A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.
Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the 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 proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of 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 GH(3) cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are 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 that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques.
Moreno
Alfredo
A
Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Ramón y Cajal, 7, E-47005 Valladolid, Spain.
SantoDomingo
Jaime
J
Fonteriz
Rosalba I
RI
Lobatón
Carmen D
CD
Montero
Mayte
M
Alvarez
Javier
J
D000165:Acridine Orange / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence; D055099:Neuroendocrine Cells / Q000378:metabolism*; D009478:Neuropeptide Y / Q000378:metabolism; D009499:Neutral Red / Q000737:chemistry; D016716:PC12 Cells; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism*
D000588:Amines; D004151:Dipeptides; D009478:Neuropeptide Y; C493330:Red DND-99; D050988:Vesicle-Associated Membrane Protein 2; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C041865:glycylphenylalanine 2-naphthylamide; D009499:Neutral Red; D000165:Acridine Orange