RT info:eu-repo/semantics/article
T1 A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes
A1 Moreno Díaz-Calderón, Alfredo
A1 Santo Domingo Mayoral, Jaime
A1 Fonteriz García, Rosalba Inés
A1 Domínguez Lobatón, María Carmen
A1 Montero Zoccola, María Teresa
A1 Álvarez Martín, Javier
K1 Calcio en el organismo
AB Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidicfluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according tothe pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein(EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteinsand acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesiclemovements and their fusion with the plasma membrane. We have now investigated in detail the degreeof colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange,neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH3 cells. We find that all the acidicdyes labelled the same population of vesicles. However, that population was largely different from theone labelled by the targeted proteins, with very little colocalization among them, in all the cell typesstudied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesiclesare not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide(GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting thatthey could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly differentsets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicledynamics is studied using these techniques.
PB Elsevier Inc.
SN 1047-8477
YR 2010
FD 2010
LK http://uvadoc.uva.es/handle/10324/5974
UL http://uvadoc.uva.es/handle/10324/5974
LA eng
NO Journal of Structural Biology, 2010, vol. 172, p. 261-269
NO Producción Científica
DS UVaDOC
RD 27-dic-2024