V-ATPase modulates exocytosis in neuroendocrine cells through the activation of the ARNO-Arf6-PLD pathway and the synthesis of phosphatidic acid

Abstract

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids. Recent groundbreaking research has emphasized the critical role of lipid localization at exocytotic sites and validated the essentiality of fusogenic lipids, such as phospholipase D (PLD)-generated phosphatidic acid (PA), during membrane fusion. Nevertheless, the regulatory mechanisms synchronizing the synthesis of these key lipids and neurosecretion remain poorly understood. The vacuolar ATPase (V-ATPase) has been involved both in vesicle neurotransmitter loading and in vesicle fusion. Thus, it represents an ideal candidate to regulate the fusogenic status of secretory vesicles according to their replenishment state. Indeed, the cytosolic V1 and vesicular membrane-associated V0 subdomains of V-ATPase were shown to dissociate during the stimulation of neurosecretory cells. This allows the subunits of the vesicular V0 to interact with different proteins of the secretory machinery. Here, we show that V0a1 interacts with the Arf nucleotide-binding site opener (ARNO) and promotes the activation of the Arf6 GTPase during the exocytosis in neuroendocrine cells. When the interaction between V0a1 and ARNO was disrupted, it resulted in the inhibition of PLD activation, synthesis of phosphatidic acid during exocytosis, and changes in the timing of fusion events. These findings indicate that the separation of V1 from V0 could function as a signal to initiate the ARNO-Arf6-PLD1 pathway and facilitate the production of phosphatidic acid, which is essential for effective exocytosis in neuroendocrine cells.

Keywords: V-ATPase; chromaffin cells; membrane fusion; neurosecretion; phospholipase D; phospholipids.

FIGURE 1

Increased V0a1–ARNO interaction after chromaffin cell stimulation. Chromaffin cells expressing V0a1-NowGFP and ARNO-shadowG were stimulated at different times with 10 µM of nicotine. Fluorescence decays of V0a1-NowGFP were recorded by fluorescence lifetime imaging microscopy (FLIM) and analyzed with a one-component exponential function. (A) Representative lifetime color-coded images of granular V0a1-NowGFP from two cells at different times after stimulation (Bar = 10 µm). Fluorescence lifetime values of V0a1-NowGFP were represented using a color code scale ranging from 1900 ps (orange) to 2400 ps (blue). Dots correspond to V0a1-NowGFP-containing granules. (B) V0a1-NowGFP lifetime distribution for different times after stimulation from cells recorded in (A). Blue boxes highlight a decrease in the longer lifetime values after cell stimulation. (C) Plot of individual mean lifetime values obtained from n = 30 cells in three independent preparations. Medians with an interquartile range are illustrated in red. *p < 0.05 compared to stimulation time 0.

FIGURE 2

Interfering with V0a1–ARNO interaction reduces Arf6 activation after PC12 cell stimulation. Distribution of the MT2-mCherry probe in PC12 cells co-expressing GFP (A) or V0a1Nt-GFP (B) in resting condition or after a 10-min stimulation with a [59 mM] K⁺ solution. After fixation, cells were immunostained for SNAP25. Masks indicate the co-localization between MT2-mCherry and SNAP25 signals. Bar = 5 µm. (C) Levels of MT2-mCherry in the subplasmalemmal area were measured and presented (n > 30 cells for each condition from three independent experiments). ***p < 0.001 compared to resting condition.

FIGURE 3

Interfering with V0a1–ARNO interaction reduces PLD activation after PC12 cell stimulation. Mock-transfected cells or cells expressing GFP or V0a1Nt-GFP were kept in Locke’s solution (resting) or stimulated with a [59 mM] K⁺ solution for 10 min (stimulated). Cells were lysed on ice, and lysates were used to measure the PLD activity. Data are expressed as the mean values ± SD from independent experiments (n = 3), each obtained from quadruplicates in individual experiments. ***p < 0.001 compared to control (Mock).

FIGURE 4

Interfering with V0a1–ARNO interaction reduces PA synthesis at the plasma membrane after PC12 cell stimulation. Distribution of Spo20p–RFP probe in cells co-expressing GFP (A) or V0a1Nt-GFP (B) in resting condition or after a 10-min stimulation with a [59 mM] K⁺ solution. After fixation, cells were immunostained for SNAP25. Masks indicate the co-localization between Spo20–RFP and SNAP25 signals. Bars = 5 µm. (C) Levels of Spo20–RFP in the subplasmalemmal area were measured and presented (n > 30 cells for each condition from three independent experiments). ***p < 0.001 compared to resting condition.

FIGURE 5

Interfering with V0a1–ARNO interaction affects individual spike kinetic parameters from chromaffin cells. (A) Scheme showing the different parameters of amperometric spikes that were analyzed. (B–E) Chromaffin cells in culture expressing GFP (control) or V0a1Nt-GFP (V0a1) or untransfected cells treated with 750 nM of FIPI for 1 h were stimulated with a local application of 100 µM of nicotine for 10 s and catecholamine secretion was monitored using carbon-fiber amperometry. (B) Histogram illustrates the number of amperometric spikes recorded per cell. (C) Average spike profile for cells expressing GFP (Control) or V0a1Nt-GFP (V0a1) or treated with FIPI (FIPI). Parameters of individual spikes including spike half width (D), spike amplitude (E), and spike charge (F) are expressed as the mean ± S.D. (n > 75 cells for each condition from three independent cell cultures). ***p < 0.001 compared to control.

FIGURE 6

Interfering with V0a1–ARNO interaction affects pre-spike foot kinetic parameters from chromaffin cells. Individual pre-spike parameters from cells recorded in Figure 5 include pre-spike duration (A), pre-spike amplitude (B), and charge (C). Data are expressed as the mean ± S.D. (n > 75 cells for each condition from three independent cell cultures). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control.