microKBA dataset

microKBA - microfluidic Kinesin Bead Assay This dataset contains raw and post-processed bead tracking data used to derive results and figures presented in the following manuscript: Marta Urbanska, Annemarie Lüdecke, Wim J. Walter, Antoine M. van Oijen, Karl E. Duderstadt, Stefan Diez Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors Small 2021, 2007388, https://doi.org/10.1002/smll.202007388 BiorXiv, https://doi.org/10.1101/2020.08.11.245910 Abstract Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, we describe an alternative, highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors. We applied tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilized a large field-of-view to simultaneously track the velocities, run lengths and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads. Importantly, the 16-μm long DNA tethers between the motors and the beads significantly reduced the vertical component of the applied force pulling the motors away from the microtubule. Our approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors. Content description **Final Analysis Files** * microKBA_Analysis.zip contains a slim version of post-processed tracking data used for obtaining final results and figures presented in the manuscript, the data is accompanied by MatLab scripts necessary to generate the final results and figures, the content of this folder is also available on github https://github.com/MartaUrb/microKBA/, see microKBA_Analysis_DirStructure for more details on content * microKBA_Analysis_DirStructure.txt describes content of microKBA_Analysis **Tracking Results** *microKBA_TrackingResults_DirStructure.txt describes directory structure of the zips below Following zip files contain raw and post-processed tracking results from individual experiments, see microKBA_TrackingResults_DirStructure for more details: * 141111_fc2_20ulpermin.zip * 141112_fc2_20ulpermin.zip * 150123_fc1_40ulpermin.zip * 150123_fc2_10ulpermin.zip * 150126_fc2_30ulpermin.zip * 150128_fc1_10ulpermin.zip * 150128_fc2_30ulpermin.zip * 150130_fc3_50ulpermin.zip **Software** * MatLab Functions.zip two MatLab functions necessary for running the post-tracking data filtering scripts * s_m_b.jar Single Molecule Biophysics plug-in for ImageJ used for generating and post-processing tracking results. This plug-in was developed by Karl E. Duderstadt, if you are interested in using a second generation of this tracking software called Molecular Archive Suite, mars, please visit the GitHub page of Duderstadt Lab https://github.com/duderstadt-lab/mars-coremicroKBA——微流控驱动蛋白微珠实验（microfluidic Kinesin Bead Assay） 本数据集包含原始及后处理后的微珠追踪数据，用于支撑下述学术论文中的研究结果与配图： 作者：Marta Urbanska、Annemarie Lüdecke、Wim J. Walter、Antoine M. van Oijen、Karl E. Duderstadt、Stefan Diez 论文标题：基于微流控的单个细胞骨架马达蛋白高通量力谱检测 期刊：Small 2021, 2007388，https://doi.org/10.1002/smll.202007388 预印本发布于BioRxiv：https://doi.org/10.1101/2020.08.11.245910 ## 摘要 细胞骨架马达蛋白可将化学能转化为机械功，以驱动核心细胞生命活动。光镊（optical trapping）实验为解析这类分子机器的负载响应机制提供了关键见解，但此类力谱（force spectroscopy）实验的通量通常仅支持单次单测量。本文报道一种高通量微流控替代方案，可快速获取细胞骨架马达蛋白的力依赖型运动参数。我们通过DNA锚定微珠（DNA-tethered beads）为步进式驱动蛋白-1（kinesin-1）马达施加可调谐的流体动力载荷，利用大视场（large field-of-view）同时追踪数百个单个驱动蛋白-1分子在不同阻性与辅助性载荷下的运动速度、行进距离与相互作用时长。值得注意的是，马达与微珠之间的16μm长DNA锚定链（DNA tether）可显著降低将马达拉离微管（microtubule）的外力垂直分量。本方法可便捷拓展至其他分子系统，为细胞骨架马达蛋白的并行化单分子力谱（single-molecule force spectroscopy）研究提供了全新手段。 ## 内容说明 ### 最终分析文件 * **microKBA_Analysis.zip**：包含用于生成论文最终结果与配图的精简版后处理追踪数据，配套提供生成最终结果与图表所需的MatLab脚本。该文件夹内容同时开源至GitHub仓库https://github.com/MartaUrb/microKBA/，详细目录结构请参考`microKBA_Analysis_DirStructure.txt`。 * **microKBA_Analysis_DirStructure.txt**：说明`microKBA_Analysis.zip`的目录内容构成。 ### 追踪结果 * **microKBA_TrackingResults_DirStructure.txt**：说明下述压缩包的目录结构。 以下压缩包包含单次独立实验的原始与后处理追踪结果，详细说明请参考`microKBA_TrackingResults_DirStructure.txt`： * 141111_fc2_20ulpermin.zip * 141112_fc2_20ulpermin.zip * 150123_fc1_40ulpermin.zip * 150123_fc2_10ulpermin.zip * 150126_fc2_30ulpermin.zip * 150128_fc1_10ulpermin.zip * 150128_fc2_30ulpermin.zip * 150130_fc3_50ulpermin.zip ### 软件工具 * **MatLab Functions.zip**：包含运行后追踪数据过滤脚本所需的两个MatLab函数。 * **s_m_b.jar**：用于生成并后处理追踪结果的ImageJ单分子生物物理学插件，由Karl E. Duderstadt开发。若需使用该软件的第二代版本**Molecular Archive Suite（简称Mars）**，请访问Duderstadt实验室的GitHub页面：https://github.com/duderstadt-lab/mars-core