Paul Dietl

The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release

To test for a possible role of lysosomes in intracellular Ca2+ homeostasis, the effects of glycyl-L-phenylalanine-beta-naphthylamide (GPN), known to permeabilize these organelles by osmotic swelling, were studied in single MDCK cells. Fluorescence of acridine orange, rhodol green dextran, lysotracker green and FITC-dextran indicated that GPN (0.2 mmol/l) elicited a reversible permeabilization of lysosomes. Cytosolic Ca2+ ([Ca2+]i) as determined by Fura-2 fluorescence increased from 60 +/- 11 to 534 +/- 66 nmol/l (n = 41) in the presence of GPN. Whereas only a single intracellular Ca2+ release could be induced by GPN in a Ca(2+)-free perfusate, repetitive release could be evoked in Ca2+ containing solutions suggesting reuptake of Ca2+ into lysosomal stores. GPN-induced Ca2+ release was blunted after pretreatment with thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, or repeated applications of ATP inducing Ca2+ release from inositol trisphosphate (InsP3) sensitive Ca2+ stores. The effect of ATP on Ca2+ release was, however, not abolished by preceding GPN treatment. GPN-induced Ca2+ release from lysosomes was independent of InsP3 formation or Ca(2+)-induced Ca2+ release, since it was unaffected by the phospholipase C inhibitor U-73, 122 or by caffeine and ruthenium red. These results suggest that Ca2+ largely accumulates in lysosomal vesicles. Moreover, these organelles seem to be part or functionally coupled with InsP3-sensitive Ca2+ stores.

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Ficks law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Fusion-activated Ca2+ entry via vesicular P2X4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes

Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca2+ concentration ([Ca2+])c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a “fusion-activated“ Ca2+ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200–500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X4. Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca2+ influx via vesicular Ca2+ channels regulates fusion pore expansion and vesicle content release.

Threshold calcium levels for lamellar body exocytosis in type II pneumocytes

Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca2+concentration ([Ca2+]i). In fura 2-loaded cells, [Ca2+]iwas selectively elevated by flash photolysis of a cell-permeant caged Ca2+ compound ( o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca2+influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579-1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca2+]ielevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca2+]iand thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca2+. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca2+ signal below ∼300 nmol/l. Our results suggest a highly Ca2+-sensitive step in LB exocytosis.

Actin coating and compression of fused secretory vesicles are essential for surfactant secretion - a role for Rho, formins and myosin II

Secretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required.

Inhibition of store-operated calcium entry contributes to the anti-proliferative effect of non-steroidal anti-inflammatory drugs in human colon cancer cells

Non‐steroidal anti‐inflammatory drugs (NSAIDs) inhibit proliferation and angiogenesis in colorectal cancer. We examined a possible involvement of store‐operated calcium (SOC) entry in human colon carcinoma cells (HRT‐18), which require calcium for proliferation. Acetyl‐salicylic‐acid (ASA), mefenamic acid (MEF) and sulindac sulfide (SUS) inhibited cell proliferation with the following order of potency: SUS > MEF >> ASA. SUS but not MEF and ASA induced apoptosis following low‐dose treatment. Furthermore, SUS and MEF significantly altered the cell cycle distribution. The ability of NSAIDs to inhibit SOC entry was assessed by measuring the intracellular calcium concentration ([Ca2+]i) in response to calcium store depletion using the endoplasmic calcium ATPase inhibitor thapsigargin. SUS and MEF, but not ASA significantly inhibited SOC entry. A causal link between SOC entry inhibition and anti‐proliferative activity was tested using the inorganic SOC entry inhibitor La3+ and the specific organic inhibitor N‐1‐n‐octyl‐3,5‐bis‐(4‐pyridyl)triazole (DPT). Both La3+ and DPT inhibited cell proliferation and SOC entry. Analogous to MEF, the anti‐proliferative effect of DPT was mediated by cell cycle arrest and not by induction of apoptosis. These data indicate a role of SOC entry for cell proliferation in cancer cells and suggest a novel anti‐proliferative NSAID mechanism in addition to its known influence on lipid metabolism.

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.

Formation of cellular projections in neural progenitor cells depends on SK3 channel activity

Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1‐ethyl‐2‐benzimidazolinone (1‐EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50 = 2 ± 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+‐channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non‐differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1‐EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre‐incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels.

A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

1. The whole‐cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura‐2 and fluo‐3 were used to measure the whole‐cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin‐Darby canine kidney (MDCK) cells. 2. In a Ca(2+)‐free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)‐free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5‐trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)‐dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of ‐4.2 +/‐ 0.7 pA pF‐1. This Iin was unaffected by inositol 1,3,4,5‐tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N‐methyl‐D‐glucamine+ (NMG+), aminomethylidin‐trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl‐ with gluconate‐ or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG‐induced elevation of [Ca2+]i. SK&F 96365 (1‐[3‐(4‐methoxyphenyl)‐propoxyl]‐1‐(4‐methoxy‐phenyl)‐ethyl‐1H‐im idazole) HCl did not inhibit the TG‐induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365‐induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent methods, stimulates La(3+)‐inhibitable Ca2+ entry in MDCK cells. Ca2+ entry is at least, in part, mediated by a cation current, which is highly, but not exclusively, selective for Ca2+ over Na+ and insensitive to SK&F 96365.

A device for simultaneous live cell imaging during uni-axial mechanical strain or compression

Mechanical stimuli control multiple cellular processes such as secretion, growth, and differentiation. A widely used method to investigate cell strain ex vivo is stretching an elastic membrane to which cells adhere. However, simultaneous imaging of dynamic signals from single living cells grown on elastic substrates during uni-axial changes of cell length is usually hampered by the movement of the sample along the strain axis out of the narrow optical field of view. We used a thin, prestrained, elastic chamber as growth substrate for the cells and deformed the chamber with a computer-controlled stretch device. An algorithm that compensates the lateral displacement during stretch kept any selected point of the whole chamber at a constant position on the microscope during strain or relaxation (compression). Adherent cells or other materials that adhere to the bottom of the chamber at any given position could be imaged during controlled positive (stretch) or negative (compression) changes of cell length. The system was tested on living alveolar type II cells, in which mechanical effects on secretion have been intensively investigated in the past.