Acidic dense core secretory vesicles in Paramecium

Abstract

The Paramecium cell produces thousands of dense core secretory vesicles (PDCSV) composed of matrices surrounded by vesicle membranes which are eventually docked in a resting state below the plasma membrane, poised for secretagogue-stimulated exocytosis. In this study I have researched whether these secretory vesicles constitute acidic compartments, the mechanism responsible for their acidity and the interaction of Ca{dollar}\sp{lcub}2+{rcub}{dollar} with these vesicles and their Ca{dollar}\sp{lcub}2+{rcub}{dollar}-sensitive dense cores. Acridine orange (AO), an agent known to accumulate in acidic compartments, was used for the first time in a living cell to microscopically demonstrate that PDCSV's were acidic. The use of secretory mutants provided proof that the fluorescent bodies were secretory vesicles and that the proton gradient was required early in the maturation of the vesicle. Protonophores and chloroquine dissipated the vesicular AO, indicating a proton gradient which was recoverable when cells were washed. Protonophore induced gradient dissipation did not affect exocytosis indicating that the proton gradient is not required for exocytosis. The treatment of cells with sodium azide demonstrated that the proton gradient was generated by an ATP-dependent mechanism. Cell fractionation techniques were developed to isolate PDCSV's with intact membrane, for the first time, from tam8, tam6 and nd6 secretory mutant cells (demonstrated by thin-section and negative stain electron microscopy). In vitro assays with these isolated PDCSV's were then formulated using AO and ({dollar}\sp{lcub}14{rcub}{dollar}C) -methylamine. Only in the presence of 5 mM Mg{dollar}\sp{lcub}2+{rcub}{dollar}-ATP did isolated PDCSV's accumulate AO or ({dollar}\sp{lcub}14{rcub}{dollar}C) -methylamine, thus demonstrating an ATP-dependent proton pump. Further use of ({dollar}\sp{lcub}14{rcub}{dollar}C) -methylamine estimated the intra-vesicular pH at 6.45. Using the AO assay with isolated PDCSV's and a series of specific proton pump inhibitors, the PDCSV proton pump was classified as of the vacuolar-type (V-type). This was confirmed by using polyclonal antibodies against proton pump polypeptides from diverse sources which cross-reacted with Paramecium proteins in PDCSV western blots. Thus by three independent methods, evidence is presented for a functioning V-type proton pump in isolated PDCSV's. A final study using isolated vesicles and dense cores determined that secretory mutant tam8 is defective in dense core expansion and tam6 is defective in secretory membrane Ca{dollar}\sp{lcub}2+{rcub}{dollar} exclusion.