Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.
In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579-1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.
Haller
T
T
Department of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria. thomas.haller@uibk.ac.at
Dietl
P
P
Pfaller
K
K
Frick
M
M
Mair
N
N
Paulmichl
M
M
Hess
M W
MW
Furst
J
J
Maly
K
K
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D005089:Exocytosis; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008561:Membrane Fusion; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D011650:Pulmonary Alveoli / Q000378:metabolism* / Q000648:ultrastructure; D011663:Pulmonary Surfactants / Q000378:metabolism; D011726:Pyridinium Compounds / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D022142:Secretory Vesicles / Q000648:ultrastructure*
C073804:FM1 43; D005456:Fluorescent Dyes; D011663:Pulmonary Surfactants; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D000255:Adenosine Triphosphate; D002118:Calcium