The detection of the precise movement of a vesicle during transport in a live cell provides key information for the intracellular delivery process. Here we report a novel numerical method for analyzing three-dimensional vesicle movement. Since the vesicle moves along a linear cytoskeleton during the active transport, our method first detects the orientation and position of the cytoskeleton as a linear section based on angle correlation and linear regression, after noise reduction. Then, the precise vesicle movement is calculated using vector analysis, in terms of rotation angle and translational displacement. Using this method, various vesicle trajectories obtained via high spatiotemporal resolution microscopy can be understood..

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The transport dynamics of endocytic vesicles in a living cell contains essential biomedical information. Although the movement mechanism of a vesicle by motor proteins has been revealed, understanding the precise movement of vesicles on the cytoskeleton in a living cell has been considered challenging, due to the complex 3D network of cytoskeletons. Here, we specify the shape of the 3D interaction between the vesicle and microtubule, based on the theoretically estimated location of the microtubule and the vesicle trajectory data acquired at high spatial and temporal precision. We detected that vesicles showed more frequent direction changes with either in very acute or in obtuse angles than right angles, on similar time scales in a microtubule network. Interestingly, when a vesicle interacted with a relatively longer (> 400 nm) microtubule filament, rotational movement along the axis of the microtubule was frequently observed. Our results are expected to give in-depth insight into understanding the actual 3D interactions between the intracellular molecule and complex cytoskeletal network.

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