.1中的polyLM聚合缓冲液或步骤2.2中的对照聚合缓冲液）加入到终浓度为100μg/ mL的层粘连蛋白等分试样中（即向50μg等分试样中加入0.5mL），并通过移液轻轻混合。</li>
	<li>如果使用荧光标记的层粘连蛋白进行可视化，则重新配制并以 1:50 wt/wt 的比例加入（例如 2 μg/100 μg），并通过移液轻轻混合。 注意:如果使用层粘连蛋白作为细胞附着的底物，请按照步骤2.6-2.8操作:</li>
	<li>在适当的缓冲液中稀释后，立即将层粘连蛋白转移到培养塑料或玻璃器皿上，并在37°C的细胞培养箱中孵育过夜。 请注意，涂覆盖玻片时，加入足够大的体积以产生平衡液滴（通常，13 mm圆形盖玻片使用50μL），并在37°C的加湿室中保存过夜。</li>
	<li>使用无菌技术27，用1x磷酸盐缓冲盐水（PBS）洗涤。</li>
	<li>小心地取出液体。不要让表面完全干燥。 注意:如果使用层粘连蛋白覆盖细胞以诱导牛奶蛋白，请按照步骤2.9-2.12操作:</li>
	<li>培养乳腺上皮细胞。在补充有 2% FBS、0.01 mg/mL 胰岛素、0.005 μg/mL EGF、0.05 mg/mL 庆大霉素和 0.05 g/mL 正霉素的 DMEM-F12 中培养 MepG 抗肌萎缩聚糖敲入 （DgKI） 或 MepG 抗肌萎缩聚糖敲除细胞 （DgKO）。</li>
	<li>在孔板或盖玻片底部培养皿中以 10,500 个细胞/cm2 的速度接种用于层粘连蛋白刺激的 DgKO 和 DgKI 细胞</li>
	<li>将稀释的层粘连蛋白在37°C的细胞培养箱中孵育30分钟。</li>
	<li>将polyLM等分试样以2,000× g 离心20分钟，但由于蛋白质实体的尺寸较小，并且需要更高的速度才能有效收集，因此在20,000× g 的中性pH下离心LM20分钟。</li>
	<li>使用无菌技术27，轻轻除去上清液，并重悬于补充有3μg/mL催乳素，2.8μM氢化可的松和5μg/mL胰岛素的适当体积的DMEM-F12诱导培养基中</li>
	<li>立即将层粘连蛋白转移到细胞培养基中以刺激细胞。我们已经成功地用50-800μg/mL Ln1刺激细胞</li>
</ol>3. 使用分离的糖蛋白进行层粘连蛋白聚合
<ol><li>将重组 nidogen-1 和 Ln1 在冰箱中的冰上轻轻解冻过夜。</li>
	<li>使用低蛋白结合尖端和试管并使用无菌技术，在细胞培养基中以 1:1 wt/wt 的比例将 Ln1 与 nidogen-1 混合。使用细胞培养基中的纯 Ln1 作为对照。</li>
	<li>如果使用荧光标记的层粘连蛋白进行可视化，则以 1:50 wt/wt 的比例（例如，2 μg/100 μg）复溶并添加。用移液器轻轻混合。</li>
	<li>在37°C的细胞培养箱中孵育Ln1-nidogen或对照Ln130分钟。</li>
	<li>将Ln1-nidogen共聚物以2,000× g 离心30分钟，将对照Ln1以20,000× g 离心30分钟。</li>
	<li>轻轻除去上清液并重悬于细胞培养基中，终浓度为 100-800 μg 总蛋白/mL。</li>
	<li>立即用于刺激细胞，或转移到盖玻片上，并在37°C湿润的细胞培养箱中粘附过夜。</li>
	<li>从盖玻片中取出培养基，用 1x PBS 洗涤盖玻片，然后在室温下用 10% 福尔马林（即 4% 甲醛在 PBS 中）固定 15 分钟。</li>
</ol>4. 使用抗肌萎缩聚糖表达细胞的层粘连蛋白聚合
<ol><li>培养乳腺上皮细胞。在补充有 2% FBS、0.01 mg/mL 胰岛素、0.005 μg/mL EGF、0.05 mg/mL 庆大霉素和 0.05 g/mL 正霉素的 DMEM-F12 中培养 MepG 抗肌萎缩聚糖敲入 （DgKI） 或 MepG 抗肌萎缩聚糖敲除细胞 （DgKO）。</li>
	<li>由于生长速率的差异，以 5,000 个细胞/cm2 接种 DgKI 细胞，以 8,000 个细胞/cm2 接种 DgKO 细胞。在37°C湿润培养箱中培养，每2-3天更换一次培养基，每4-5天使用严格的无菌技术传代一次。使用血细胞计数器或自动细胞计数器27 仔细计数细胞，以确保适当的生长速率。</li>
	<li>在孔板或盖玻片底部培养皿中以 10,500 个细胞/cm2 的强度接种用于层粘连蛋白刺激的细胞并培养 2 天，使用 DgKI 细胞产生分形 Ln1 和 DgKO 细胞作为阴性对照。在冰箱中将 Ln-1 在冰上轻轻解冻过夜，然后与补充有 3 μg/mL 催乳素、2.8 μM 氢化可的松和 5 μg/mL 胰岛素的 DMEM-F12 诱导培养基混合。对于 35 mm 培养皿，使用 1 mL 含有 50-800 μg Ln1 的诱导培养基。</li>
	<li>如果使用荧光 Ln1，则以 50:1 wt/wt 的比例混合。</li>
	<li>从细胞中取出培养基，并用补充有 3 μg/mL 催乳素、2.8 μM 氢化可的松和 5 μg/mL 胰岛素的 DMEM-F12 层粘连蛋白和诱导培养基覆盖</li>
	<li>培养细胞 2 天，然后用 10% 福尔马林固定。</li>
</ol>5. 通过光学显微镜表征层粘连蛋白微观结构
<ol><li>收集固定样品，并在室温下用1x PBS洗涤3次5分钟。</li>
	<li>在室温下，用0.5%Triton X-100,3%牛血清白蛋白，10%山羊血清和40μg/ mL抗小鼠阻断抗体片段在1x PBS中封闭和透化样品1小时。 注意: Triton X-100 是危险的。戴上手套和护目镜进行处理并妥善处理。</li>
	<li>将稀释度为1:100的抗层粘连蛋白抗体与1x PBS与0.5%Triton X-100和3%BSA混合，并在室温下或在4°C下在加湿室中加入样品2小时（通常使用底部带有湿实验室湿巾的尖端盒）。</li>
	<li>取出一抗，用 1x PBS 和 0.5% Triton X-100 和 3% BSA 洗涤 3 次。</li>
	<li>在含有0.5%Triton X-100和3%BSA的1x PBS样品中以1:500的稀释度向样品中加入适当的二抗，并在室温下在加湿室中避光孵育。</li>
	<li>去除二抗，用 1x PBS 和 0.5% Triton X-100 和 3% BSA 洗涤 3 次。</li>
	<li>对于含有细胞的样品，加入 6 μM 鬼笔环肽 45 分钟，然后用含有 0.5% Triton X-100 和 3% BSA 的 PBS 洗涤 3 次，然后用 PBS 洗涤 3 次。然后用 0.1 μg/mL DAPI 在 PBS 中染色 5 分钟。</li>
	<li>如果合适，可以挂载样品。</li>
	<li>使用高分辨率共聚焦显微镜对 ln-1 结构进行成像。使用配备浸没物镜的激光扫描显微镜（水浸复消色差计40X/1.1NA或油浸复消色差计63X/1.4NA）。</li>
</ol>6. 通过盒数维度表征层粘连蛋白结构
<ol><li>使用 Matlab 工具箱或 ImageJ 中提供的箱计数维度算法分析分形属性。这些方法对层粘连蛋白的图像进行阈值测量，并在对数对数上绘制含有 Ln1 的盒数与盒的大小。这些参数之间关系的斜率是盒数维数:非分形几何的维数为 0、1 或 2，而分形几何的非整数维数为28。 注意:酸处理、糖蛋白处理和 DgKI 细胞附着的 Ln1 的盒计数分形维数均为 ~1.7，而对照 Ln1 的分形维数为 2.0。</li>
</ol>7. 通过电子显微镜表征层粘连蛋白构建体
<ol><li>将层粘连蛋白重悬于细胞培养基中，并在潮湿的37°C环境中吸附到二氧化硅晶片上90分钟。</li>
	<li>将基质固定在 pH 7.2 的 0.1 M 二甲胂酸钠缓冲液中的 2.5% 戊二醛中。 注意:戊二醛是危险的，应在通风橱中处理，并戴上手套和护目镜，并作为危险废物处理。</li>
	<li>在pH 7.2的0.1M二甲胂酸钠缓冲液中的1%四氧化锇中建立后缀基质。 注意: 四氧化锇是危险的，应在通风橱中处理，并戴上手套和护目镜，并妥善处理。二甲胂酸盐是危险的，应在通风橱中处理，并戴上手套和护目镜，并妥善处理。</li>
	<li>在pH 7.2的二甲胂酸钠缓冲液中洗涤3次。</li>
	<li>随着乙醇浓度的增加而脱水。</li>
	<li>溅射涂层基质上一层薄薄的金。</li>
	<li>使用扫描电子显微镜进行图像处理。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Palmero, C. 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=The+follicular+thyroid+cell+line+PCCL3+responds+differently+to+laminin+and+to+polylaminin,+a+polymer+of+laminin+assembled+in+acidic+pH.">The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.</a> Molecular and Cellular Endocrinology. 376 (1), 12-22 (2013).</li><li>Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polylaminin+recognition+by+retinal+cells.">Polylaminin recognition by retinal cells.</a> Journal of Neuroscience Research. 92 (1), 24-34 (2014).</li><li>Leal-Lopes, C., Grazioli, G., Mares-Guia, T. R., Coelho-Sampaio, T., Sogayar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerized+laminin+incorporation+into+alginate-based+microcapsules+reduces+pericapsular+overgrowth+and+inflammation.">Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.</a> Journal of Tissue Engineering and Regenerative Medicine. 13 (10), 1912-1922 (2019).</li><li>Paccola Mesquita, F. C., Hochman-Mendez, C., Morrissey, J., Sampaio, L. C., Taylor, 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=Laminin+as+a+Potent+Substrate+for+Large-Scale+Expansion+of+Human+Induced+Pluripotent+Stem+Cells+in+a+Closed+Cell+Expansion+System.">Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.</a> Stem Cells International. 2019, 9 (2019).</li><li>Walker, J. M., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , 61-67 (2002).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Techniques. 7th edition. , (2016).</li><li>Mandelbrot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Fractal Geometry of Nature. , (1982).</li><li>Schittny, J. C., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+short+arm+domains+of+basement+membrane+laminin+are+critical+for+its+self-assembly.">Terminal short arm domains of basement membrane laminin are critical for its self-assembly.</a> Journal of Cell Biology. 110 (3), 825-832 (1990).</li><li>Schuger, L., Yurchenco, P., Relan, N. K., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+fragment+E4+inhibition+studies:+basement+membrane+assembly+and+embryonic+lung+epithelial+cell+polarization+requires+laminin+polymerization.">Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.</a> International Journal of Developmental Biology. 42 (2), 217-220 (1998).</li><li>Colognato, H., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function:+the+laminin+family+of+heterotrimers.">Form and function: the laminin family of heterotrimers.</a> Developmental Dynamics. 218 (2), 213-234 (2000).</li><li>Kabaeva, Z., Meekhof, K. E., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemma+instability+during+mechanical+activity+in+Large+myd+cardiac+myocytes+with+loss+of+dystroglycan+extracellular+matrix+receptor+function.">Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function.</a> Human Molecular Genetics. 20 (17), 3346-3355 (2011).</li></ol>

这项工作由劳伦斯利弗莫尔国家实验室在美国能源部的主持下根据合同DE-AC52-07NA27344进行。IM 版本 LLNL-JRNL-799443。

层粘连蛋白是上皮、肌肉和神经器官系统中ECM的关键元素，在体外已被证明在调节细胞功能分化中起着重要作用。越来越多的证据表明，向细胞展示的层粘连蛋白结构调节其信号传导和由此产生的细胞表型15,16，因此控制 Ln1 微观结构的方法应该对在这些领域工作的基础生物学家以及寻求概括体外正常行为的组织工程师感兴趣。更一般地说，已知各种细?...

与生长因子或细胞因子不同，细胞外基质 （ECM） 蛋白可以组装成结构网络。由于ECM功能障碍是许多疾病的关键因素，因此细胞从ECM组成、微观结构和生物力学中感知和转导信号的机制是治疗开发的有吸引力的靶标。越来越多的证据表明，这些网络的微观结构在这些蛋白质如何向细胞发出信号方面起着重要作用。迄今为止，ECM 微观结构与细胞功能之间的这种联系已在胶原 I1、纤连蛋白2 和纤维蛋白3 中得到证明。
层粘连蛋白-111 （Ln1） 是一种三聚体十字形蛋白质，是细胞外基质的关键结构成分，可接触上皮细胞4、肌肉细胞5 和神经细胞6。在乳腺中，Ln1 是乳腺上皮细胞功能分化为产奶细胞所必需的 7,8,9，Ln1 对于诱导肿瘤逆转至关重要 10,11。Ln1 和其他层粘连蛋白是肌肉中皮质肌动蛋白网络和肌节组织发育所必需的12,13，以及神经细胞迁移和神经突生长13 所必需的。因此，了解层粘连蛋白向细胞发出信号的机制是一个活跃的研究领域。
Ln1 包含多种受体的多个粘附结构域，包括整合素、突聚糖、肌营养不良聚糖、LBP-110 和 LamR（图 1A）4,14。含有层粘连蛋白 α1 的 c 末端球状结构域的 E8 片段是结合抗肌萎缩聚糖和整合素 α6β1 和 α3β115 所必需的，并且是上皮细胞功能分化所必需的15,16。相比之下，短臂显示出不同的生物活性17，这意味着在网络形成后识别这些不同Ln1结构域的可用性可以改变细胞行为。
我们最近证明，排列成聚合物网络的 Ln1 表现出分形特性（即在多个长度尺度上自相似的结构）18。与缺乏这种排列的 Ln1 相比，分形网络对上皮细胞发出的信号不同19。该分形网络表示特定的组装结构，其中长臂优先暴露于单元18。由于这种不同的长臂显示，上皮细胞经历功能分化为产奶细胞，具有组织良好的紧密连接和肌动蛋白应力纤维的抑制 19。
我们描述了从短臂-短臂结合相互作用中生成 Ln1 网络的 3 种方法，这些方法显示了 Ln1 长臂的高显示:1） 通过与酸性缓冲液的无细胞组装，2） 通过与糖蛋白共孵育，或 3） 通过与细胞表面抗肌萎缩聚糖的相互作用。这三种方法通过三种截然不同的机制产生相似的层粘连蛋白微观结构，从而在实验设计中具有灵活性。先前的研究表明，这三种方法可以代表生物学的稳健性:在乳腺上皮细胞中，细胞表面抗肌萎缩聚糖、糖蛋白或人工结构的层粘连蛋白都可以诱导功能分化19。因此，研究人员可以根据研究对象在这些方法之间进行选择:表达抗肌萎缩聚糖的细胞对 Ln-1 微观结构不敏感，因为细胞膜中的抗肌萎缩聚糖诱导正确的聚合成分形网络19,20。基质凝胶是层粘连蛋白和糖蛋白的混合物21，代表了Ln-1最经济的来源，并提供了诱导抗肌萎缩聚糖敲除乳腺细胞分化所需的微观结构20，但含有具有批次间变异性的复杂蛋白质混合物。PolyLM 代表了提供此功能的最干净、最还原的系统，但与Matrigel 19 相比，诱导乳腺细胞分化的成本更高，功效更低。
这三种方法具有扩散限制聚集的分形网络结构特征18,19，并且与多个实验系统中的聚集层粘连蛋白相比，显示出生物活性的改变22,23,24,25。使用这些方法的未来研究可以识别上皮分化所需的受体和信号传导，并确定在异常微环境20（例如肿瘤）中恢复细胞中的正常细胞-基质相互作用。

<table><tbody><tr><td>10% formalin</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Anti-Laminin primary antibody</td><td>Sigma Aldrich</td><td>L9393</td><td></td></tr><tr><td>Anti-Rabbit Alexafluor 488 secondary antibody</td><td>Thermo</td><td>A32731</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>CaCl2</td><td>Sigma Aldrich</td><td>C4901 or similar</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Thermo</td><td>Heracell 150 or similar</td><td></td></tr><tr><td>Centrifuge for microcentrifuge tubes</td><td>Eppendorf</td><td>5418 or similar</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 710 or equivalent</td><td></td></tr><tr><td>Coverslips</td><td>Thermo</td><td>25CIR-1 or similar</td><td></td></tr><tr><td>DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td><td>Thermo</td><td>11330107</td><td></td></tr><tr><td>EGF (Epidermal Growth Factor)</td><td>Sigma Aldrich</td><td>11376454001</td><td></td></tr><tr><td>Fluorescently Labeled Laminin</td><td>Cytoskeleton Inc</td><td>LMN02</td><td></td></tr><tr><td>Gentamicin</td><td>VWR</td><td>VWRV0304-10G</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>HCl</td><td>Sigma Aldrich</td><td>320331 or similar</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888-5G</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>16634-50mg</td><td></td></tr><tr><td>MepG Dystroglycan Knockout Cells</td><td>LBNL</td><td>N/A</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>S8045 or similar</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>ant-nr-1</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>Sigma Aldrich</td><td>75633</td><td></td></tr><tr><td>Ovine Prolactin</td><td>Los Angeles Biomedical Research Institute</td><td>Ovine Pituitary Prolactin</td><td></td></tr><tr><td>Phalloidin</td><td>Thermo</td><td>A22287</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo</td><td>10010023 or similar</td><td></td></tr><tr><td>Purified Laminin-111</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>Recombinant human Nidogen-1, carrier free</td><td>R&amp;amp;D systems</td><td>2570-ND-050</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma Aldrich</td><td>S2889 or similar</td><td></td></tr><tr><td>Sodium Cacodylate</td><td>Sigma Aldrich</td><td>C0250</td><td></td></tr><tr><td>Sterile filters</td><td>Millipore</td><td>SLGP033RS or similar</td><td></td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>252859 or similar</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Ultra-low protein binding tubes</td><td>VWR</td><td>76322-516, 76322-522 or similar</td><td></td></tr><tr><td>Ultrapure water</td><td>Thermo</td><td>15230253 or similar</td><td></td></tr></tbody></table>

generating a fractal microstructure of laminin-111 to signal to cells

层粘连蛋白-111 （Ln1） 是上皮、肌肉和神经系统中细胞外基质的重要组成部分。我们之前已经证明，Ln1的微观结构改变了它向细胞发出信号的方式，这可能是因为Ln1组装到网络中暴露了不同的粘附结构域。在该协议中，我们描述了三种生成聚合Ln1的方法。

层粘连蛋白-111是一种三聚体蛋白，在其短臂末端具有三个自组装结构域（图1A）。如果处理得当，短臂可以聚合成六边形晶格（图1B，1C），在更大的空间尺度上显示出扩散限制的聚集体分形特性（图2）。在含钙的中性pH缓冲液中孵育的层粘连蛋白形成用抗Ln1抗体可视化的小聚集体（图2A），其?...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

我们描述了三种生成具有分形特性的 Ln1 聚合物的方法，与未聚合的 Ln1 相比，这些聚合物向细胞发出的信号不同。

生成层粘连蛋白-111的分形微观结构以向细胞发出信号

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Research

JoVE Journal

Bioengineering

924 次观看.  Texas Heart Institute. 我们描述了三种生成具有分形特性的 Ln1 聚合物的方法，与未聚合的 Ln1 相比，这些聚合物向细胞发出的信号不同。

视频: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

小角度 x 射线散射溶液中大分子的结构研究

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

生物化学

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

微图案牵引显微镜的图案生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

使用 PRIMO 微模式系统对蛋白质的光诱导分子吸附，研究细胞对细胞外基质蛋白的反应

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

神经科学

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...