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

Wedge resection versus lobectomy in T1 lung cancer patients: a propensity matched analysis

Objectives: Performing wedge resection rather than lobectomy for primary lung cancer remains controversial. Recent studies demonstrate no survival advantage for non-anatomical resection compared to lobectomy in patients with early-stage lung cancer. The objective of this study was to investigate whether in patients with T1 tumours, non-anatomical wedge resection is associated with equivalent survival to lobectomy. Methods: This was a retrospective cohort study of patients who underwent lung resection at the Lancashire Cardiac Centre between April 2005 and April 2018. Patients were subjected to multidisciplinary team discussion. The extent of resection was decided by the team based on British Thoracic Society guidelines. The primary outcome was overall survival. Propensity matching of patients with T1 tumours was also performed to determine whether differences in survival rates exist in a subset of these patients with balanced pre-operative characteristics. Results: There were 187 patients who underwent non-anatomical wedge resection and 431 patients who underwent lobectomy. Cox modelling demonstrated no survival difference between groups for the first 1.6 years then a risk of death 3-fold higher for wedge resection group after 1.6 years (HR 3.14, CI 1.98–4.79). Propensity matching yielded 152 pairs for which 5-year survival was 66.2% for the lobectomy group and 38.5% for the non-anatomical wedge group (SMD = 0.58, p = 0.003). Conclusions: Non-anatomical wedge resection was associated with significantly reduced 5-year survival compared to lobectomy in matched patients. Lobectomy should remain the standard of care for patients with early-stage lung cancer who are fit enough to undergo surgical resection

Surfactant secretion in LRRK2 knock-out rats : changes in lamellar body morphology and rate of exocytosis

Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca2+ release upon ATP treatment and significant triggering of LB exocytosis. These findings are in line with the strong Ca2+-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca2+ signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP

A small key unlocks a heavy door : the essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air–liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air–liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air–liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air–liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air–liquid interface

Vesicular calcium channels as regulators of the exocytotic post-fusion phase

Regulated secretion is a fundamental cellular process in many different types of eukaryotic cells with Ca2+-triggered exocytosis taking centre stage. Elevations of the cytoplasmic Ca2+ concentration ([Ca2+]c) regulate multiple steps from vesicle fusion with the plasma membrane to fusion pore dilation and subsequent retrieval of spent vesicles. The general view is that the rise in [Ca2+]c initiates during the pre-fusion stage and either results from Ca2+-influx via Ca2+ channels in the plasma membrane or from release from intracellular Ca2+-stores. However, there is increasing evidence that exocytosis of secretory vesicles triggers additional, localised Ca2+ signals via insertion of vesicle-associated Ca2+ channels into the cell surface. These restricted Ca2+ signals following fusion are ideally suited for regulating the post-fusion fate of individual secretory vesicles. In invertebrates they have been shown to trigger compensatory endocytosis.  Recently we have reported that exocytosis of lamellar bodies in alveolar type II epithelial cells results in a localized Ca2+-influx via vesicular P2X4 receptors which regulates fusion pore expansion and vesicle content release. This finding expands the emerging picture that post-fusion Ca2+ entry via vesicle-associated Ca2+ channels plays a central role for regulated exocytosis

Chemotopy of amino acids on the olfactory bulb predicts olfactory discrimination capabilities of zebrafish Danio rerio

Amino acids reliably evoke strong responses in fish olfactory system. The molecular olfactory receptors (ORs) are located in the membrane of cilia and microvilli of the olfactory receptor neurons (ORNs). Axons of ORNs converge onspecific olfactory bulb (OB) glomeruli and the neural responses of ORNs expressing single Ors activate glomerular activity patterns typical for each amino acid. Chemically similar amino acids activate more similar glomerular activity patterns then chemically different amino acids. Differential glomerular activity patterns are the structural basis for amino acid perception and discrimination. We studied olfactory discrimination in zebrafish Danio rerio (Hamilton 1822) by conditioning them to respond to each of the following amino acidsČ L-Ala, L-Val, L-Leu, L-Arg, and L-Phe. Subsequently, zebrafish were tested for food searching activities with 18 nonconditioned amino acids. The food searching activity during 90 s of the test period was significantly greater after stimulation with the conditioned stimulus than with the nonconditioned amino acid. Zebrafish were able to discriminate all the tested amino acids except L-Ile from L-Val and L-Phe fromL-Tyr. We conclude that zebrafish have difficulties discriminating amino acid odorants that evoke highly similar chemotopic patterns of activity in the OB

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

Mechanical strain of alveolar type II cells in culture: changes in the transcellular cytokeratin network and adaptations

Mechanical forces exert multiple effects in cells, ranging from altered protein expression patterns to cell damage and death. Despite undisputable biological importance, little is known about structural changes in cells subjected to strain ex vivo. Here, we undertake the first transmission electron microscopy investigation combined with fluorescence imaging on pulmonary alveolar type II cells that are subjected to equibiaxial strain. When cells are investigated immediately after stretch, we demonstrate that curved cytokeratin (CK) fibers are straightened out at 10% increase in cell surface area (CSA) and that this is accompanied by a widened extracellular gap of desmosomes–the insertion points of CK fibers. Surprisingly, a CSA increase by 20% led to higher fiber curvatures of CK fibers and a concurrent return of the desmosomal gap to normal values. Since 20% CSA increase also induced a significant phosphorylation of CK8-ser431, we suggest CK phosphorylation might lower the tensile force of the transcellular CK network, which could explain the morphological observations. Stretch durations of 5 min caused membrane injury in up to 24% of the cells stretched by 30%, but the CK network remained surprisingly intact even in dead cells. We conclude that CK and desmosomes constitute a strong transcellular scaffold that survives cell death and hypothesize that phosphorylation of CK fibers is a mechano-induced adaptive mechanism to maintain epithelial overall integrity