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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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Late states of fusion pores. (A) LSM images of fused LBs at the cell periphery before (a) and at different times (b–d, corresponding to the asterisks in B; bleaching was at time 0) after a FRAP measurement. (B) Minor changes in IFM 1–43 recovery rates of LBs 1, 2, and 4, measured at ∼10, 20, and 30 min after initial fusion. (C) Mean time constants of individual LBs (n = 8 ± SD) after repeated FRAP measurements. (D) Progressive increase in IFM 1-43 recovery of LB no. 3 in A within three subsequent measurements. Data were fit by Eq. A4 (solid lines). Arrow denotes transition of the fusion pore diameter (see text). Bar, 10 μm.
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fig4: Late states of fusion pores. (A) LSM images of fused LBs at the cell periphery before (a) and at different times (b–d, corresponding to the asterisks in B; bleaching was at time 0) after a FRAP measurement. (B) Minor changes in IFM 1–43 recovery rates of LBs 1, 2, and 4, measured at ∼10, 20, and 30 min after initial fusion. (C) Mean time constants of individual LBs (n = 8 ± SD) after repeated FRAP measurements. (D) Progressive increase in IFM 1-43 recovery of LB no. 3 in A within three subsequent measurements. Data were fit by Eq. A4 (solid lines). Arrow denotes transition of the fusion pore diameter (see text). Bar, 10 μm.

Mentions: An example of a type II cell after stimulation is shown in Fig. 1 A. As demonstrated, FM 1-43–stained LBs remain located at the site of vesicle fusion, suggesting that fusion pore restriction impedes their release into the extracellular space. In analogy to IFM 1-43 measurements of initial fusion pores as described above, and after careful validation of the feasibility of FRAP measurements (Fig. 2, B–D), we sought to restore destained conditions of fused LBs for repeated IFM 1–43 recovery measurements. The LSM images in Fig. 4 A show part of an ATP-stimulated cell exhibiting four fused LBs. Scanning the same region with the unattenuated laser led to a disappearance of IFM 1–43, followed by recoveries of different rates that were quite stable during several repeats (Fig. 4, B and C). However, fluorescence of some LBs (LB no. 3 in Fig. 4 A and data in Fig. 4 D) developed progressively from a slow initial into a fast recovery in subsequent measurements. The model (Eq. A4) describes best (r > 0.995) the characteristics of this particular LB (1.8 μm ∅) by the supposition of a variable pore area in the observation interval of 34 min. At the beginning of the first FRAP measurement (10 min after initial fusion), the pore area is calculated to be 0.07 μm2, raising linearly to 0.48 μm2 after 200 s. 10 min later, when starting an additional FRAP experiment, the pore area was enlarged to 0.58 μm2 followed by a further expansion (arrow) up to 2.24 μm2 during the next 3 min. After beginning the last experiment in this series (30 min), full expansion of the pore (3.14 μm2) occurred. Such very large areas during late stages of secretion most likely reflect LBs during the process of release, which is accompanied by large scale surface expansions as seen by transmission electron microscopy (TEM) (see Fig. 8 C). Under these conditions (dramatic change in LB morphology), model calculations will inevitably lead to a pore area overestimation.


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)

Late states of fusion pores. (A) LSM images of fused LBs at the cell periphery before (a) and at different times (b–d, corresponding to the asterisks in B; bleaching was at time 0) after a FRAP measurement. (B) Minor changes in IFM 1–43 recovery rates of LBs 1, 2, and 4, measured at ∼10, 20, and 30 min after initial fusion. (C) Mean time constants of individual LBs (n = 8 ± SD) after repeated FRAP measurements. (D) Progressive increase in IFM 1-43 recovery of LB no. 3 in A within three subsequent measurements. Data were fit by Eq. A4 (solid lines). Arrow denotes transition of the fusion pore diameter (see text). Bar, 10 μm.
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Related In: Results  -  Collection

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fig4: Late states of fusion pores. (A) LSM images of fused LBs at the cell periphery before (a) and at different times (b–d, corresponding to the asterisks in B; bleaching was at time 0) after a FRAP measurement. (B) Minor changes in IFM 1–43 recovery rates of LBs 1, 2, and 4, measured at ∼10, 20, and 30 min after initial fusion. (C) Mean time constants of individual LBs (n = 8 ± SD) after repeated FRAP measurements. (D) Progressive increase in IFM 1-43 recovery of LB no. 3 in A within three subsequent measurements. Data were fit by Eq. A4 (solid lines). Arrow denotes transition of the fusion pore diameter (see text). Bar, 10 μm.
Mentions: An example of a type II cell after stimulation is shown in Fig. 1 A. As demonstrated, FM 1-43–stained LBs remain located at the site of vesicle fusion, suggesting that fusion pore restriction impedes their release into the extracellular space. In analogy to IFM 1-43 measurements of initial fusion pores as described above, and after careful validation of the feasibility of FRAP measurements (Fig. 2, B–D), we sought to restore destained conditions of fused LBs for repeated IFM 1–43 recovery measurements. The LSM images in Fig. 4 A show part of an ATP-stimulated cell exhibiting four fused LBs. Scanning the same region with the unattenuated laser led to a disappearance of IFM 1–43, followed by recoveries of different rates that were quite stable during several repeats (Fig. 4, B and C). However, fluorescence of some LBs (LB no. 3 in Fig. 4 A and data in Fig. 4 D) developed progressively from a slow initial into a fast recovery in subsequent measurements. The model (Eq. A4) describes best (r > 0.995) the characteristics of this particular LB (1.8 μm ∅) by the supposition of a variable pore area in the observation interval of 34 min. At the beginning of the first FRAP measurement (10 min after initial fusion), the pore area is calculated to be 0.07 μm2, raising linearly to 0.48 μm2 after 200 s. 10 min later, when starting an additional FRAP experiment, the pore area was enlarged to 0.58 μm2 followed by a further expansion (arrow) up to 2.24 μm2 during the next 3 min. After beginning the last experiment in this series (30 min), full expansion of the pore (3.14 μm2) occurred. Such very large areas during late stages of secretion most likely reflect LBs during the process of release, which is accompanied by large scale surface expansions as seen by transmission electron microscopy (TEM) (see Fig. 8 C). Under these conditions (dramatic change in LB morphology), model calculations will inevitably lead to a pore area overestimation.

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