Duno-Miranda Sebastian, Warshaw David M, Nelson Shane R
Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington, Vermont.
Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington, Vermont.
Biophys Rep (N Y). 2025 Jun 21;5(3):100221. doi: 10.1016/j.bpr.2025.100221.
The In Vitro Motility Assay (IVMA) is a widely used experimental system to study the chemical and mechanical activity of myosin and other cytoskeletal motor proteins. In the IVMA, myosin molecules are bound to a glass surface and propel fluorescently labeled actin filaments across the surface, which are recorded using video fluorescence microscopy. The length and velocity of the actin filaments offer a measurement of the chemomechanical activity of the myosin motor proteins. Although the assay itself is well suited for high-throughput application, current video analysis approaches are slow, labor intensive, and subject to human bias. To address this shortfall, we introduce ATLAS, an open-source, platform independent software package that utilizes state-of-the-art machine learning algorithms to identify fluorescently labeled actin filaments and then track and analyze their motion in the IVMA. Utilizing both experimental data and a large array of simulated actomyosin motility movies, we demonstrate that ATLAS accurately and efficiently measures both the velocity and length of actin filaments across a broad range of experimental conditions.
体外运动分析（IVMA）是一种广泛应用的实验系统，用于研究肌球蛋白和其他细胞骨架运动蛋白的化学和机械活性。在IVMA中，肌球蛋白分子与玻璃表面结合，并推动荧光标记的肌动蛋白丝穿过表面，通过视频荧光显微镜进行记录。肌动蛋白丝的长度和速度提供了对肌球蛋白运动蛋白化学机械活性的测量。尽管该分析本身非常适合高通量应用，但目前的视频分析方法速度慢、劳动强度大且容易受到人为偏差的影响。为了解决这一不足，我们引入了ATLAS，这是一个开源的、与平台无关的软件包，它利用最先进的机器学习算法来识别荧光标记的肌动蛋白丝，然后在IVMA中跟踪和分析它们的运动。利用实验数据和大量模拟的肌动球蛋白运动电影，我们证明ATLAS能够在广泛的实验条件下准确、高效地测量肌动蛋白丝的速度和长度。
