Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells

Abstract

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.

Fig. 1

Modulators of cytoskeleton and vesicle formation affect ATP release from A549 cells. a Examples of ATP release time course measured in a flow-through chamber with A549 cells treated with brefeldin A (BFA) to disrupt the Golgi complex, exocytosis inhibitors monensin and NEM, and cytochalasin D and nocodazole to disrupt the cytoskeleton. They were stimulated with 50% hypotonic stress (t = 0), and ATP release rate was expressed in pmol/min/10⁶ cells. b The peak rate of release (black bar) and total ATP secreted during 15 min of hypotonic stimulation (gray bar) were inhibited in cells treated with different agents acting on the cytoskeleton or affecting vesicle formation. The data, mean values ± SD from three to four independent experiments for each drug tested, were normalized to control values. All drugs produced significant inhibition of ATP release, indicated by asterisks (two-sample independent t test, P < 0.05)

Fig. 2

Quinacrine loading into A549 cells. Images comparing fluorescence intensity (a) and fluorescence lifetime (b), at time 0 (background autofluorescence; left-side images), and 17 min after the addition of 5 μM quinacrine (right-side images). Both intensity and lifetime images show non-uniform granular distribution of quinacrine within the cells. c Histograms of fluorescence lifetime τ; the peak on the left, which corresponds to τ = 1.5 ns, arises from cell autofluorescence, while in quinacrine-loaded cells a second peak of longer fluorescence lifetime of τ = 4.5 ns is predominant. Images were acquired with a confocal Olympus microscope equipped with a PicoQuant fluorescence lifetime system (see “Methods”), picture resolution 300 × 300 pixels (area 80 × 80 μm²). Fluorescence intensity and lifetime scale bars are shown to the right of the images. Note different fluorescence intensity scales for each image, adjusted to disclose a weak background autofluorescence in the absence of quinacrine

Fig. 3

TIRF images of A549 cells. Comparison of TIRF images of A549 cells loaded with 10 μM quinacrine (left) or 30 μM Bodipy-ATP (right). Both images show similar staining pattern. With both fluorophores, fluorescence was excited with a 488-nm laser line and observed at 512 nm, Olympus IX71 inverted microscope with ×60, NA 1.45 TIRF objective

Fig. 4

Fluorescence intensity changes of quinacrine-containing vesicles during exocytosis. a TIRF image of a single quinacrine-stained cell: the cell contour is outlined by a dotted line (left). On the right, the same cell with regions of interest placed on selected vesicles. b Sequential TIRF images of quinacrine-loaded vesicles showing the time course of fluorescence changes within ROI during 60 s. Hypotonic shock was applied at t = 0. c Three examples of fluorescence changes within single vesicles (indicated by arrows in a and b) during hypotonic shock (1) gradual loss of fluorescence signal due to photobleaching, no exocytosis; (2) abrupt loss of fluorescence attributed to exocytotic release of vesicular quinacrine and its dispersal by diffusion that was preceded by a transiently increased fluorescence likely due to vesicle recruitment deeper into the evanescent field; (3) abrupt loss of fluorescence without the preceding intensity increase. Fluorescence intensity is expressed in arbitrary units (AU)

Fig. 5

Hypotonic shock stimulates exocytosis of quinacrine-stained vesicles. a TIRF image of several cells in a field of view with 120 selected ROIs. b Examples of exocytotic events, seen as abrupt losses of fluorescence intensity taking place at different time points during stimulation by 50% hypotonic shock applied at t = 20 s. c Histogram of exocytotic events: hypotonic stimulation was applied at t = 20 s. Cumulative data from three independent experiments performed at 37°C; 367 ROI totally. d Single-vesicle fluorescence intensity changes during hypotonic shock at 20°C. For clarity, intensity traces from only 13 ROI in one cell are illustrated. Only one trace showed a step-drop of fluorescence (arrow). The data are representative of three experiments: 334 ROI totally