Department of Chemistry, Georgia State University, Atlanta, Georgia.
Department of Chemistry, Georgia State University, Atlanta, Georgia.
Biophys J. 2021 Apr 20;120(8):1378-1386. doi: 10.1016/j.bpj.2020.11.2278. Epub 2020 Dec 25.
Resolving coordinated biomolecular interactions in living cellular environments is vital for understanding the mechanisms of molecular nanomachines. The conventional approach relies on localizing and tracking target biomolecules and/or subcellular organelles labeled with imaging probes. However, it is challenging to gain information on rotational dynamics, which can be more indicative of the work done by molecular motors and their dynamic binding status. Herein, a bifocal parallax single-particle tracking method using half-plane point spread functions has been developed to resolve the full-range azimuth angle (0-360°), polar angle, and three-dimensional (3D) displacement in real time under complex living cell conditions. Using this method, quantitative rotational and translational motion of the cargo in a 3D cell cytoskeleton was obtained. Not only were well-known active intracellular transport and free diffusion observed, but new interactions (tight attachment and tethered rotation) were also discovered for better interpretation of the dynamics of cargo-motor-track interactions at various types of microtubule intersections.
解析活细胞环境中协调的生物分子相互作用对于理解分子纳米机器的机制至关重要。传统方法依赖于定位和跟踪用成像探针标记的靶标生物分子和/或亚细胞细胞器。然而，获取有关旋转动力学的信息具有挑战性，因为它更能指示分子马达的做功及其动态结合状态。在此，开发了一种双焦点视差单粒子跟踪方法，该方法使用半平面点扩散函数实时解析复杂活细胞条件下的全范围方位角（0-360°）、极角和三维（3D）位移。使用该方法，获得了货物在 3D 细胞细胞骨架中的定量旋转和平移运动。不仅观察到了众所周知的主动细胞内运输和自由扩散，而且还发现了新的相互作用（紧密附着和系绳旋转），以便更好地解释在各种类型的微管交叉处货物-马达-轨道相互作用的动力学。
