16 research outputs found
Fusion-Activated Ca2+ Entry: An âActive Zoneâ 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 decayâ=â3.2 s) rise of localized [Ca2+]c originating at the site of lamellar body fusion. [Ca2+]c increase followed with a delay of âŒ0.2â0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at âŒ10 ”m/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+âDependent Actin Coating of Lamellar Bodies after Exocytotic Fusion: A Prerequisite for Content Release or KissâandâRun
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 Ă-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 (âkiss-coat-and-releaseâ), remains in this fused state without further action (âkiss-coat-and-waitâ), or is retrieved and pushed forward in the cell on top of an actin tail (âkiss-coat-and-runâ). 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
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
Fusion Dependent Activation of Vesicular P2X4 Receptors Leads to a Volume Increase in Alveolar Type II Epithelial Cells - Coupling Secretion and Lung Fluid Homeostasis?
Molecular basis of early epithelial response to streptococcal exotoxin: role of STIM1 and Orai1 proteins: Streptolysin O activates SOC entry
Streptolysin O (SLO) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pyogenes. SLO induces diverse types of Ca2+ signalling in host cells which play a key role in membrane repair and cell fate determination. The mechanisms behind SLO-induced Ca2+ signalling remain poorly understood. Here, we show that in NCI-H441 cells, wild-type SLO as well as non-pore-forming mutant induces long-lasting intracellular Ca2+ oscillations via IP3-mediated depletion of intracellular stores and activation of store-operated Ca2+ (SOC) entry. SLO-induced activation of SOC entry was confirmed by Ca2+ add-back experiments, pharmacologically and by overexpression as well as silencing of STIM1 and Orai1 expression. SLO also activated SOC entry in primary cultivated alveolar type II (ATII) cells but Ca2+ oscillations were comparatively short-lived in nature. Comparison of STIM1 and Orai1 revealed a differential expression pattern in H441 and ATII cells. Overexpression of STIM1 and Orai1 proteins in ATII cells changed the short-lived oscillatory response into a long-lived one. Thus, we conclude that SLO-mediated Ca2+ signalling involves Ca2+ release from intracellular stores and STIM1/Orai1-dependent SOC entry. The phenotype of Ca2+ signalling depends on STIM1 and Orai1 expression levels. Our findings suggest a new role for SOC entry-associated proteins in S.âpyogenes-induced lung infection and pneumonia
Combined Atomic Force MicroscopyâFluorescence Microscopy: Analyzing Exocytosis in Alveolar Type II Cells
Hybrid atomic force microscopy (AFM)âfluorescence
microscopy
(FM) investigation of exocytosis in lung epithelial cells (ATII cells)
allows the detection of individual exocytic events by FM, which can
be simultaneously correlated to structural changes in individual cells
by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form
of exocytosis found in many non-neuronal cells. Exocytosis of LBs,
following stimulation with adenosine-5âČ-triphosphate (ATP)
and phorbol 12-myristate 13-acetate (PMA), results in a cation influx
via P2X<sub>4</sub> receptors at the site of LB fusion with the plasma
membrane (PM), which should induce a temporary increase in cell height/volume.
AFM measurements were performed in single-line scans across the cell
surface. Five minutes after stimulation, ATII cells revealed a cell
height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8%
(<i>N</i> = 9), respectively. These transient changes depend
on exocytic LBâPM fusion. Nonstimulated cells and cells lacking
LB fusions did not show a significant change in cell height/volume
(<i>N</i> = 8). In addition, a cell height decrease was
observed in ATII cells stimulated by uridine-5âČ-triphosphate
(UTP) and PMA, agonists inducing LB fusion with the PM, but not activation
of P2X<sub>4</sub> receptors. The cell height and volume decreased
by â8.6 ± 3.6% and â11.2 ± 3.9% (<i>N</i> = 5), respectively. Additionally, low force contact and dynamic
mode AFM imaging of cell areas around the nucleus after stimulation
with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography
images compared to nonfused LBs, concluding that different âdynamic
statesâ of LBs or locations from the PM are captured during
imaging