由托马斯·F.和凯特·米勒·杰弗里斯MEMORIA信托（以DEC）和美国国立卫生研究院授予R35GM119617（以DEC）这项工作是支持的。共焦显微镜成像在所述纳米材料VCU表征核心（NCC）设施执行。

1.获取Nesprin-2G传感器DNA和其它质粒DNA <ol><li>从商业来源获得Nesprin-2G TS（张力传感器），Nesprin-2G HL（无头）控制，mTFP1，金星和TSmod。传播的所有DNA的质粒，并使用标准的大肠杆菌菌株，如DH5-α，净化他们如先前12,13说明。 </li></ol> 2.转染细胞与Nesprin-2G和其它质粒DNA <ol><li>生长NIH 3T3成纤维细胞的细胞至70-90％铺满在6孔细胞培养皿中在标准的细胞培养培养箱中温（37℃）和CO 2（5％）调节。对于细胞生长培养基，用Dulbecco氏补充有10％胎牛血清的改良的Eagle培养基（DMEM）。 </li><li>在细胞培养罩，除去培养基并用约1mL降低血清细胞培养基冲洗每一个孔。添加降低血清细胞培养基中的800μL到每个孔并放置在培养箱中在6孔腔。 </li><li>吸移管700降低血清细胞培养基到1.5mL管中的脂质载体溶液35μL的μL以形成"lipomix"。通过移液混合。标记管具有"L"。 </li><li>收集6个的1.5mL管中并标记它们1到6吸取100μL的降低血清细胞培养基到每个管中。 <ol><li>从步骤1中，移液管2 Nesprin 2G-TS的微克使用质粒DNA的浓度成管1和2。移液器2 Nesprin-HL的微克到管中3和4移取1 MTFP的微克到管5移液器1微克mVenus的入管6吹打不同类型的DNA时，不要再使用移液管尖端。 </li></ol></li><li>移液管将100μL从"L"管到每个标记的管（1-6）的lipomix的，并通过反复吹打混匀。使用干净的吸管每个管。孵育10-20分钟。 </li><li>从每个标记的管加入200微升到井在6孔室具有70-90％汇合的细胞。标签的每个井的顶部与相应的DNA溶液。放置6孔室在4-6 h的培养箱中。 </li><li>吸出介质，并添加减少的血清细胞培养基中的1-2毫升冲洗。吸出降低血清细胞培养基，加入胰蛋白酶的2 mL至每个孔中，并将其放置在6孔培养皿在培养箱（5-15分钟）。 </li><li>虽然细胞在培养箱中分离，涂层6玻璃底在溶解于磷酸盐缓冲盐水（PBS）20微克/毫升的浓度与纤连蛋白的一个层观看菜肴。允许的菜涂覆在细胞培养罩（约20分钟）的表面上。 </li><li>加入2毫升的DMEM一旦细胞被分离中和胰蛋白酶。 </li><li>传送每个孔中的物质与标记的15-mL离心管中，并在摇摆转子离心机降速在90×g离心5分钟。吸出上清液并重新悬浮用移液管混合在DMEM 1000微升的各细胞沉淀。 </li><li>抽吸从纤连蛋白混合物玻璃菜肴和吸管每个细胞悬浮液的1000μL到玻璃培养皿。 </li><li>后的细胞沉淀到的玻璃盘的底部（〜15分钟），在细胞培养箱中添加另外1毫升的DMEM + 10％FBS + 1％青霉素 - 链霉素向每个孔和地点。允许细胞附着并表达传感器，用于至少18-24小时。 注:电池使用商用阳离子脂质转染试剂（见材料的表 ）转。可替代地，稳定的细胞系可以通过使用质粒与基因赋予对毒素进行选择（所述的pcDNA质粒为Nesprin-TS和-HL上可用的DNA库网站（参见材料的表 ）提供表达遗传霉素抗性细胞）。此外，病毒感染的方法（慢病毒，逆转录病毒，或腺病毒）可用于表达在，是很难转染细胞的传感器。 </li><li>除了Nesprin-TS，转染的细胞附加与Nesprin HL零为CE控制，如在以下步骤中描述2.1-2.12; TSmod也可以用作零力控制并且应当表现出相似的FRET到Nesprin-HL。 </li><li>转染细胞用mTFP1和金星，以产生频谱指纹（参见步骤4）。 注:mTFP1和金星通常表达水平高于Nesprin-TS，并且同样地，低量的DNA可能需要被转染的，以实现相似的表达水平。 </li></ol> 3.确认转染效率<ol><li>大致完成转染后18-24小时，使用倒置荧光显微镜通过荧光细胞的数目相比较，以在视图细胞（通常为5-30％）的总数来检查转染的效率。 <ol><li>使用配备有接近462纳米（mTFP1）或525纳米（金星）和近492纳米（mTFP1）或525纳米（金星）为中心的发射滤波器的激励频率的荧光显微镜。 注:另外，GFP过滤器组将捕获mTFP1的组合，并且已经NUS排放和允许的转染效率的确认。 </li></ol></li><li>转染后48个小时内的图像的活细胞。 <ol><li>可替代地，在固定的4％多聚甲醛（与钙和镁的PBS中）5分钟，储存于PBS中，视图细胞48小时后8。 注意:超过48小时，信号质量和强度衰减。固定细胞保持它们的状态，其中包括FRET中表达8;然而，细胞只应在PBS成像，如固定介质可能影响FRET 14。固定的细胞仅可相对于其他固定的细胞，因为有可能在表达的变化。 注意:多聚甲醛是有毒的。穿戴合适的个人防护装备（PPE）。 </li></ol></li></ol> mTFP1和金星荧光基团的4捕捉光谱指纹光谱分离<ol><li>在细胞培养罩，更换成像mediu细胞培养基米（HEPES缓冲的），补充有10％胎牛血清。 </li><li>放置观看碗碟的温度受控（37℃）共聚焦显微镜阶段。 </li><li>将带有mTFP1转染的细胞在油物镜的玻璃的观看菜在60X放大倍数为1.4的数值孔径。 注:此油经激光扫描显微镜中使用的60X物镜到接近于玻璃基板的折射率。油放置在物镜的顶部并且与玻璃盖玻片直接接触。 </li><li>定位mTFP1表达细胞与458 nm激发源，并在500纳米为中心的发射带通滤波器。 </li><li>随着视场荧光细胞，选择（在这里所使用的软件"LAMBDA模式"）的光谱检测模式和捕获分光图像（ 图3-1）;包括除了使用10度纳米的增量（ 图3-3）458nm处的所有频率。选择明亮fluoresc在电池（20像素的半径）ENT区域（ROI）。 注意:mTFP1表达ROI的频谱形状应保持在单元相对恒定。如果该形状变化很大，重新调整激光和增益的设置，以改善信噪比。 </li><li>通过单击添加荧光ROI的意思是，归一化强度的光谱数据库"光谱保存到数据库中。" </li><li>优化激光功率和增益，使得良好的信噪比得以实现。设定适用于不同的设备而有所不同。使用非荧光，未转染细胞作为背景参考，提高增益和功率，直到所述细胞是明亮的，但不超过检测器（饱和点）的动态范围。背景细胞不应该在平均后检测的荧光。 </li><li>重复此过程，金星转染的细胞，但下列情况除外: <ol><li>确保激励频率是在515纳米和该带通滤波器是CE定位金星表达细胞时接近530纳米ntered。 </li><li>在"光谱模式"，使用的515nm的，而不是458纳米的激发频率。 </li></ol></li></ol> 5.捕获未混合的图像<ol><li>切换拍摄模式为"光谱分离。" </li><li>添加金星和mTFP1的光谱指纹到解混信道（ 图3中 ，箭头-2）。 </li><li>设置的激发激光回458纳米氩源。 </li><li>放置60X油浸物镜上面的Nesprin-TS观看菜。 </li><li>聚焦在荧光细胞与足够亮表达后，调整增益和激光功率来优化信噪比。 注意:由于Nesprin-TS的FRET效率接近20％，未混合的金星图像应比未混合mTFP1图像基本上调光器。一旦可接受的功率和增益设置已被迭代地确定，这些参数必须保持所有的图像恒定上校ured。 </li><li>捕捉的最小Nesprin-TS细胞的15-20图像具有相对类似的亮度和避免过度像素饱和（饱和的所有像素的图像处理过程中被除去）。 </li><li>重复使用相同的成像参数Nesprin-HL细胞的图像捕获处理。 </li></ol> 6.图像处理和比率的图像分析<ol><li>使用开源的ImageJ（斐济）软件（http://fiji.sc/），使用BioFormats Reader中打开本机格式的图像。 </li><li>前处理的图像，并计算使用先前建立的方案15的比率的图像。 </li></ol>

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The authors have nothing to disclose.

一种方法和跨Nesprin-2G，在核LINC复合体的蛋白质的机械张力的活细胞成像演示，在上面概述的。在此之前的工作，各种技术，如微吸管，磁珠术和显微激光烧蚀，已经被用于对细胞核申请应变，并测量它的体积的材料特性16，17，18。 然而，直到我们最近的工作，还没有研究已经直接表明拉力在活细胞中2直接转移到一个LINC复杂的蛋白质。 先前，我们的实验室证明Nesprin2G，被称为迷你Nesprin2G，的修改版本可以被工程化以表达称为TSmod（ 图2C）2 FRET力探针。 TSmod先前显示动态测定拉伸为CES在活细胞和在不同的承重蛋白3，4，6，8。它进一步表明，迷你Nesprin-2G是从内源性Nesprin-2G区分相对于其定位，对细胞骨架，和能力的已知作用营救核定位11,19。使用与TSmod（Nesprin-TS）的迷你Nesprin-2G蛋白，已表明表达传感器NIH3T3成纤维细胞有不能结合到肌动蛋白2相对于Nesprin-HL，张力的基线水平。此外，蜂窝式收缩的使用抑制剂或肌球蛋白的活化剂的调制可以通过Nesprin-TS 2来检测。 有许多在本协议的关键步骤。混淆Nesprin-TS与Nesprin-HL（DNA或表达细胞）不readiLY检测，因为两者同样定位于核膜，并会导致混乱的结果。由于Nesprin-HL是一个没有力控制，测量FRET应该比Nesprin-TS上的平均水平。谨慎，测序分离的DNA构建体，并仔细标记细胞被劝告。第二，要仔细优化的共焦显微镜检测器的增益和激光功率，以改善信噪比和减少漂白是重要的。为了恰当地测量FRET，这些参数必须在图像采集过程中保持不变。此外，优化检测器增益或功率为暗淡或非常明亮的表达细胞是次优的，因为中值表达细胞将倾向于掉出检测器的动态范围的。表达的传感器的高浓度的某些细胞倾向于在核质和内质网发荧光，从而使得难以解决核膜，这大概是与问候FO更有趣的区域RCE。选择单元格略低的表达水平趋于解决传感器定位这个问题。 虽然比例光谱成像是测量FRET相对简单和快速的方式，但它确实的FRET效率，因为受体信号渗出至20不输出单元。没有的FRET效率单位，这是不可能的FRET比率索引转换成校准力测量。由于这个限制，只有相对定量力可以比较出来。 FRET效率可以通过更复杂的方法，例如荧光寿命成像显微术（FLIM）或受体漂白来确定。绝对力（在PN级）可以从FRET效率来估计，如先前所描述3,8。 相较于以前的方法来测量核的机械性质或基于核0的位移N个外部施加的力，测量FRET从Nesprin-TS具有核膜16，17，18的一个组件上非侵入性地探测张力的优点。该传感器输出与个别Nesprin-2G分子的应变相关的荧光信号。相比之下，微吸管需要使用的工具细腻活细胞工作时。共聚焦激光烧蚀导致局部细胞损伤，并且需要一个脉冲激光源。磁性扭转术转移力到经由细胞骨架核膜，不能用于测量特定蛋白质的力，除非将被加上Nesprin-2G张力传感器。 虽然已经证明，基于肌动蛋白的拉力被传递到Nesprin-2G中，LINC复合物的组分，它仍然是未知的拉伸或压缩力多少被施加到其它结构LINC蛋白，例如SUN-1或SUN-2。未来的工作将在克隆FRET力探讨了这些推定的承重LINC蛋白进一步阐明与蛋白质级别分辨率核力学被引导。这提出了自己的另一个问题是，在FRET变化可能是无关的张力变化的，而是反映了Nesprin-2G齐聚或Nesprin-2G的构象变化或者变化。在细胞pH的变化可以影响所述传感器的荧光并改变测得的FRET。所述Nesprin-2G HL传感器代表了一些变化的良好控制（与该传感器观察到的FRET的变化最有可能无关的力），和分子间FRET对照构建体可以被开发以评估低聚（见下文）。此外，nesprin-2G TS的表达受CMV启动子，这导致过度驱动，并且表达的这种高水平的可能潜在地诱导生物的伪像。用C最新进展上光定期间隔开短回文重复序列（CRISPR）已经允许同源定向修复（HDR）接近插入较大的DNA序列插入基因组中21。这种方法可被用来修改内源性Nesprin-2G（或其他蛋白质），以包括TSmod。这将使用适当的内源性启动子，从而减少了过表达伪影的电位提供的力传感器的表达。 TSmod和其它弹性FRET探针也可以用于开发其他蛋白质，这可能随后在类似的协议作为本文中描述的可以使用新的传感器。任何新的力传感器的发展的一个主要问题是确定的FRET传感器内部的插入位点。首先，插入的位点必须在经受机械负荷的蛋白质的区域。这些区域可以通过检查其中骨架连接蛋白与蛋白相互作用来鉴定。其次，插入大的（〜50 kDa）的TSmod必须不会破坏所研究的蛋白的生物学功能。一个以前开发的"迷你"形式Nesprin-2G的显示，桥接到SUN结合KASH结构域肌动蛋白结合CH域足以在受伤的单层11，19 Nesprin-2G核运动，这表明TSmod的插入可能不会破坏蛋白质的功能。传感器的生物学功能的确认需要用于蛋白质功能的功能测定法（其中所述力传感器可以示于耗尽内源蛋白的细胞以拯救的特定功能）。第三，在蛋白质低聚的变化可以改变蛋白质之间的FRET（称为分子间FRET 22），这将导致在没有强制相关FRET变化。这方面的一个仔细的分析往往需要专门的分子间FRET结构是特别报告齐聚。

力敏感，遗传编码的FRET传感器最近出现的用于在活细胞中测量基于拉伸力，提供深入了解的机械力跨蛋白1，2，3，4施加的重要工具。有了这些工具，研究人员能够以非侵入性图像内的力量在使用传统的荧光显微镜的活细胞。这些传感器由通过弹性肽3分离的FRET对（供体和受体荧光蛋白，最频繁蓝色供体和黄色受体）的。在对比C-或N-末端标记，该传感器被插入到测量跨蛋白质传递的机械力的蛋白质的内部位点，表现为一个分子应变计。增加跨越传感器的结果的机械张力在FRET-P之间的距离增加空气，导致降低FRET 3。其结果是，该FRET是负相关的拉伸力。 这些基于荧光的传感器已被用于粘着斑蛋白（纽蛋白3和踝蛋白4），细胞骨架蛋白（α辅肌动蛋白5），和细胞-细胞连接蛋白显影（E-钙粘蛋白6，7，VE-钙粘蛋白8，和PECAM 8）。在这些生物传感器中最常用的和充分表征的弹性连接体被称为TSmod和由40个氨基酸，（GPGGA）8，其从所述蜘蛛丝蛋白衍生鞭的重复序列组成。 TSmod已经显示出表现为线性弹性纳米弹簧，与FRET响应要求1至拉伸力3 5对-N。鞭状的不同长度可以被用于改变所述动态řTSmod FRET力灵敏度9的安格。除了鞭状，血影蛋白重复5和绒毛头戴受话器肽（称为HP35）4已被用作在类似力的生物传感器4 FRET对之间的弹性的肽。最后，最近的一份报告显示，TSmod也可用于检测压缩力10。 我们最近开发了用于细胞核-细胞骨架（LINC）复合蛋白Nesprin2G的接头的力传感器，通过使用TSmod插入称为迷你Nesprin2G（ 图2C），其行为类似于内源性一个先前开发的截短的蛋白Nesprin2G Nesprin-2G 11。在LINC复杂包含了从外面导致细胞核内，连接细胞质细胞骨架的核纤层的多种蛋白质。 Nesprin-2G是结构蛋白结合到两个肌动蛋白骨架在细胞质和核周空间SUN蛋白质。使用我们的生物传感器，我们能够证明Nesprin-2G是受制于NIH3T3成纤维细胞肌动球蛋白2依赖紧张。这是第一次力在核LINC复杂的跨蛋白直接测量，它很可能成为了解力在力学生物学的核心作用的重要工具。 下面的协议提供的如何使用Nesprin-2G力传感器，包括在哺乳动物细胞中Nesprin张力传感器（Nesprin-TS）的表达Nesprin-细胞的FRET图像的表达，以及采集和分析了详细的方法TS。使用配备有光谱检测器的倒共焦显微镜，如何测量敏化发射的描述使用FRET光谱分离，并设置比例FRET成像。输出比率的图像可以被用来制造相对QUAntitative力比较。虽然该协议的重点是Nesprin-TS的成纤维细胞中的表达，这是很容易适应其它哺乳动物细胞，包括细胞系和原代细胞。此外，该协议，因为它涉及到图像采集和分析FRET可以容易地适用于已经被用于其它蛋白开发了其他基于FRET的力生物传感器。

<table><tbody><tr><td>Nesprin-TS DNA</td><td>Addgene</td><td>68127</td><td>Retrieve from https://www.addgene.org/68127/</td></tr><tr><td>Nesprin-HL DNA</td><td>Addgene</td><td>68128</td><td>Retrieve from https://www.addgene.org/68128/</td></tr><tr><td>mTFP1 DNA</td><td>Addgene</td><td>54613</td><td>Retrieve from https://www.addgene.org/54613/</td></tr><tr><td>mVenus DNA</td><td>Addgene</td><td>27793</td><td>Retrieve from https://www.addgene.org/27793/</td></tr><tr><td>TSmod DNA</td><td>Addgene</td><td>26021</td><td>Retrieve from https://www.addgene.org/26021/</td></tr><tr><td>Competent Cells</td><td>Bioline</td><td>BIO-85026</td><td></td></tr><tr><td>Liquid LB Media</td><td>ThermoFisher</td><td>10855001</td><td>https://www.thermofisher.com/order/catalog/product/10855001</td></tr><tr><td>Solid LB Bacterial Culture Plates</td><td>Sigma-Aldrich</td><td>L5667</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&amp;amp;region=US</td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>Spectrophotometer</td><td>Biorad</td><td>273 BR 07335</td><td>SmartSpec Plus</td></tr><tr><td>quartz cuvette</td><td>Biorad</td><td>1702504</td><td>Cuvette for SmartSpec Plus</td></tr><tr><td>DNA isolation kit</td><td>Macherey-Nagel</td><td>740412.5</td><td>NucleoBond Xtra Midi Plus</td></tr><tr><td>6-well cell culture dish</td><td>Falcon-Corning</td><td>353046</td><td>Multiwell 6-well Polystrene Culture Dish</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium, (DMEM) cell media</td><td>Gibco</td><td>11995-065</td><td>DMEM(1x)</td></tr><tr><td>Bovine Serum</td><td>Life Technologies</td><td>16170-078</td><td></td></tr><tr><td>reduced serum cell media</td><td>Gibco</td><td>31985-070</td><td>Reduced Serum Medium, &quot;optimem&quot;</td></tr><tr><td>Lipid Carrier Solution</td><td>invitrogen</td><td>11668-019</td><td>Lipid Reagent, &quot;Lipofectamine 2000&quot;</td></tr><tr><td>1.5 mL sterile plastic tube</td><td>Denville</td><td>c2170</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td>0.25% Trypsin-EDTA (1x)</td></tr><tr><td>glass-bottom microscope viewing dish</td><td>In Vitro Scientific</td><td>D35-20-1.5-N</td><td>35 mm Dish with 20 mm Bottom Well #1.5 glass</td></tr><tr><td>Fibronectin</td><td>ThermoFisher</td><td>33016015</td><td>fibronectin human protein, plasma</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>14190-144</td><td>Dulbecco&#039;s Phosphate Buffered Saline</td></tr><tr><td>15 mL sterile centrifuge tube</td><td>Greiner bio-one</td><td>188261</td><td></td></tr><tr><td>swinging rotor centrifuge&amp;nbsp;</td><td>Thermo electron</td><td>Centra CL2</td><td>Swinging rotor thermo electron 236</td></tr><tr><td>cell culture biosafety hood</td><td>Forma Scientific</td><td>1284</td><td></td></tr><tr><td>climate controlled cell culture incubator</td><td>ThermoFisher</td><td>3596</td><td></td></tr><tr><td>inverted LED widefield fluorescent microscope</td><td>Life technologies</td><td>EVOS FL</td><td></td></tr><tr><td>Clear HEPES buffered imaging media</td><td>Molecular Probes</td><td>A14291DJ</td><td></td></tr><tr><td>Fetal bovine Serum</td><td>Life technologies</td><td>10437-028</td><td></td></tr><tr><td>Temperature Controlled-Inverted confocal w/458 and 515 nm laser sources&amp;nbsp;</td><td>Zeiss</td><td>&amp;nbsp;LSM 710-w/spectral META detector</td><td></td></tr><tr><td>Outgrowth Media</td><td>Newengland Biolabs</td><td>B9020s</td><td></td></tr><tr><td>NIH 3T3 Fibroblasts</td><td>ATCC</td><td>CRL-1658</td><td></td></tr></tbody></table>

a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

按照上述协议，质粒DNA从DNA库中获取并转化入大肠杆菌细胞中。表达该传感器DNA 的大肠杆菌从LB /氨苄青霉素平板上选择并在液体LB肉汤扩增。以下载体的扩增，DNA质粒使用标准，市售的DNA提取试剂盒纯化为TRIS-EDTA缓冲液中。使用分光光度计，纯化的DNA被量化为微克/毫升（ 图1A，1-2天）的标准浓度。 使用NIH3T3成纤维细胞，将细胞在6孔塑料细胞培养皿中生长至70-90％铺满。要插入的质粒DNA到NIH3T3细胞，进行脂质介导的转染质粒。可商购转染试剂（参见材料的表 ）被用作脂质容器DNA。两个重复所述Nesprin-TS和Nesprin-HL传感器转染到4个细胞孔（ 图1B）。在用于FRET摄像准备，mTFP1和金星单荧光团构建体转染到NIH3T3细胞（ 图1B）。转染后，将细胞转移到玻璃底查看与高倍率，倒共焦显微镜兼容菜肴。 在继续共焦成像，一个简单的倒宽视场荧光显微镜被用于验证转染效率。使用10X物镜0.25，许多细胞在典型的场的视图的数值孔径表示的Nesprin-TS（ 图2）。一般情况下，传感器周围的局部核膜，与Nesprin-2G 2的内源性的定位是一致的。无力控制传感器，Nesprin-HL，遵循类似的表达模式2。 <p类="jove_content" FO:保持-together.within页="1"&gt;的Nesprin传感器TS和HL，活细胞中表达单一荧光团构建体（mTFP1或金星）的转染效率的确认后，用倒置共聚焦成像显微镜。使用所述光谱检测模式，mTFP1和金星光谱指纹被记录在光谱分离数据库（ 图3，箭头-2和图4A）。以下指纹，捕获参数切换到解混模式，以装载作为部件（ 图4D）的mTFP1和金星光谱。原始图像被获取作为光谱分辨图像栈（ 图4）。 最后，光谱分辨图像堆栈被导入到ImageJ的开源软件进行处理。对图像进行预处理，并利用既定的程序15进行了计算比率的图像。 <p类="jove_content" FO:保持-together.within页="1"&gt;由于FRET比率是相对测量，每次实验必须比较两个或更多的条件。此外，由于激光和摄像机检测器设置可以影响FRET，所有条件应在相同的成像会话获取。通常情况下，我们使用武力不敏感的无头无尾或控制，它代表零力控制，作为基线我们的实验。因为FRET比率的图像可以是有噪声的，通常有必要采取对于每种条件的多个图像（5-20​​）。对于有在FRET大的差异的条件下，这将是在FRET比率图像视觉上可辨别的。在FRET用于Nesprin张力传感器的最显着的差异是未图案化的和细长的细胞（ 图5）之间。虽然小区之间变化，这两个条件之间的FRET的变化是如此显着进一步的分析可以不需要。然而，其他条件往往有迷你MAL改变FRET;它们可能不是单独的图像之间在视觉上可辨别的，而是需要的数据在聚合进行分析。要确定是否这些变化是显著，对于每个图像中位数FRET比率被计算并在视觉上表示为一组直方图。通过条件之间比较直方图，它变得更容易看到FRET实验组之间微妙的变化。在图6A和6B中 ，各单个电池的FRET直方图是通过在层叠直方图的彩色表示。海绵诱癌素A处理的细胞（ 图6B）显示出向左移FRET直方图相对于与肌球蛋白抑制剂ML7（ 图6A）处理的细胞中一个。 <img alt="图1" src="/files/ftp_upload/54902/54902fig1.jpg" /> 图1: 基于时间的实验流程图。 （A）与获得SENS开始或质粒DNA， 大肠杆菌载体转化用DNA，选择和扩增（1-2天）。从浓缩大肠杆菌 LB肉汤，质粒DNA分离和定量（第3天）。使用纯化的DNA质粒，NIH3T3成纤维细胞转染在六孔格式（B），并转移到玻璃底培养皿是用倒置共聚焦显微镜（第3天）不兼容。为了验证转染的成功，细胞都配备有适当的过滤器集（第4天）一个宽视场荧光显微镜下观察。在成功转染队伍，将细胞成像在配备有光谱检测器共焦显微镜。使用光谱分离，mTFP1和金星通道在采集过程中分离，并且保存为光谱分辨图像栈（天数4-5）。最后，图像被预处理ImageJ中，并且比率图像被计算的。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54902/54902fig1large.jpg" target="_blank">PLE</a>ASE点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54902/54902fig2.jpg" /> 图2: 转染的NIH3T3成纤维细胞的代表性图像。 （A）转染的NIH3T3成纤维细胞用GFP表达filterset Nesprin-TS的放大10倍。 （B）的边界框的部分从传感器A.表达的分解图如下核膜和经常延伸到细胞质中。 （C）Nesprin-TS和-HL控制以及它们如何结合到肌动蛋白和SUN结合结构域。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54902/54902fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54902/54902fig3.jpg" /> 图3:Spectral指纹教程利用共聚焦软件。从20像素的半径在该高亮显示的区域的感兴趣有色十字线划定获得的归发射光谱。 （箭头-1）光谱检测模式以捕获图像。 （箭头-2）"光谱分离"设置为归一化谱添加到光谱数据库。 （箭头-3）的激励频率。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54902/54902fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54902/54902fig4.jpg" /> 图4: 实时光谱分离和共聚焦软件的输出。 （A）临界成像参数来再现实时，未混合的图像。 （箭头-1）的激励频率。 （箭头-2）活光谱分离模式。在未混合的金星信道（B）核图像。 （ <str<html>翁&gt; C）核中未混合mTFP1通道图像。 （D）金星和mTFP1的图像相结合。 （箭头-3），其中被表达Nesprin-TS的细胞的核膜。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54902/54902fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54902/54902fig5.jpg" /> 图5: 在图案化和未图案化的马-达氏犬肾（MDCK）细胞Nesprin-TS FRET比率的图像。 MDCK细胞稳定表达Nesprin-TS进行成像于10微米范围内的纤连蛋白线（A）或非图案化的聚二甲基硅氧烷（PDMS）膜（B）。核膜目视掩蔽并加工成FRET比率。着色比例尺代表用于未图案化的图案化和核中FRET比率振幅。e.jove.com/files/ftp_upload/54902/54902fig5large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/54902/54902fig6.jpg" /> 图6: 从加工成比例FRET图像Nesprin-TS表达细胞和聚集数据的直方图分析。 （A）的代表性，与肌球蛋白ML7抑制剂，由个体细胞核的颜色编码处理Nesprin-TS的FRET比率图像的归一化叠加柱状图。 （B）代表，用海绵诱癌素A，细胞收缩的活化剂处理过的Nesprin-TS的FRET比率图像的归一化叠加柱状图。传说把每一个分析，核其对直方图的位置。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54902/54902fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

a-protocol-for-using-frster-resonance-energy-transfer-fret-force

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.5K 次观看.  Virginia Commonwealth University. A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex. 

视频: A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

科研

生物化学

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

生物工程

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

活植物细胞中的Förster共振能量转移测量

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

福斯特共振能量转移映射:阐明全球结构特征的新方法

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...