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

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

Ca2+‐Dependent Actin Coating of Lamellar Bodies after Exocytotic Fusion: A Prerequisite for Content Release or Kiss‐and‐Run

Type II pneumocytes secrete surfactant, a lipoprotein-like substance reducing the surface tension in the lung, by regulated exocytosis of secretory vesicles termed lamellar bodies (LBs). This secretory process is characterized by a protracted postfusion phase in which fusion pores open slowly and may act as mechanical barriers for release. Combining dark-field with fluorescence microscopy, we show in ß-actin green fluorescent protein-transfected pneumocytes that LB fusion with the plasma membrane is followed by actin coating of the fused LB. This is inhibited by cytoplasmic Ca2+ chelation or the phospholipase D inhibitor C2 ceramide. Actin coating occurs by polymerization of actin monomers, as evidenced by staining with Alexa 568 phalloidin. After actin coating of the fused LB, it either shrinks while releasing surfactant (“kiss-coat-and-release”), remains in this fused state without further action (“kiss-coat-and-wait”), or is retrieved and pushed forward in the cell on top of an actin tail (“kiss-coat-and-run”). In the absence of actin coating, no release or run was observed. These data suggest that actin coating creates a force needed for either extrusion of vesicle contents or retrieval and intracellular propulsion

TGF-β1 increases permeability of ciliated airway epithelia via redistribution of claudin 3 from tight junction into cell nuclei

TGF-β1 is a major mediator of airway tissue remodelling during atopic asthma and affects tight junctions (TJs) of airway epithelia. However, its impact on TJs of ciliated epithelia is sparsely investigated. Herein we elaborated effects of TGF-β1 on TJs of primary human bronchial epithelial cells. We demonstrate that TGF-β1 activates TGF-β1 receptors TGFBR1 and TGFBR2 resulting in ALK5-mediated phosphorylation of SMAD2. We observed that TGFBR1 and -R2 localize specifically on motile cilia. TGF-β1 activated accumulation of phosphorylated SMAD2 (pSMAD2-C) at centrioles of motile cilia and at cell nuclei. This triggered an increase in paracellular permeability via cellular redistribution of claudin 3 (CLDN3) from TJs into cell nuclei followed by disruption of epithelial integrity and formation of epithelial lesions. Only ciliated cells express TGF-β1 receptors; however, nuclear accumulations of pSMAD2-C and CLDN3 redistribution were observed with similar time course in ciliated and non-ciliated cells. In summary, we demonstrate a role of motile cilia in TGF-β1 sensing and showed that TGF-β1 disturbs TJ permeability of conductive airway epithelia by redistributing CLDN3 from TJs into cell nuclei. We conclude that the observed effects contribute to loss of epithelial integrity during atopic asthma

An evaluation study of various excitation signals for electrical impedance spectroscopy

Electrical Impedance Spectroscopy (EIS) is a popular method for investigating tissue properties. Implementing the signal generator for EIS measurements with a suitable excitation signal type is thereby one of the two system components. The choice of the excitation signal defines the measurement speed, signal-to-noise ratio (SNR), total area and power consumption of the system, and many more properties. Signal types such as analog single-tone, analog multi-tone, linear feedback shift registers (LFSR), and single-tone Sigma Delta Modulated (SDM) are proposed in the state of the art. In this work an EIS setup is implemented and successfully tested with all the mentioned signal types to evaluate their properties on impedance models as well as in vitro cell layers. It is proposed to combine the SDM with the multi-tone excitation signal yielding a very versatile excitation generator. Multi-tone SDM are as fast as analog multitones, while benefiting from a binary output and thus less system complexity. The implemented EIS setup is used to perform EIS measurement for biological samples. The results show a very good matching with the reference for all the excitation signals

2-APB and capsazepine-induced Ca2+ influx stimulates clathrin-dependent endocytosis in alveolar epithelial cells

Calcium as a second messenger influences many cellular and physiological processes. In lung, alveolar type II (ATII) cells sense mechanical stress and respond by Ca2+ dependent release of surfactant, which is essential for respiratory function. Nevertheless, Ca2+ signaling mechanisms in these cells - in particular Ca2+ entry pathways are still poorly understood. Herein, we investigated pharmacological properties of non-voltage-gated Ca2+ channel modulators in ATII and NCI-H441 cells and demonstrate that 2-Aminoethoxydiphenyl-borinate (2-APB) and capsazepine (CPZ) activate Ca2+ entry with pharmacologically distinguishable components. Surprisingly, 2-APB and CPZ activated clathrin dependent endocytosis in ATII and NCI-H441 cells, which was dependent on Ca2+ entry. The internalized material accumulated in non-acidic granules distinct from surfactant containing lamellar bodies (LB). LB exocytosis was not observed under these conditions. Our study demonstrates that 2-APB/CPZ induces Ca2+ entry which unlike ATP- or stretch-induced Ca2+ entry in ATII cells does not activate exocytosis but an opposing endocytotic mechanism