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

Irina Akopova, Sabina Tatur, Mariusz Grygorczyk, R. Luchowski, Ignacy Gryczynski, Zygmunt Gryczynski, Julian Borejdo, Ryszard Grygorczyk

Research output: Contribution to journalArticle

41 Scopus citations

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.

Original languageEnglish
Pages (from-to)59-70
Number of pages12
JournalPurinergic Signalling
Volume8
Issue number1
DOIs
StatePublished - Mar 2012

Keywords

  • ATP exocytosis
  • ATP release
  • Mechano-sensitive nucleotide release
  • Purinergic signaling
  • TIRF microscopy

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