Publications (32) View all
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Article: Deuterium Oxide Dilution: A Novel Method to Study Apical Water Layers and Transepithelial Water Transport
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ABSTRACT: Lung epithelia regulate the water flux between gas filled airways and the interstitial compartment in order to maintain organ function. Current methodology to assess transepithelial water transport is limited. We present a novel D2O dilution method to quantify sub-micro liter volumes of aqueous solutions on epithelial cell layers. Evaluating D2O / H2O mixtures using mid-infrared (3-15 µm) attenuated-total-reflection (ATR) spectroscopy, a resolution of 0.06% vol. / vol. change, corresponding to 24 nL, was achieved. Using this method we demonstrate that water transport across NCI-H441 lung epithelial cell layers and apical surface (ASL) volumes are coupled to dexamethasone dependent amiloride-sensitive ion transport. Yet, contrary to current dogma, electrogenic transport is not rate-limiting for water transport. This clearly indicates the need to directly assess net water rather than ion transport across epithelial cell layers. The presented D2O dilution method enables such direct and quick quantification of transepithelial water transport with high resolution.Analytical Chemistry 04/2013; · 5.86 Impact Factor -
Article: Fusion-activated cation entry (FACE) via P2X4 couples surfactant secretion and alveolar fluid transport.
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ABSTRACT: Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X(4) receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (I(sc)) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in I(sc). Overexpressing dominant-negative P2X(4) abrogated this effect by ∼50%, whereas potentiating P2X(4) lead to ∼80% increase in I(sc). Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE.-Thompson, K. E., Korbmacher, J. P., Hecht, E., Hobi, N., Wittekindt, O. H., Dietl, P., Kranz, C., Frick, M. Fusion-activated cation entry (FACE) via P2X(4) couples surfactant secretion and alveolar fluid transport.The FASEB Journal 01/2013; · 5.71 Impact Factor -
Article: Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells.
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ABSTRACT: Keratin filaments impart resilience against mechanical extension of the cell. Despite the pathophysiological relevance of this function, very little is known about the mechanical properties of intermediate filaments in living cells and how these properties are modulated. We used keratin mutants that mimic or abrogate phosphorylation of keratin 8-serine(431) and keratin 18-serine(52) and investigated their effect on keratin tortuousness after cell stretch release in squamous cell carcinoma cells. Cells transfected with the wild-type keratins were used as controls. We can show that keratin dephosphorylation alters the stretch response of keratin in living cells since keratin tortuousness was abolished when phosphorylation of keratin18-serine(52) was abrogated. Additional experiments demonstrate that keratin tortuousness is not simply caused by a plastic overextension of keratin filaments because tortuousness is reversible and requires an intact actin-myosin system. The role of actin in this process remains unclear, but we suggest anchorage of keratin filaments to actin during stretch that leads to buckling on stretch release. Dephosphorylated keratin18-serine(52) might strengthen the recoil force of keratin filaments and hence explain the abolished buckling. The almost exclusive immunolabeling for phosphorylated keratin18-serine (52) in the cell periphery points at a particular role of the peripheral keratin network in this regard.-Fois, G., Weimer, M., Busch, T., Felder, E. T., Oswald, F., von Wichert, G., Seufferlein, T., Dietl, P., Felder, E. Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells.The FASEB Journal 12/2012; · 5.71 Impact Factor -
Article: Combined atomic force microscopy-fluorescence microscopy: analyzing exocytosis in alveolar type II cells.
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ABSTRACT: Hybrid atomic force microscopy (AFM)-fluorescence 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-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) 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% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 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 "dynamic states" of LBs or locations from the PM are captured during imaging.Analytical Chemistry 06/2012; 84(13):5716-22. · 5.86 Impact Factor -
Article: Spatio-temporal aspects, pathways and actions of Ca(2+) in surfactant secreting pulmonary alveolar type II pneumocytes.
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ABSTRACT: The type II cell of the pulmonary alveolus is a polarized epithelial cell that secretes surfactant into the alveolar space by regulated exocytosis of lamellar bodies (LBs). This process consists of multiple sequential steps and is correlated to elevations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) required for extended periods of secretory activity. Both chemical (purinergic) and mechanical (cell stretch or exposure to an air-liquid interface) stimuli give rise to complex Ca(2+) signals (such as Ca(2+) peaks, spikes and plateaus) that differ in shape, origin and spatio-temporal behavior. This review summarizes current knowledge about Ca(2+) channels, including vesicular P2X4 purinoceptors, in type II cells and associated signaling cascades within the alveolar microenvironment, and relates stimulus-dependent activation of these pathways with distinct stages of surfactant secretion, including pre- and postfusion stages of LB exocytosis.Cell calcium 05/2012; 52(3-4):296-302. · 4.29 Impact Factor
