Elsevier

Neuroscience

Volume 146, Issue 2, 11 May 2007, Pages 659-669
Neuroscience

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

https://doi.org/10.1016/j.neuroscience.2007.02.039Get rights and content

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.

Section snippets

Isolation and culture of bovine chromaffin cells

Chromaffin cells were isolated from bovine adrenal glands following collagenase digestion and they were separated from the debris and erythrocytes by centrifugation on Percoll gradients as described elsewhere (Almazan et al 1984, Gil et al 1998). Cells were maintained in 35 mm Petri dishes as monolayer cultures with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 10 μM cytosine arabinoside, 10 μM 5-fluoro-2′-deoxyuridine, 50 IU/ml penicillin and 50 μg/ml

Fluorescence reflection microscopy enables chromaffin granules to be observed in living cells in the absence of fluorescence labeling

Chromaffin granules contain a matrix that efficiently packs catecholamines in a collapsed polymer of chromagranins (Fischer-Colbrie et al., 1987) and therefore, it is theoretically possible to visualize these dense vesicles by using reflection fluorescence. In this way, we observed a punctuate pattern of fluorescence emission in the cytoplasm of chromaffin cells using a confocal microscope under conditions where part of the wavelength used for excitation is not blocked in the light emission

Fluorescence reflection combined with transmitted light microscopy provides a unique method to visualize dense granules in different areas of chromaffin cells

Studies of vesicle movement are commonly based on the fluorescent labeling of these structures and the use of epifluorescence microscopy (Kaether et al 1997, Steyer et al 1997, Oheim et al 1998, Oheim and Stuhmer 2000). While these valuable studies generate information regarding granule transport, they also present limitations such as the labeling of a limited proportion of the vesicle population, the uncertain estimation of granule size due to the scattering of epifluorescence and the possible

Acknowledgments

This work was supported by grants from the Ministry of Science and Technology (MST, BMC2002-00845 and Ministry of Education and Culture (MEC) of Spain/Fondos FEDER(BFU2005-02154/BFI), and the Generalitat Valenciana (GRUPOS 03/040 and ACOMP06/036). I.L. was recipient of fellowships from the MEC of Spain. D.G. was a fellow of the MST of Spain.

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