Fusion-Activated Ca2+ Entry: An “Active Zone” of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes

Miklavc, P; Frick, M; Wittekindt, OH; Haller, T; Dietl, P

Abstract

Background

Ca2+ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca2+ concentration ([Ca2+]c) in the prefusion phase, the occurrence and significance of Ca2+ signals in the postfusion phase have not been described before.

Methodology/Principal Findings

We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca2+- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca2+]c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t1/2 of decay = 3.2 s) rise of localized [Ca2+]c originating at the site of lamellar body fusion. [Ca2+]c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca2+ from, or addition of Ni2+ to the extracellular solution, strongly inhibited these [Ca2+]c transients, whereas Ca2+ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca2+]c. Both effects were reduced by the non-specific Ca2+ channel blocker SKF96365.

Conclusions/Significance

Fusion-activated Ca2+ entry (FACE) is a new mechanism that leads to [Ca2+]c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca2+ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions.

Citation

Miklavc, P., Frick, M., Wittekindt, O., Haller, T., & Dietl, P. (2010). Fusion-Activated Ca2+ Entry: An “Active Zone” of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes. PLoS ONE, 5(6), e10982. https://doi.org/10.1371/journal.pone.0010982

• Journal Article Type: Article
• Publication Date: Jun 8, 2010
• Deposit Date: Oct 24, 2016
• Publicly Available Date: Oct 24, 2016
• Journal: PLoS ONE
• Electronic ISSN: 1932-6203
• Publisher: Public Library of Science
• Volume: 5
• Issue: 6
• Pages: e10982
• DOI: https://doi.org/10.1371/journal.pone.0010982
• Publisher URL: http://dx.doi.org/10.1371/journal.pone.0010982