Cellular neuroscienceVesicle movements are governed by the size and dynamics of F-actin cytoskeletal structures in bovine 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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Captivating New Roles of F-Actin Cortex in Exocytosis and Bulk Endocytosis in Neurosecretory Cells
2016, Trends in NeurosciencesCortical F-Actin affects the localization and dynamics of SNAP-25 membrane clusters in chromaffin cells
2013, International Journal of Biochemistry and Cell BiologyThe small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells
2012, Biochemical and Biophysical Research CommunicationsCitation Excerpt :In fact, other groups have shown that Cdc42 enhanced the F-actin remodeling on the cell surface [4,13], and that the F-actin localized preferentially to the cortical region forming a peripheral barrier limiting vesicle access to the plasma membrane [14]. Furthermore, the F-actin network is a key element in vesicle transport during exocytosis [15,16]. Thus, the decrease in plasma membrane-docked vesicles caused by inhibition of Cdc42 activity might be ascribed to either the suppression of vesicle docking by actin barrier formation or the attenuation of vesicle transport or biogenesis due to inhibition of actin remodeling.