Dynamic single-vesicle tracking of cell-bound membrane vesicles on resting, activated, and cytoskeleton-disrupted...

The composition, structure, production, motion, fate, and functions of cell-bound membrane vesicles pre-existing in the plasma membrane of cells are poorly understood. Here, single-vesicle tracking of individual cell-bound membrane vesicles in the plasma membrane of endothelial cells treated with or without various reagents was performed to investigate the motion of cell-bound membrane vesicles. The efficacy of each of these reagents was confirmed prior to single-vesicle tracking. Via single-vesicle tracking, we found that oxLDL, TNF-α, and VEGFα significantly increased the average number of cell-bound membrane vesicles per cell, implying that cell activation by oxLDL, TNF-α, and VEGFα could trigger the production of cell-bound membrane vesicles. It was also found that oxLDL, TNF-α, VEGFα, LPS, and MβCD but not LDL could significantly affect the motion speed of cell-bound membrane whereas none of them could significantly influence the displacement (moving range) of cell-bound membrane vesicles. The single-vesicle tracking further revealed that the average number of cell-bound membrane vesicles per cell and the mean speed/displacement of individual cell-bound membrane vesicles could be dramatically altered by the cytoskeleton-disrupting reagents (cytochalasin D and nocodazole). The data imply that the production and movement of cell-bound membrane vesicles are probably controlled by intracellular cytoskeletons and capable of being affected by multiple conditions e.g. cell activation, membrane fluidity alteration, and others.