Nearly neutral secretory vesicles in Drosophila nerve terminals

Abstract

The acidity of mammalian secretory vesicles drives concentration and processing of their contents. Here, pH-sensitive green fluorescent protein (GFP) variants show that the > or =30-fold (H+) difference between secretory vesicles (pH < or = 5.7) and the cytoplasm (pH = 7.2) in mammalian cells is not present in peptidergic and small synaptic vesicles of the Drosophila neuromuscular junction. First, we find that fluorescence from Topaz-tagged atrial natriuretic factor, a peptidergic vesicle pH indicator, is only modestly affected by collapsing the H+ gradient in type III synaptic boutons. Quantitation shows that peptidergic vesicles are nearly neutral (pH = 6.74 +/- 0.05), even when temperature is elevated. Furthermore, small synaptic vesicles in glutamatergic synaptic boutons, studied with synaptophluorin, are as alkaline as peptidergic vesicles. Finally, yellow fluorescent protein measurements show that cytoplasmic pH is only slightly different than in mammals (pH = 7.4). Thus, the marked acidity of mammalian secretory vesicles is not conserved in evolution, and a modest vesicular H+ gradient is sufficient for supporting neurotransmission.

FIGURE 1

(A) ANF-Tpz responses in type III boutons. (1) Peptide fluorescence under control conditions in Ca²⁺-free saline; (2) approximate doubling of fluorescence after collapsing the vesicular pH gradient at pH 7.2; (3) increase in signal after setting the vesicular pH to 10.5. Bar is 2 μm. (B) The ammonium effect is reversible at pH 7.2 (n = 4). (C) Synaptophluorin responses in type I boutons. (1) Pseudo-color representation of synaptophluorin fluorescence under control conditions, which includes the vesicular and surface signals; (2) vesicular signal revealed after quenching surface fluorescence with pH 5.5 medium; (3) vesicular signal at pH 7.2. Obtained by subtracting surface signal (1 − 2) from total signal after setting the pH to 7.2 in all compartments with ammonium (4). (5) Total synapto-phluorin signal after setting the pH to 9.2, which was used with 4 to calculate the pK of the indicator. Bar is 2 μm. (D) Cytoplasmic YFP responses in type I boutons. (Left) Fluorescence increase after setting the pH to 7.8. Bar is 1 μm. (Right) Change in YFP fluorescence versus cytoplasmic pH; n = 4 for pH 7.2, 4 for pH 7.4, and 3 for pH 7.8.