这项工作得到了美国国家科学基金会研究生研究奖学金的支持。DGE-1745301 （S.Y.）、皮尤-斯图尔特学者奖 （S.C.）、Searle 学者奖 （S.C.）、Shurl 和 Kay Curci 基金会研究补助金 （SC）、Merkin 创新种子补助金 （SC）、Mallinckrodt 研究补助金 （SC） 和 Margaret E.早期医学研究信托基金 2024 年赠款（南卡罗来纳州）。SC 还得到了 NIH/NCI 的支持，奖项编号为 P30CA016042。

1. 细胞中蛋白质的标记
<ol><li>在所需细胞系中表达与 HaloTag 融合的目标蛋白。</li>
	<li>使用转座子或病毒转导在与 1.1 相同类型的细胞中稳定表达带有 Halo 标签的 H2B。</li>
</ol>2. 盖玻片的制备
<ol><li>在使用盖玻片进行细胞培养之前，清洁盖玻片以去除自发荧光污染物。<ol><li>将直径为 25 mm 的 #1.5 盖玻片安装在陶瓷染色架上，然后放入聚丙烯容器中。</li>
		<li>将盖玻片完全浸入1M的KOH溶液中并超声处理1小时。</li>
		<li>用双蒸水（ddH2O）冲洗盖玻片三次。</li>
		<li>将盖玻片完全浸入100%乙醇中并超声处理1小时。</li>
		<li>将盖玻片完全浸入用ddH2O稀释的20%乙醇中，并在4°C下储存直至使用。</li>
	</ol></li>
</ol>3. 显微镜细胞的制备
注意:在生物安全柜中执行本节中的步骤，以防止细胞污染。
<ol><li>将细胞铺在清洁的盖玻片上。如果要瞬时表达 Halo 标记的蛋白质，请在成像前 2 天进行。如果 Halo 标记的蛋白质稳定或内源性表达，则在成像前 1 天进行。<ol><li>使用无菌镊子将盖玻片放入 6 孔板（每个细胞样品一个盖玻片/孔）中，并让残留的乙醇蒸发。</li>
		<li>用适当数量的细胞铺板每个孔，以在成像时达到 70% 的汇合度。用具有相同靶汇合度稳定表达 H2B-Halo 的细胞再铺一块孔。</li>
		<li>仅当瞬时表达感兴趣的 Halo 标记蛋白时，才执行此步骤。将细胞在37°C和5%CO2下孵育24小时。然后，使用适当的转染试剂并遵循制造商的指南，用编码目标标记蛋白的质粒转染细胞。</li>
		<li>将细胞在37°C和5%CO2下孵育24小时。</li>
	</ol></li>
	<li>用不可光活化的 Halo 配体（例如 JFX549）和可光活化的 Halo 配体（例如 PA-JF646）对表达目标 Halo 标记蛋白的细胞进行染色。仅用光活化 Halo 配体染色表达 H2B-Halo 的细胞。 注意:此处列出的 Halo 配体浓度应用作起点;最佳浓度将取决于目标蛋白的表达水平和缩合物局部浓度。<ol><li>对于表达目标 Halo 标记蛋白的每个细胞样品，将 100 nM JFX549 Halo 配体35 和 20 nM PA-JF646 Halo 配体36 稀释在 500 μL 适当的细胞培养基中。对于每个表达 H2B-Halo 的样品，在 500 μL 适当的细胞培养基中仅稀释 20 nM PA-JF646 Halo 配体。</li>
		<li>对于每个孔，吸出现有培养基，加入 2 mL 1x PBS，然后吸出 PBS（在方案的其余部分称为"冲洗"），并替换为含有适当 Halo 配体的培养基。</li>
		<li>在37°C和5%CO2下孵育1小时。</li>
		<li>用 PBS 冲洗四次，然后用不含 Halo 配体的新鲜培养基替换。</li>
		<li>在37°C和5%CO2下孵育15分钟。</li>
		<li>重复步骤 3.2.4 和 3.2.5 三次（总共四个冲洗孵育周期）。</li>
		<li>使用无菌镊子将装有细胞的盖玻片转移到与显微镜载物台兼容的细胞培养盖玻片室中。</li>
		<li>将无酚红培养基加入腔室并进行成像。在37°C和5%CO2 下孵育不立即成像的样品，直到需要。</li>
	</ol></li>
</ol>4. 单分子成像
注意:测量H2B和目标蛋白停留时间的独立实验应在多（≥3）天内进行，以产生具有统计学意义的结果。在显微镜上对细胞样品进行成像之前，使用0.1μm染色微球（材料表）或类似的校准标准品对准两个相机。
<ol><li>准备显微镜。<ol><li>打开显微镜和控制显微镜的计算机。</li>
		<li>在显微镜上打开活细胞培养组件（加热器和CO2），并将其设置为37°C和5%CO2。让系统达到平衡。</li>
		<li>将一滴适当的油滴在显微镜上的 100 倍 TIRF 油浸物镜上，然后将细胞样品加载到显微镜载物台上。</li>
	</ol></li>
	<li>识别要成像的单元格。<ol><li>在明场照明下对细胞样品进行成像，并调整 z 位置，直到细胞聚焦。</li>
		<li>识别具有健康细胞形态特征的细胞。</li>
		<li>裁剪视场 （FOV），使其仅捕获目标细胞核。</li>
		<li>使用激光照明并调整TIRF角度，直到达到具有最佳单分子信噪比的HILO照明41 。</li>
	</ol></li>
	<li>成像活细胞中的 H2B-Halo。 注意:本节中使用的激光功率特定于此实验，并作为示例包含在内。应根据讨论中列出的标准选择适当的激光功率。<ol><li>按如下方式设置采集配置。将曝光时间设置为 500 毫秒。在每次 500 ms 曝光期间，用 9.1 mW、640 nm 光束激发 PA-JF646 标记的 H2B-Halo 分子。在帧之间的死区时间（此处使用的成像系统为158.01 μs），用111 μW，405 nm光束光活化分子。</li>
		<li>在这些设置下连续捕获 2000 帧。当光活化分子的数量不足时，在采集过程中逐渐增加 405 nm 光束的功率。</li>
	</ol></li>
	<li>对活细胞中感兴趣的 Halo 标记蛋白进行成像。<ol><li>使用长通二向色镜在两个相机之间分离发射波长，一个用于检测 PA-JF646，另一个用于检测 JFX549。在摄像机前使用适当的发射滤光片。</li>
		<li>使用 4.3.1 中描述的相同采集配置，并进行以下修改。添加一个额外的 JFX549 通道，以跟踪 Halo 标记的蛋白质缩合物随时间推移的位置。每 10 秒在 JFX549 通道中采集一帧（500 ms，2.1 mW，561 nm 激发），同时保持对 PA-JF646 通道的采集。这将导致渗漏到PA-JF646通道中，这将在采集后的数据分析中得到处理。</li>
		<li>在这些设置下连续捕获 2000 帧。当光活化分子的数量不足时，在采集过程中逐渐增加 405 nm 光束的功率。</li>
	</ol></li>
</ol>5. 单分子成像数据分析
注意:第 5 节中使用的参数特定于此实验，并作为示例包含在内。应根据讨论中列出的标准选择适当的参数。
<ol><li>准备原始成像数据进行处理。<ol><li>对于目标蛋白质的数据，使用ImageJ或类似的图像处理软件将原始成像数据文件中的每个通道转换为独立的.tif文件。对于H2B的数据，以同样的方式将原始成像数据文件中的单通道转换为.tif文件。 注意:根据所使用的采集软件，冷凝水电影中可能存在空帧，因为每 10 秒只拍摄一个冷凝水跟踪帧。空帧应填充最新的冷凝水帧（某些采集软件会自动执行此操作）。此外，如果在这些缩合物跟踪帧期间，从缩合物 （JFX549） 通道到单分子 （PA-JF646） 通道有明显的渗漏，则相应的单分子帧应替换为最近的单分子帧。</li>
		<li>如果由于上述原因，任一电影中有任何帧需要替换，请通过修改（如有必要）并运行 pretracking_comb.txt（Chong 等人 1 中可用的 ImageJ 宏）并按照提示将其替换为该电影中的最新帧。</li>
	</ol></li>
	<li>使用SPT软件生成单粒子轨迹，例如，SLIMfast39，Teves等人42中可用的MTT算法38的GUI实现。<ol><li>从 5.1.2 加载包含 SLIMfast 中单分子电影的文件，方法是单击 Load > Imagestack。</li>
		<li>设置参数（本地化错误率:10-6;通货紧缩循环:3） 通过单击 OPT > 工作表:本地化进行定位。</li>
		<li>设置参数（峰值发射:664 nm; 滞后时间:500 ms），通过单击 OPT > Sheet: Acquisition 进行采集。</li>
		<li>在单个帧中可视化所有分子的定位，以确保选择的参数是适当的（TEST LOC）。如果不合适，请修改步骤 5.2.2 和 5.2.3 中的参数并重复此步骤。</li>
		<li>生成一个文件，其中包含每帧中所有分子的定位 （LOC ALL）。</li>
		<li>在 SLIMfast 中从 5.2.5 加载文件，方法是单击 > SLIMfast >加载粒子数据。</li>
		<li>设置参数（最大预期扩散系数:0.1μm2/s; 最大参赛者数量:5）通过单击 OPT生成轨迹 > 表:跟踪。</li>
		<li>生成一个包含所有分子轨迹的文件 （GEN TRAJ）。</li>
	</ol></li>
	<li>使用 Drosopoulos 等人 43 中提供的 evalSPT39 过滤掉短于 tmin（2.5 秒）的轨迹，以解释导致人为缩短轨迹的跟踪误差。<ol><li>将文件从 5.2.8 加载到 evalSPT 中（+ > 5.2.8 中的文件>正常）。</li>
		<li>设置参数（最小:5 帧; max:从 5.2.8 开始的文件的最大轨迹长度）以过滤掉短于 2.5 秒的轨迹。</li>
		<li>生成包含所有过滤轨迹的文件 （EXPORT DATA）。</li>
	</ol></li>
	<li>仅针对目标蛋白质的轨迹遵循此步骤。将轨迹分类为冷凝水和冷凝水外的轨迹，然后提取平均冷凝水内停留时间。 注意:本节的所有代码都可以在 Chong et al.1 中找到。<ol><li>对 JFX549 通道中采集的影片的所有帧进行阈值，以生成一个延时短片，该短片填充了时间演进的二进制蒙版，通过运行 ImageJ 宏、细胞核和聚类 mask_v2.txt，突出显示凝聚物位置，并按照提示进行操作。</li>
		<li>重新格式化 5.3.3 中生成的单分子轨迹。使用 MATLAB 脚本 ConvertASCII_SlowTracking_css3.m，并根据需要调整文件名、路径和参数。（参数: 曝光，0.5秒; 分辨率，0.16 μm/像素）。</li>
		<li>使用 MATLAB 脚本 categorization_v4.m 根据给定分子在凝聚物 F 中度过的寿命分数对轨迹进行排序，并根据需要调整文件名和路径。</li>
		<li>仅使用冷凝水轨迹继续。</li>
	</ol></li>
	<li>提取 kobs，s 和 kpb 并计算校正后的平均停留时间 <img alt="Equation 6" src="/files/ftp_upload/66169/66169eq05.jpg" style="margin: auto;">。<ol><li>使用 MATLAB 脚本 PLOT_ResidenceHist_css.m 从目标缩合物蛋白轨迹中提取 kobs，s，并根据需要调整文件名、路径和参数。 （参数: 曝光，0.5秒; StartFrameForFit，5 帧）。</li>
		<li>使用 MATLAB 脚本 PLOT_ResidenceHist_css.m 从 H2B 轨迹中提取 kpb，并根据需要调整文件名、路径和参数。 （参数: 曝光，0.5秒; StartFrameForFit，5 帧）。</li>
		<li>计算与其缩合物特异性结合的目标蛋白质的校正平均停留时间， <img alt="Equation 6" src="/files/ftp_upload/66169/66169eq05.jpg" style="margin: auto;">如下所示 <img alt="Equation 7" src="/files/ftp_upload/66169/66169eq07v3.jpg" style="margin: auto;"> 注意:应执行对照以验证足够长以模糊移动颗粒的不同暴露时间，例如300ms和800ms，仍然导致目标蛋白质的平均停留时间相同。</li>
	</ol></li>
</ol>

基于活细胞单分子成像的蛋白质相互作用定量

作者没有什么可透露的。

这里介绍的协议是为Irgen-Gioro等人40中研究的系统而设计的。根据应用的不同，可以修改方案的某些组件，例如，生成荧光标记细胞系的方法、荧光标记系统以及使用的盖玻片样式。细胞中蛋白质的光晕标记可以使用两种策略来完成，具体取决于哪种策略更适合给定的实验。1） 外源表达:将目标蛋白融合到 HaloTag 中，并在靶细胞系中使用转染瞬时表达或使用转座子或病毒转导稳定表达融合。2）基因组编辑:使用基因组编辑技术（例如CRISPR44）在靶细胞系中编码目的内源性蛋白质的基因位点敲入HaloTag。每种方法的优点和缺点在别处讨论45，但简而言之，外源性表达策略耗时较少，但会产生具有广泛表达水平的细胞群，这些表达水平通常是非生理性的。基因组编辑策略需要更长的时间，但可以在其天然表达水平上标记内源性表达的目标蛋白。
此外，该协议没有明确要求使用 HaloTag;相反，它与任何能够同时对细胞中的相同蛋白质进行单分子和集成标记的标签兼容。因此，可以修改该协议以与其他自标记标签（如 SNAP-tag46）一起使用。最后，可以使用用于细胞培养的预清洁商业玻璃底培养皿，例如MatTek培养皿（MatTek Life Sciences，P35G-1.5-20-C），代替盖玻片室，前提是它们与显微镜物镜和浸油兼容;但是，盖玻片可以在使用前立即更彻底地清洁，因此是可取的。
虽然上述对方案的修改是可选的，但必须优化实验和分析参数，即HaloTag配体的浓度，激光功率，定位和跟踪参数以及tmin。应选择不可光活化的 HaloTag 配体的浓度，使缩合物标记得足够密集，以便能够生成掩模。应选择光活化 HaloTag 配体的浓度和光活化激光功率，以便对蛋白质分子进行标记和光活化，以产生高质量的单粒子轨迹，因为每帧中过多的定位将导致不准确的轨迹生成。对于代表性结果中显示的实验，每帧通常少于五个定位。激发激光功率应保持在足够低的水平，以尽量减少快速光漂白，但应保持足够高，以精确定位单个分子。单分子定位和跟踪参数应根据激发密度和目标蛋白质的预期扩散动力学进行调整。最后，应在 tmin 范围内计算 的<img alt="Equation 6" src="/files/ftp_upload/66169/66169eq05.jpg" style="margin: auto;">值（从 0 秒开始，以曝光时间递增）。的<img alt="Equation 6" src="/files/ftp_upload/66169/66169eq05.jpg" style="margin: auto;">值应收敛于某个阈值 tmin;此 tmin 应在步骤 5.3 中使用。
这里概述的方法以传统技术无法达到的精度量化了缩合物中蛋白质的相互作用动力学。此外，它可以在活细胞中以最小的样品扰动做到这一点。这一点至关重要，因为IDR的相互作用行为，包括其凝结物的形成，高度依赖于其局部环境40。
Irgen-Gioro 等人 40 的代表性结果表明，该方法能够提取与其自组装 LLPS 缩合物结合的蛋白质的停留时间。重要的是，该方法可以很容易地扩展到通过同型或异型相互作用与任何类型的缩合物结合的任何蛋白质。此外，它不仅限于测量经历LLPS的蛋白质的相互作用动力学。已知可引起尤文肉瘤的融合癌蛋白 EWS::FLI1 已被证明可形成局部高浓度枢纽，在其转录激活和致癌转化功能中起重要作用 1,2。虽然这些枢纽的形成是由 EWS::FLI1 的瞬时、选择性和多价 IDR-IDR 相互作用驱动的，但到目前为止，仍然没有令人信服的证据表明它们是真正的 LLPS 缩合物 1,2。即便如此，我们还是使用这里介绍的方法测量了 EWS::FLI1 在其中心的平均停留时间，并表明特定残基的突变及其 IDR 的缺失显着破坏了 EWS::FLI1 与其中心1 的结合。
尽管该方法在表征缩合物中蛋白质的结合动力学方面具有通用性，但在特定情况下选择蛋白质结合模型时应谨慎行事。现有的生化数据支持这样一种观点，即双分量指数分布通常是许多系统1,37,40,42的合适模型，但相同的分布也被证明是蛋白质结合在其他系统中的不恰当表示，其中替代模型如三分量指数47,48,49和幂律分布50更好地匹配实验观察结果。为了避免得出不准确的结论，人们应该仔细选择和激励一个模型，验证拟合的质量是否支持模型的使用，并明智地解释结果。

越来越多的研究表明，生物分子凝聚物在细胞组织和众多细胞功能中起着重要作用，例如转录调控1、2、3、4、5、DNA 损伤修复6、7、8、染色质组织9、10、11、12、X 染色体失活 13,14,15 和细胞内信号转导 16,17,18。此外，生物分子缩合物的失调与许多疾病有关，包括癌症19,20,21和神经退行性疾病22,23,24,25,26。缩合物的形成通常由瞬时、选择性和多价蛋白质-蛋白质、蛋白质-核酸或核酸-核酸相互作用驱动27。在某些条件下，这些相互作用会导致液-液相分离（LLPS），这是一种密度跃迁，可局部富集无膜液滴中的特定生物分子。这种多价相互作用通常由蛋白质的固有无序区域 （IDR） 介导 1,28,29。鉴于凝聚物在分子水平上的普遍存在，在分子水平上对这些相互作用的生物物理表征对于我们理解许多健康和异常的细胞功能至关重要。尽管基于共聚焦荧光显微镜的技术，例如光漂白后的荧光恢复（FRAP）30,31,32，已被广泛用于定性地表明凝聚物与周围细胞环境之间的分子交换是动态的，但使用传统的共聚焦显微镜或单分子通常无法量化凝聚物中特定生物分子的相互作用动力学没有专门数据分析方法的显微镜。该协议中描述的单颗粒跟踪（SPT）技术基于活细胞单分子显微镜33，并提供了一种独特而强大的工具来量化缩合物中特定蛋白质之间的相互作用动力学。用于此类测量的 SPT 读数是目标蛋白质在缩合物中的平均停留时间。
该协议可分为两部分 - 数据采集和数据分析。成像数据采集的第一步是在细胞中表达与 HaloTag34 融合的目标蛋白质。这样就可以用两个荧光团标记感兴趣的蛋白质，其中大多数蛋白质分子用不可光活化的荧光团（例如，JFX549 Halo 配体35）标记，其中一小部分用光谱不同的、可光活化的荧光团（例如，PA-JF646 Halo 配体36).这允许同时采集细胞中的所有凝聚物位置，并采集目标蛋白质与凝聚物结合和解结合的单分子电影。同时，相同类型的细胞被修饰以稳定表达带有晕标记的H2B，这是一种在染色质上基本不动的组蛋白。然后用 PA-JF646 Halo 配体对细胞进行染色，以实现 H2B 的单分子成像。正如下面将详细讨论的那样，该实验解释了光漂白的贡献，以便能够精确定量目标蛋白质的相互作用动力学。然后，必须在干净的盖玻片上培养用于成像实验的细胞，用HaloTag配体染色，并组装到活细胞成像室中。从那里，样品在能够进行双通道成像和单分子检测的全内反射荧光 （TIRF） 显微镜上在高度倾斜和层压光学片 （HILO） 照明下成像。然后将发射物分成两个摄像头，一个跟踪冷凝物位置，一个跟踪单个分子。采集以较长的整合时间（大约数百毫秒）进行，以模糊自由扩散的蛋白质，并且仅捕获由于与细胞中的稳定结构结合而移动性较差的蛋白质37。
数据分析的第一步是使用已建立的单粒子跟踪 （SPT） 算法38,39 在电影的每一帧中定位单个蛋白质分子，并在其可检测的生命周期内将定位组装成每个分子的轨迹。然后，通过将分子在整个轨迹中的定位与相应时间所有凝聚物的定位进行比较，将轨迹分为代表凝聚物内部分子的轨迹和表示凝聚物外部分子的轨迹 1.
接下来，使用所有冷凝水轨迹的长度生成生存曲线 （1 - CDF）。然后通过将存活曲线拟合到以下蛋白质结合的双组分指数模型来提取分子的表观平均停留时间，
<img align="center" alt="Equation 1" src="/files/ftp_upload/66169/66169eq01.jpg" style="margin: auto;">,
其中 A 为非特异性结合分子的分数， kobs，ns 和 kobs，s 分别为非特异性结合分子和特异性结合分子的观察到的解离速率。从这里开始只考虑 kobs，s 。蛋白质解离 ktrue，s 和荧光团 kpb 的光漂白的动力学有助于 kobs，s 作为
<img alt="Equation 2" src="/files/ftp_upload/66169/66169eq02.jpg" style="margin: auto;">;
因此，为了分离蛋白质解离的影响，测量了前面提到的细胞系中H2B-Halo的特异性解离速率。
<img alt="Equation 3" src="/files/ftp_upload/66169/66169eq03.jpg" style="margin: auto;">
H2B 是一种稳定整合到染色质中的蛋白质，在单分子电影采集的时间尺度上经历最小的解离37。其比解离速率等于 PA-JF646 Halo 配体的光漂白速率，或
<img alt="Equation 4" src="/files/ftp_upload/66169/66169eq04.jpg" style="margin: auto;">.
目标 <img alt="Equation 5" src="/files/ftp_upload/66169/66169eq05.jpg" style="margin: auto;">蛋白的平均缩合物停留时间为
<img alt="Equation 6" src="/files/ftp_upload/66169/66169eq06.jpg" style="margin: auto;">.
显示了Irgen-Gioro等人40 的代表性结果，其中应用该协议来证明将寡聚结构域与IDR融合会导致IDR在其缩合物中的停留时间更长。这一结果表明，添加的寡聚化结构域稳定了驱动LLPS的IDR的同型相互作用。原则上，具有略微修改方案的相同方法可用于表征参与任何类型缩合物形成的任何蛋白质的同型或异型相互作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 &#181;m TetraSpeck microsphere</td>
			<td>Invitrogen</td>
			<td>T7279</td>
			<td>Single-molecule imaging</td>
		</tr>
		<tr>
			<td>25 mm Diameter, #1.5 Coverslips</td>
			<td>Marienfeld Superior</td>
			<td>111650</td>
			<td>Preparation of coverslips</td>
		</tr>
		<tr>
			<td>593/40 nm bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-593/40-25</td>
			<td>Single-molecule imaging</td>
		</tr>
		<tr>
			<td>676/37 nm bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-676/37-25</td>
			<td>Single-molecule imaging</td>
		</tr>
		<tr>
			<td>6-Well TC Plate</td>
			<td>Genesee</td>
			<td>25-105MP</td>
			<td>Preparation of cells for microscopy</td>
		</tr>
		<tr>
			<td>Cell Line: U-2 OS</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td>Labeling of proteins in cells</td>
		</tr>
		<tr>
			<td>ConvertASCII_SlowTracking_css3 
			.m</td>
			<td></td>
			<td></td>
			<td>Analysis of single-molecule imaging data: Available in Chong et al., 2018</td>
		</tr>
		<tr>
			<td>Coverglass Staining Rack</td>
			<td>Thomas</td>
			<td>24957</td>
			<td>Preparation of coverslips</td>
		</tr>
		<tr>
			<td>Deuterated Janelia Fluor 549 (JFX549)</td>
			<td>Janelia Research Campus</td>
			<td></td>
			<td>Preparation of cells for microscopy</td>
		</tr>
		<tr>
			<td>DMEM, Low Glucose</td>
			<td>Gibco</td>
			<td>10-567-022</td>
			<td>Labeling of proteins in cells: Growth media used: DMEM with 5% fetal bovine serum, 1% penstrep</td>
		</tr>
		<tr>
			<td>Eclipse Ti2-E Inverted Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Single-molecule imaging</td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>Lab Alley</td>
			<td>EAP200-1GAL</td>
			<td>Preparation of coverslips</td>
		</tr>
		<tr>
			<td>evalSPT</td>
			<td></td>
			<td></td>
			<td>Analysis of single-molecule imaging data: Available in Drosopoulos et al., 2020</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Cytiva</td>
			<td>SH30396.03</td>
			<td>Labeling of proteins in cells: Growth media used: DMEM with 5% fetal bovine serum, 1% penstrep</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>Analysis of single-molecule imaging data</td>
		</tr>
		<tr>
			<td>Ikon Ultra CCD Camera</td>
			<td>Andor</td>
			<td>X-13723</td>
			<td>Single-molecule imaging</td>
		</tr>
		<tr>
			<td>Longpass dichroic beamsplitter</td>
			<td>Semrock</td>
			<td>Di02-R635-25x36</td>
			<td>Single-molecule imaging: Red/Far Red beamsplitter</td>
		</tr>
		<tr>
			<td>LUN-F Laser Unit</td>
			<td>Nikon</td>
			<td></td>
			<td>Single-molecule imaging: 405/488/561/640</td>
		</tr>
		<tr>
			<td>MatTek glass-bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td>Preparation of cells for microscopy: 35 mm, #1.5 coverslip dish for cell culture.</td>
		</tr>
		<tr>
			<td>NIS-Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Single-molecule imaging: Microscope acquisition software</td>
		</tr>
		<tr>
			<td>nucleus and cluster mask_v2.txt</td>
			<td></td>
			<td></td>
			<td>Analysis of single-molecule imaging data: Available in Chong et al., 2018</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15-140-122</td>
			<td>Labeling of proteins in cells: Growth media used: DMEM with 5% fetal bovine serum, 1% penstrep</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Thermo Fisher Scientific</td>
			<td>18912014</td>
			<td>Labeling of proteins in cells</td>
		</tr>
		<tr>
			<td>Photoactivatable Janelia Fluor 646 (PA-JF646)</td>
			<td>Janelia Research Campus</td>
			<td></td>
			<td>Preparation of cells for microscopy</td>
		</tr>
		<tr>
			<td>PLOT_ResidenceHist_css.m</td>
			<td></td>
			<td></td>
			<td>Analysis of single-molecule imaging data: Available in Chong et al., 2018</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Mallinckrodt Chemicals</td>
			<td>6984-06</td>
			<td>Preparation of coverslips</td>
		</tr>
		<tr>
			<td>pretracking_comb.txt</td>
			<td></td>
			<td></td>
			<td>Analysis of single-molecule imaging data: Available in Chong et al., 2018</td>
		</tr>
		<tr>
			<td>SLIMfast</td>
			<td></td>
			<td></td>
			<td>Analysis of single-molecule imaging data: Available in Teves et al., 2016</td>
		</tr>
		<tr>
			<td>Stage-top incubation system</td>
			<td>Tokai Hit</td>
			<td></td>
			<td>Single-molecule imaging: For live-cell imaging</td>
		</tr>
		<tr>
			<td>TwinCam dual emission image splitter</td>
			<td>Cairn Research</td>
			<td></td>
			<td>Single-molecule imaging</td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Branson</td>
			<td>5800</td>
			<td>Preparation of coverslips</td>
		</tr>
	</tbody>
</table>

single-molecule measurement of protein interaction dynamics within biomolecular condensates

通过液-液相分离 （LLPS） 形成的生物分子缩合物被认为在细胞组织和越来越多的细胞功能中至关重要。表征活细胞中的LLPS也很重要，因为异常凝聚与许多疾病有关，包括癌症和神经退行性疾病。LLPS 通常由内在无序蛋白质之间的选择性、瞬时性和多价相互作用驱动。非常令人感兴趣的是参与LLPS的蛋白质的相互作用动力学，通过测量它们的结合停留时间（RT），即它们在缩合物中结合的时间，可以很好地总结出来。在这里，我们提出了一种基于活细胞单分子成像的方法，该方法使我们能够测量缩合物中特定蛋白质的平均RT。我们同时可视化单个蛋白质分子及其与之相关的缩合物，使用单颗粒跟踪 （SPT） 绘制单分子轨迹，然后将轨迹拟合到蛋白质-液滴结合模型以提取蛋白质的平均 RT。最后，我们展示了代表性结果，其中应用这种单分子成像方法比较蛋白质在其 LLPS 缩合物下融合和未融合到寡聚结构域时的平均 RT。该协议广泛适用于测量参与LLPS的任何蛋白质的相互作用动力学。

在这里，我们介绍了Irgen-Gioro等人40的代表性结果，我们使用该SPT协议比较了两种蛋白质在各自的自组装LLPS缩合物中的相互作用动力学。TAF15（TATA-box 结合蛋白相关因子 15）含有一个 IDR，在人细胞中过表达时可以发生 LLPS。我们假设将 TAF15（IDR） 与形成 24 个亚基寡聚体的 FTH1（铁蛋白重链 1）融合将导致更稳定的同型蛋白质-蛋白质相互作用，从而驱动 LLPS。为了验证这一假设，我们在 U2OS 细胞中瞬时表达 Halo-TAF15（IDR） 或 TAF15（IDR）-Halo-FTH1 融合，并按照上述方案对每种蛋白质进行双色单分子成像。 图1A显示了TAF15（IDR）-Halo-FTH1电影的代表性帧。在PA-JF646通道中检测到的分子被定位，这些定位被组装成轨迹。然后，通过与突出冷凝水位置的二元掩模进行比较，在冷凝水内和冷凝水外种群之间对生成的轨迹进行分类（图1B）。虽然为了获得良好的能见度，我们只显示了一小部分轨迹，但与凝聚物结合和结合在凝聚物外的分子的轨迹之间有明显的区别。接下来，根据双分量指数模型拟合冷凝水轨迹长度的生存曲线（图1C）。最后，提取光漂白校正后的平均停留时间，并绘制两种蛋白质的图（图1D）。结果表明，Halo-TAF15（IDR）在LLPS缩合物中的平均停留时间为10.23 s±1.10 s，而TAF15（IDR）-Halo-FTH1的平均停留时间为64.15 s±11.65 s。这一结果表明，在TAF15（IDR）中添加寡聚结构域确实可以稳定蛋白质-缩合物结合。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66169/66169fig01.jpg"> 图 1:基于 SPT 的方法解决了蛋白质在其缩合物中停留时间的差异。 答:TAF15（IDR）-Halo-FTH1双色单分子电影的代表性帧。用较高浓度的非光活化染料（100 nM JFX549，黄色）和较低浓度的光活化染料（20 nM PA-JF646，品红色）标记蛋白质以观察单个蛋白质，从而实现SPT。 Halo-TAF15（IDR）采集相同成像条件下的胶片，H2B-Halo采集单通道PA-JF646胶片。一条白色虚线勾勒出原子核的轮廓。B.代表性的合并图像覆盖了凝结物位置（灰色）和轨迹（多色）的二进制掩模。C.拟合双分量指数模型的TAF15（IDR）-Halo-FTH1的代表性生存曲线。D.Halo-TAF15在各自缩合物中的平均停留时间明显短于TAF15（IDR）-Halo-FTH1。在三天内进行的独立实验中测量的 20 个细胞中平均每种蛋白质的值。误差作为平均值的标准误差传播，星号表示两种蛋白质停留时间的显著差异（p<0.05，Wilcoxon 秩和检验）。该图经Irgen-Gioro等人许可改编40。<a href="https://www.jove.com/files/ftp_upload/66169/66169fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells at JoVE.com

作者聚焦:活人细胞中蛋白质-凝聚物动力学的评估

许多固有的无序蛋白质已被证明参与高动态生物分子凝聚物的形成，这种行为对许多细胞过程很重要。在这里，我们提出了一种基于单分子成像的方法，用于量化活细胞中生物分子缩合物中蛋白质相互作用的动力学。

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 ...

single-molecule-measurement-protein-interaction-dynamics-within

Universidad San Sebastian

Research

JoVE Journal

Biology

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

3.0K 次观看.  California Institute of Technology. 1 我们的实验室旨在了解固有无序蛋白质区域 3 的相互作用行为 2 （interaction behaviors），以及它们如何在健康和患病人类细胞的转录调节 4 中发挥作用。5 在我们的实验室中，我们开发和使用新颖的 6 单分子显微镜技术 7 与分子生物学、8 生化和蛋白质组学方法 9 相结合来研究生物分子凝聚物。10 该协议能够量化动力学 11 通过特定的蛋白质 12 与活?...

视频: 作者聚焦:活人细胞中蛋白质-凝聚物动力学的评估

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, ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

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 ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

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 ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...