AbstractResolving 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 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 well-known active intracellular transport and free diffusion were observed but new interactions, tight attachment and tethered rotation, were discovered for better interpretation of the dynamics of cargo-motor-track interactions at various types of microtubule intersections.Significance StatementTranslation and rotational motion of cargo during pauses at the microtubule intersections in living cells were revealed by high-accuracy three-dimensional single particle rotational tracking. The current study demonstrates the potential of studying coordinated interactions in living cellular environments by resolving characteristic rotational motions.
Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS
