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

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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 Ca2+-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.

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Acknowledgments

This study was supported in part by the Canadian Institutes of Health Research, the Canadian Cystic Fibrosis Foundation (RG) and the National Institutes of Health (USA): NIH-5R21CA149897-02 (ZG) and NIH-HL090786 (JB).

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Authors and Affiliations

  1. Research Centre, Centre hospitalier de l’Université de Montréal (CRCHUM)—Hôtel-Dieu, 3850 St. Urbain St., Montreal, QC, Canada, H2W 1T7

    Sabina Tatur, Mariusz Grygorczyk & Ryszard Grygorczyk

  2. Department of Medicine, Université de Montréal, Montreal, Quebec, Canada

    Ryszard Grygorczyk

  3. Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas, Fort Worth, TX, USA

    Irina Akopova, Rafał Luchowski, Ignacy Gryczynski, Zygmunt Gryczynski & Julian Borejdo

  4. Department of Cell Biology and Anatomy, University of North Texas, Fort Worth, TX, USA

    Ignacy Gryczynski

  5. Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, USA

    Zygmunt Gryczynski

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  1. Irina Akopova
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  2. Sabina Tatur
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Correspondence to Ryszard Grygorczyk.

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Akopova, I., Tatur, S., Grygorczyk, M. et al. Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells. Purinergic Signalling 8, 59–70 (2012). https://doi.org/10.1007/s11302-011-9259-2

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