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Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

Haller T, Dietl P, Pfaller K, Frick M, Mair N, Paulmichl M, Hess MW, Furst J, Maly K - J. Cell Biol. (2001)

Bottom Line: Proc.Natl.Acad.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria. thomas.haller@uibk.ac.at

ABSTRACT
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.

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Fusion pore dynamics revealed by continuous photobleaching. (A) Exemplified IFM 1-43 recordings obtained from released LBs, LBs within fused vesicles, and LBs within transiently (as in Fig. 2 D) fused vesicles. Steady-state IFM 1-43 reflects a balanced rate between dye bleaching and replenishment. (B) Confocal z sections of a single fused LB at times indicated in the upper diagram during a continuous photobleaching experiment (identical LSM gain setting in 1–3). Despite the apparent disappearance of fluorescence during steady state, the LB was still visible by contrast enhancement (IFM 1-43 >0). The shift in steady-state IFM 1-43 reflects spontaneous fusion pore expansion. (Bottom right) IFM 1-43 tracings of two LBs from the same cell, demonstrating the differential effect of ionomycin on the steady-state IFM 1-43.
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fig5: Fusion pore dynamics revealed by continuous photobleaching. (A) Exemplified IFM 1-43 recordings obtained from released LBs, LBs within fused vesicles, and LBs within transiently (as in Fig. 2 D) fused vesicles. Steady-state IFM 1-43 reflects a balanced rate between dye bleaching and replenishment. (B) Confocal z sections of a single fused LB at times indicated in the upper diagram during a continuous photobleaching experiment (identical LSM gain setting in 1–3). Despite the apparent disappearance of fluorescence during steady state, the LB was still visible by contrast enhancement (IFM 1-43 >0). The shift in steady-state IFM 1-43 reflects spontaneous fusion pore expansion. (Bottom right) IFM 1-43 tracings of two LBs from the same cell, demonstrating the differential effect of ionomycin on the steady-state IFM 1-43.

Mentions: Although FRAP could be applied repeatedly, we aimed at a more continuous approach to record fusion pore dynamics (Fig. 5 A). IFM 1-43 data were obtained from LSM image stacks of FM 1-43–stained LBs, acquired at regular intervals using intermediate laser transmission (32%). At this light intensity, each image stack should lead to partial bleaching only. Indeed, in released LBs (Fig. 5 A), IFM 1-43 dropped initially, followed by a steady state, which most likely corresponds to a dynamic equilibrium between bleaching and replenishment of unbleached dye. When the same protocol was applied to transiently fused vesicles lacking access to extracellular FM 1-43 (Fig. 2 D), IFM 1-43 dropped to zero. Correspondingly, in the intermediate situation (exocytotic fusion), IFM 1-43 attained a value >0 indicating a limited rate of dye replenishment through the fusion pore. Thus, it has to be concluded that any change in the status of the fusion pore (both closure or expansion) should result in a corresponding shift in the steady-state IFM 1-43. This modified FRAP protocol provides a continuous assessment of fusion pore dynamics. For each data point, a stack of eight confocal images was captured within a defined space below and above the measured LB. This excludes the possibility that IFM 1-43 changes are due to changes in LB location relative to the focal plane, or to exocytosis of additional vesicles in the vicinity of the one observed.


Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

Haller T, Dietl P, Pfaller K, Frick M, Mair N, Paulmichl M, Hess MW, Furst J, Maly K - J. Cell Biol. (2001)

Fusion pore dynamics revealed by continuous photobleaching. (A) Exemplified IFM 1-43 recordings obtained from released LBs, LBs within fused vesicles, and LBs within transiently (as in Fig. 2 D) fused vesicles. Steady-state IFM 1-43 reflects a balanced rate between dye bleaching and replenishment. (B) Confocal z sections of a single fused LB at times indicated in the upper diagram during a continuous photobleaching experiment (identical LSM gain setting in 1–3). Despite the apparent disappearance of fluorescence during steady state, the LB was still visible by contrast enhancement (IFM 1-43 >0). The shift in steady-state IFM 1-43 reflects spontaneous fusion pore expansion. (Bottom right) IFM 1-43 tracings of two LBs from the same cell, demonstrating the differential effect of ionomycin on the steady-state IFM 1-43.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198834&req=5

fig5: Fusion pore dynamics revealed by continuous photobleaching. (A) Exemplified IFM 1-43 recordings obtained from released LBs, LBs within fused vesicles, and LBs within transiently (as in Fig. 2 D) fused vesicles. Steady-state IFM 1-43 reflects a balanced rate between dye bleaching and replenishment. (B) Confocal z sections of a single fused LB at times indicated in the upper diagram during a continuous photobleaching experiment (identical LSM gain setting in 1–3). Despite the apparent disappearance of fluorescence during steady state, the LB was still visible by contrast enhancement (IFM 1-43 >0). The shift in steady-state IFM 1-43 reflects spontaneous fusion pore expansion. (Bottom right) IFM 1-43 tracings of two LBs from the same cell, demonstrating the differential effect of ionomycin on the steady-state IFM 1-43.
Mentions: Although FRAP could be applied repeatedly, we aimed at a more continuous approach to record fusion pore dynamics (Fig. 5 A). IFM 1-43 data were obtained from LSM image stacks of FM 1-43–stained LBs, acquired at regular intervals using intermediate laser transmission (32%). At this light intensity, each image stack should lead to partial bleaching only. Indeed, in released LBs (Fig. 5 A), IFM 1-43 dropped initially, followed by a steady state, which most likely corresponds to a dynamic equilibrium between bleaching and replenishment of unbleached dye. When the same protocol was applied to transiently fused vesicles lacking access to extracellular FM 1-43 (Fig. 2 D), IFM 1-43 dropped to zero. Correspondingly, in the intermediate situation (exocytotic fusion), IFM 1-43 attained a value >0 indicating a limited rate of dye replenishment through the fusion pore. Thus, it has to be concluded that any change in the status of the fusion pore (both closure or expansion) should result in a corresponding shift in the steady-state IFM 1-43. This modified FRAP protocol provides a continuous assessment of fusion pore dynamics. For each data point, a stack of eight confocal images was captured within a defined space below and above the measured LB. This excludes the possibility that IFM 1-43 changes are due to changes in LB location relative to the focal plane, or to exocytosis of additional vesicles in the vicinity of the one observed.

Bottom Line: Proc.Natl.Acad.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria. thomas.haller@uibk.ac.at

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus