这个项目是支持的科研能力增强格兰特（Bowling Green州立大学）。

1。制备细胞单分子实验系统<ol><li>至少提前一周，从股票开始解冻在37 ° C，在一个25厘米2培养瓶中将用5 mL媒体的HeLa细胞株的新鲜培养，细胞在37 ° C范围内5％的CO 2 24小时的孵化器和分裂，至少有3次，以提供细胞的最佳条件。然而，细胞不应该被分割的20倍以上，由于其增长速度的倾向显着放缓。以前HeLa细胞基因工程有GFP（绿色荧光蛋白）融合到核膜孔膜蛋白的聚甲醛121。这是必要的核膜的精确定位。 </li><li>实验前一天，旨在为单个分子实验的细胞应置于无菌培养皿中的蒸压盖滑传播。盖玻片应暂停在4.5克/ L葡萄糖，862 mg / L的GlutaMAX的我，15毫克/毫升酚红，100 U / mL青霉素，100μg/mL为辅的DMEM（贝科改良Eagle培养基，大岛，NY）链霉素和10％新生牛血清。一种文化的量应该是每1ML盖玻片，在表面均匀分布。这些培养皿中的盖玻片，然后放入CO 2培养箱，实验前24小时不停。为了一个细胞培养适合单分子实验的盖玻片上的细胞汇合不应超过70％，提供了一个良好的洗涤缓冲液和蛋白的核运输中发生的利益自由。 </li><li>在实验的日子，流商会，构建了顶盖滑两个垫片硅脂线。它是必不可少的，以确保细胞不会干在准备。我们通常喜欢有两院提出的封面上滑，让两个不同的实验条件。顶盖单用钻石刀切割约6平方毫米被放置在一个Kimwipe倒挂的玻璃盖滑的小条子。两行硅脂精细沿着他与一个10毫升的塑料注射器，封口膜紧密连接到控制油脂珠的大小一个枪头盖玻片边缘。 </li><li>对HeLa细胞的涂层幻灯片，然后滴干和家庭加工铝框 - 脾气，这有助于举行的幻灯片在显微镜样品架安装在油脂作为粘合剂。 </li><li>尖镊子，然后倒在细胞涂层的幻灯片，然后拿起两条线的油脂顶盖单，形成了一个流动室。脂盖玻片边缘轻轻按一个比较坚定执着。 </li><li>由于我们一个细胞涂幻灯片上的两个独立的商会，这是必须提供良好的密封，以防止交叉污染。为此应用于商会从顶部向底部的幻灯片之间除了密封硅脂的几行。几行的油脂也被放在分庭的外侧。 </li><li> DMEM培养液，然后添加到媒体流室，防止细胞干燥。解决方案流升任一小块滤纸吸收的液体流室的出口侧。稳定的手中，解决方案可以同时添加微管的流室，用一只手的一侧，而另一边滤纸吸收。 </li><li>施工后，用湿棉签拭子，被冲走的幻灯片的底部与水的两倍，然后用100％的乙醇的两倍。 </li><li>一旦安装在显微镜幻灯片，洗净，用2 × 25μL导入缓冲区（20毫米HEPES，110毫米KOAc，5毫米NaOAc，2毫米MgOAc，1毫米EGTA，pH值7.3）的细胞。为1〜2分钟，2 × 25μL的40μg/ mL的进口缓冲区毛地黄皂苷透细胞，然后再洗净，用2 × 25μL导入缓冲区（了20毫米HEPES，110毫米KOAc，5毫米NaOAc，2毫米MgOAc，1毫米EGTA，pH值7.3），辅以1.5％聚乙烯吡咯烷酮（PVP，360 kDa的）。这可能是必要的，以监视在通透明亮的领域真正的使用时间。 PVP被列入毛地黄皂苷后，所有进口的缓冲液，以防止核渗透肿胀。完整的细胞核，连同控制细胞质环境，提供了一个大大简化运输系统，使我们能够阐明复杂的核运输一步一步。上合奏和系统进行单分子实验提供了核运输中有着深刻的见解。 </li></ol> 2。制备核运输实验必需的蛋白质<ol><li>通过传统的细菌转化大肠杆菌 （JM109）的特殊菌株以过度表达的基因负责工程感兴趣的上述蛋白质的生产和保存在股票，在-80 ° C （免入息审查贷款计划的2xGFP pTrcHisB载体由Invitrogen）。一个loopful或〜50μL荧光或货物表达大肠杆菌细胞的蛋白质冷冻库存转移到LB培养基5ML除了与氨苄青霉素5μL（ü 1000）一夜之间在37 °彗星在225 RPM用颤抖的有氧和成长。 </li><li> 24小时后转入500毫升LB培养基500μL饱和文化和动摇在37℃孵化器为5-6小时后，除了250μLIPTG（异丙基 - D - 1 -硫代半乳糖苷）和另一孵育5-6个小时。这是必要的，因为IPTG是乳糖的代谢物，触发lac操纵子，lacZ基因与基因的兴趣和IPTG取代的转录，然后用于诱导基因表达。 </li><li>然后纺文化是在冷却的高速离心15分钟，在12000 RPM和被丢弃上清。细胞，然后悬浮在40毫升的量应该是每一个颗粒1克的10毫升的基础上CelLytic乙（B在CelLytic快速，高效提取的蛋白质，是适合亲和纯化及分析的结果。） 28μL巯基乙醇的加入打破二硫键和400μLphenylmethanesulphonyl氟停用蛋白酶消化细胞裂解后的蛋白质的利益。还增加了一个“鸡尾酒蛋白酶”（400μL0.2 mg / mL的胃酶抑素A。的400μL0.2 mg / mL的亮肽素; 400μL2 mg / ml的胰蛋白酶抑制剂），以确保蛋白提取物不分析前降低为目标利益。然后，我们添加400μL1M咪唑，是用来洗脱势必镍离子附着在微珠表面层析柱和脱氧核糖核酸酶我1MM，用于不必要的单，双链DNA降解成5 phosphodinucleotide和寡核苷酸的标签蛋白片段。 </li><li>在黑暗的混合搅拌30分钟冰，让足够的反应时间，自旋为60分钟，在10000 RPM的冷却离心机。收获上清，并准备为色谱执行与Ni - NTA（镍，氮基酸Superflow Qiagen公司）在离心管离心纺丝最大速度为3分钟，用裂解缓冲液洗涤，重复纺纱机和洗衣机，直到混合物是输液明确任何蓝色色调。由此产生的混合物搅拌均匀，慢慢地在冰浴60分钟黑暗的容器。 </li><li>执行柱层析流通过12分数如下。第一部分的流量通过10 mL上清液用Ni - NTA在上一步中混合。二是裂解缓冲液10毫升（50毫米的钠磷酸盐，300毫米20毫米氯化钠，咪唑，pH值8.0），第三个10毫升缓冲液（10毫米咪唑，100毫米氯化钠，pH 8.0），第四，其余0.5 mL的洗脱缓冲液（pH值8.0，氯化钠100毫米，250毫米咪唑） </li><li>准备的SDS - PAGE聚丙烯酰胺凝胶，并准备通过混合4μL的一小部分，1μL5X SB（300MM三6.8，25％的BME，10％SDS，50％的甘油）我们的样品。对于标准，我们使用了由Invitrogen 100 kb的DNA阶梯。在加载之前，我们煮5分钟，我们的样本。加载后，我们跑了60分钟的凝胶在120V。然后，我们的染色，destained并终于在2个小时的特殊凝胶在80C少雨干燥。 </li><li>然后，我们查看凝胶的结果，并设的分数，取得最好的结果。根据我们的利益的蛋白质，应分别位于频段的一种蛋白质的大小。这部分应选择MonoQ和Superdex 200（Amersham公司法玛西亚）进一步蛋白质纯化。 </li><li>纺纱与孔径大小的10K Nanosep过滤器最集中的分数，在14 5分钟的转速，洗衣机洗脱缓冲液，每次集中执行。这是必要的，以确定最终的蛋白浓度，利用BSA进一步蛋白标记过程检测。 </li><li>免入息审查贷款计划的2xGFP内在绿色荧光不能被用于核运输中，由于其光稳定性差及与细胞自体荧光光谱重叠的单分子成像。因此，我们的标签与货物分子在室温下2小时超过50 mM磷酸钠，150 mM氯化钠半胱氨酸反应的有机染料（Alexa555或Alexa的647酰亚胺分子探针）后，减少与TCEP磷酸三（2 - 羧）20分钟。淬火与2 - 巯基乙醇的反应，和蛋白质溶液透析除去免费的染料。 </li></ol> 3。单分子显微镜<ol><li>我们的显微镜设置包括配备1.4 NA 100X油浸泡目标（UPLSAPO 100XO，奥林巴斯）奥林巴斯IX81，一个35兆瓦633 nm的氦氖激光器（梅勒斯Griot），与绿色荧光蛋白过滤汞灯成立，芯片倍增增益电荷耦合器件摄像机（级联128 +，罗珀科学）和THË Slidebook软件包（智能成像创新）数据采集和处理。一个光学斩波器（新港）是用于产生激光激发的关闭模式。绿色荧光蛋白和Alexa647荧光汞灯和633 nm激光激发。绿色和红色荧光发射的目标是一致的分色过滤器（Di01-R405/488/561/635-25x36，Semrock）和排放过滤器（NF01-405/488/561/635-25X5的过滤，收集。 0，Semrock）和相同的CCD相机的成像。 </li><li>在单分子实验中，我们使单货分子荧光光漂白时间比运输时间长。要确定的漂白时间，0.1纳米的货物分子镀在玻璃盖滑和30分钟后，由于非特异性吸收，成为动弹不得。然后，测量荧光的时间当然是。取决于蛋白质感兴趣的光漂白时间会有所不同cysteins能够反应与马来酰亚胺，因此光照强度和光照面积的基础上，应调整，以免诱发了初步的漂白和确保货物与Alexa的647显示标记的分子荧光的时间比货物与NPC的互动。 </li><li>在通透细胞的进口实验，货物和必要的运输辅助因子加在一起。典型的进口货物反应中1毫米的GTP，Importin 0.5微米，0.5微米Importin - 1，2微米跑和1微米被动运输便利的交通和10KDa葡聚糖0.1纳米NTF2 ..批量实验，荧光货物浓度&gt; 100纳米〜100时为单分子检测。这些浓度&gt; 99％的货物是有望形成复合物与Importin和Importin。运输率监测epifluorescence（如果只是相对的利率是很重要的的）或共聚焦荧光显微镜（如果绝对率分别为所需）。 </li><li>核膜的立场首先是由宽领域epifluorescence显微镜从事HBO汞灯与GFP的过滤器设置一个图片的NE。在一行或一列的像素强度大约是垂直的NE与高斯拟合。特别设置的像素强度的高斯峰位置被认为是该行和列的东北位置。一系列如高斯峰位置，然后配合一个二次多项式，在整个图像中产生的NE的路径。 </li><li>一旦NE是局部的，它是必不可少的摆脱所有利用633 nm的氦氖激光的自体荧光，直到它完全光漂白，通常超过60秒就足够了。 </li><li>样品分子，然后将保持在1.5％的PVP溶液和混合物中添加一个25升的体积流室，然后通过应用滤纸芯分拖。再来一次护理后应采取的进口货物蛋白除了右后核膜本地化通透细胞以来，重点可以剧烈运动容易被打乱。 </li><li>只要货物分子的加入，633 nm的氦氖激光器是用来照亮分子的过境运输轨迹。大约5 Hz的一个光学斩波器（新港）应从事，以避免由于激光的高功率密度，以及允许一个新的部分非光漂白单分子方法的NE的漂白。应采取一系列的单分子易位的视频与高速CCD摄像头，可以上浮到2500赫兹。 </li></ol> 4。代表性的成果正确执行，我们的协议允许我们收集了一系列的视频展示了单货，在通透的HeLa POM 121细胞的核孔复合体相互作用的分子易位。 （图1）。我们的目标图像和跟踪通过毛地黄皂苷，通透的HeLa细胞中稳定表达GFP - POM121全国人大葡聚糖分子的瞬态运输步骤。细胞培养与低浓度的标记的葡聚糖分子。纳入集中核的赤道平面与清晰的图像了488纳米汞灯的光激发GFP荧光的绿色环。一个GFP - NE的长时间曝光，使我们获得一个很好的信号噪声比（SNR），以本地化的NE的中间平面。此前发现10 kDa的葡聚糖分子通过NE约2毫秒内弥漫了约20-40纳米的空间分辨率由500或1000赫兹的检测帧速率。这样的时空分辨率，允许捕获这是不够的澄清，更多的空间信息被动扩散通过全国人民代表大会的只有一到两个葡聚糖分子的扩散步骤。为了捕捉在全国人民代表大会更好spati的葡聚糖分子更细的步骤otemporal决议，我们已经进行了两大改进：（一）适应更快的检测，2500赫兹帧频捕捉到更加精细的扩散步骤;及（ii）雇用了更高的照明光密度从单个分子，以激发更多的光子，获得一个更高的空间分辨率。这些改进使我们捕捉到整个东北alexa647 -葡聚糖分子标记的瞬态扩散的多个步骤，辐射100 千瓦 /厘米2 633 nm激光时空分辨率的12 nm和400毫秒。 如图1所示，一系列静止图像的葡聚糖分子的典型交通活动记录。通过跟踪个别过境葡聚糖分子的空间运动轨迹，扩散的罚款通过NE步骤区别开来。我们发现，有大约30％的交互事件与NE已经认识到始发和目的地车厢。其中，从细胞质移动到东北的葡聚糖分子有两个目标：通过NE后扩散核或类似情况的东北出口事件发生相互作用后回迁到细胞质：葡聚糖分子从细胞核开始进入细胞质后弥漫原子核相互作用与NE。这样的图像系列包含关于全国人民代表大会，以及对分子的停留时间通过全国人民代表大会时间信息的单个分子的空间位置的信息。葡聚糖分子的运动轨迹，通过分析周围的东北地区，运输时间，效率和入口的频率才能确定。我们工作中的运输时间，可以被认定为进口或出口时。他们的平均停留时间葡聚糖分子在接受进口或出口的NPC被定义为。进口或出口的效率被定义为完整的进口或出口事件的完整和流产的事件的总和的百分比。进口或出口入口的频率是指分子在第二次与NE交互的数量。 <img src="/files/ftp_upload/2040/2040fig1.jpg" alt="图1" /> 图1。葡聚糖分子的典型交通事件，通过NE选择的视频帧。 （A）细胞质对核事件。一个单分子右旋糖酐（红点）开始从细胞质中，与NE（绿色像素线）相互作用，并在细胞核中结束。事件的轨迹（蓝线和开放点）进行了测定，并与NE（黑线）叠加。绿色和红色的线代表从NE 100纳米距离的细胞质和核方。 C时，细胞​​质中。 N的原子核。比例尺：2毫米。 （B）细胞质，细胞质事件。 （C）细胞核的核事件。 （D）细胞核，细胞质事件。

<ol>
	<li>Ma, J., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Snapshots+of+Three-Dimensional+Distribution+of+Transient+Interactions+in+Nuclear+Pore+Complex.">Single Molecule Snapshots of Three-Dimensional Distribution of Transient Interactions in Nuclear Pore Complex.</a> Proc. Natl. Acad. Sci. USA. , (2010).</li><li>Sarma, A., Goryaynov, A., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Imaging+of+the+Calcium-Regulated+Nuclear+Pore+Permeability.">Single Molecule Imaging of the Calcium-Regulated Nuclear Pore Permeability.</a> , .</li><li>Yang, W., Musser, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+single+molecules+transiting+through+nuclear+pore+complexes+using+narrow-field+epifluorescence+microscopy.">Visualizing single molecules transiting through nuclear pore complexes using narrow-field epifluorescence microscopy.</a> Methods. 39, 316-328 (2006).</li><li>Yang, W., Gelles, J., Musser, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+single-molecule+translocation+through+nuclear+pore+complexes.">Imaging of single-molecule translocation through nuclear pore complexes.</a> Proc. Natl. Acad. Sci. USA. 101, 12887-12892 (2004).</li></ol>

必须小心，以确保在实验过程中适当的NE和单分子共定位。同样重要的是提供一个高的信噪比，尤其是当信号为低由于恶劣的标签或漂白。定位精度是有限的背景噪声（焦荧光，CCD读出噪声，暗电流和其他因素引起的）。

single-molecule imaging of nuclear transport

实用的单分子荧光显微镜方法已被证明是一个很大的利用理解在过去十年中的生物反应。与细胞内深的高时间分辨率和空间分辨率的分子间相互作用的调查一直具有挑战性的，因为从单个分子所产生的固有的微弱信号。杨等人最近的作品。证明窄场epifluorescence显微镜允许在单分子水平的可视化的核质运输。通过单分子的方法，如核运输的时间和效率的重要动力学，信号依赖和独立的货运分子，已获得。在这里，我们描述的方法途径提高0.4毫秒和12 nm的时空分辨率的协议。提高了分辨率，使我们捕捉到半完整的细胞通过核孔复合物（NPC）在瞬态的主动转运和被动扩散事件。我们期望使用这种方法，在阐明细胞内的其他具有约束力的和贩运活动。

Watch this Scientific Journal Video about Single-Molecule Imaging of Nuclear Transport at JoVE.com

Single-Molecule Imaging of Nuclear Transport

单分子显微镜approch成核运输提供了新的见解。

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

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13.3K 次观看.  Bowling Green State University. 单分子显微镜approch成核运输提供了新的见解。

视频: Single-Molecule Imaging of Nuclear Transport

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

基于活细胞单分子成像的蛋白质相互作用定量

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

作者聚焦：活人细胞中蛋白质-凝聚物动力学的评估

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

生物分子缩合物中蛋白质相互作用动力学的单分子测量

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...