Vesicle movements are governed by the size and dynamics of F-actin cytoskeletal structures in bovine chromaffin cells

Abstract

Dense vesicles can be observed in live bovine chromaffin cells using fluorescent reflection confocal microscopy. These vesicles display a similar distribution, cytoplasmic density and average size as the chromaffin granules visualized by electron microscopy. In addition, the acidic vesicles labeled with Lysotracker Red comprised a subpopulation of the vesicles that are visualized by reflection fluorescence. A combination of fluorescence reflection and transmitted light images permitted the movements of vesicles in relation to the cortical cytoskeleton to be studied. The movement of vesicles located on the outside of this structure was restricted, with an apparent diffusion coefficient of 1.0±0.4×10−4 μm2/s. In contrast, vesicles located in the interior moved much more freely and escaped from the visual confocal plane. Lysotracker labeling was more appropriate to study the movement of the faster moving vesicles, whose diffusion coefficient was five times higher. Using this type of labeling we confirmed the restriction on cortical movement and showed a clear relationship between vesicle mobility and the kinetics of cytoskeletal movement on both sides of the cortical cytoskeleton. This relationship was further emphasized by studying cytoskeletal organization and kinetics. Indeed, an estimate of the size of the cytoskeletal polygonal cages present in the cortical region and in the cell interior agreed well with the calculation of the theoretical radius of the cages imprisoning vesicle movement. Therefore, these data suggest that the structure and kinetics of the cytoskeleton governs vesicle movements in different regions of chromaffin cells.