ROLE OF DEFORMATION-INDUCED LIPID TRAFFICKING IN THE PREVENTION OF PLASMA MEMBRANE STRESS FAILURE

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1 Online Supplement for: ROLE OF DEFORMATION-INDUCED LIPID TRAFFICKING IN THE PREVENTION OF PLASMA MEMBRANE STRESS FAILURE METHODS Experimental Design for Determination of Plasma Membrane Breaks Two fluorescently labeled molecules, excluded from the intracellular compartment with an intact plasma membrane, were used as markers of resealed and nonresealed plasma membrane breaks. When deformation-induced breaks occur, these molecules are internalized along a concentration gradient. Cells were incubated with FDx (molecular weight = 70 kda; Fluka, Milwaukee, WI) for 10 minutes at 37 C and then strained as described below. After cell deformation, cells were washed with 1X PBS and incubated for 10 minutes with propidium iodide (650 Da; Sigma, St. Louis, MO) or in a few experiments with unesterified calcein (985 Da; Molecular Probes, Eugene, OR). Cells with homogenous intracellular distribution of FDx or nuclear labeling of propidium iodide were counted in five low power (40X) microscope fields in each culture well for each experiment and tabulated as a percent of the total cells counted. FDx positive cells were considered wounded and resealed and propidium iodide positive cells as wounded and nonresealed. Alveolar Epithelial Cell Culture Cell lines. Human A549 (passages 78 88) and rat L2 (passages 27 40) alveolar epithelial cells (American Type Culture Collection, Rockville, MD) were grown on six-well culture plates (Bioflex; Flexcell International Corporation, McKeesport, PA). The base of these culture plates consists of a flexible silicoelastic membrane with a surface area of 9.6 cm 2. Its surface was impregnated with collagen I and used for both cell injury and lipid trafficking experiments. Ham s F12K containing L-glutamine (2 mm), fetal calf serum (10%), and penicillinstreptomycin-amphotericin B (100 U/mL, 100 µg/ml, 25 µg/ml, respectively; Sigma) was used as a growth medium. Cells were passaged and seeded at a density of 20,800 cells/cm 2 (200,000 cells/well) 48 hours before each experiment, resulting in a 95% 98% confluent monolayer of cells.

2 E2 Primary cell culture. Rat Type II alveolar epithelial cells were harvested as previously described (E1). Briefly, Sprague-Dawley rats ( g) were anesthetized with intraperitoneal pentobarbital (70 mg/kg of body weight), followed by 300 U of heparin, and the trachea was cannulated. The lungs were lavaged multiple times with salt solutions and once with elastase solution (30 U/mL). Subsequently, the heart and lungs were excised en bloc, the heart and trachea removed, and the remaining lung tissue minced with DNAse (Type I, 250 mg/ml). The remaining mixture was then filtered (150 mm pore size then 15 mm pore size; SefarAmerica Inc., Kansas City, MO) and centrifuged and the cells plated on IgG-coated petri dishes for 1 hour. Nonadherent cells were then plated (2 x 10 6 cells/well) onto Bioflex wells as noted above. Cell Strain Devices Two strain devices were used, both having been previously described (E2, E3). For cell injury experiments we used a live cell strain system able to produce precise strain rates and amplitudes while maintaining cells in an environment equivalent to an incubator (E2). Strain amplitudes of 3% 25% and rates of 1% 140% per second were studied. For lipid trafficking experiments, Bioflex wells were placed in a rubber sealed mold with loading posts and fixed to the confocal microscope movable stage mount (E3). The membrane and, in turn, the attached cells were then deformed from below by a portable vacuum device connected to the Delrin mold. A single stretch of 25% (± 2.8%) was applied to the cells and held on average for 90 seconds to allow for focussing and image acquisition. Labeling of Lipid Membranes The methods have been previously described (E3). Briefly, intracellular membranes of A549 and/or Day 5 ATII cells were fluorescently labeled by incubating with 2 µm FM1 43 for 3 hours at 37 C. Cells were washed with 1X PBS before stretching and imaging for determination of lipid trafficking. The fluorescence intensity of the cell decreases after exocytosis to the plasma membrane. In separate experiments, lipid trafficking was also measured after treatment with (1) cold temperature (4 C); (2) plasma membrane cholesterol depletion (β-methyl-cyclodextrin, Sigma, 10 µm, 10-minute incubation at 37 C); and (3) cytoskeletal altering agents (Cytochalasin D, Paclitaxel, and colchicine, Sigma, 1 µm, 1 hour at 37 C) immediately after FM1-43 loading and compared with untreated cells. Cholesterol depletion was confirmed by cholesterol

3 E3 quantification of the cell after β-methyl-cyclodextrin treatment using a method previously described by Leppimaki and colleagues (E4). Conditions of 4 C were obtained using a pumpregulated circulating bath system developed in our laboratory whereby 1X PBS was passed through cooled tubing and then circulated through the culture well. Accurate temperatures were confirmed using a thermometer probe placed in the culture well (Control Co., Friendswood, TX). Cell Imaging For cell injury experiments, cells were viewed immediately after injurious deformation using a 40X water immersion objective lens (NA = 0.75, Carl Zeiss Inc., Thornwood, NY) attached to an upright microscope (BH2, Olympus, Melville, NY). Fluorescent labels were excited with blue (λ 488 nm) and green light (λ 540 nm) to visualize FDx or calcein and propidium iodide, respectively. For lipid trafficking experiments cells were imaged using an argon ion laser scanning confocal microscope (Olympus Fluoview3) mounted on an upright microscope (BH2, Olympus) after membrane lipid labeling. One micron optical sections were obtained using an infinity corrected 40X water immersion objective lens (NA = 0.8) (Olympus). Fluorescent labels were excited with blue (λ = 488 nm) laser light and emission wavelengths collected simultaneously by green (λ = nm) and red (λ > 605 nm) filters (Olympus) for FM1 43. At each optical section plane, images were digitized at 8-bit resolution and stored in arrays of 512 x 512 pixels. Fluorescence Quantitation For lipid trafficking experiments, membrane fluorescent intensity measurements were calculated using ANALYZE, an image display and manipulation package (Mayo Foundation, Rochester, MN) (E5). A single image slice through the middle of the cell was acquired in both its undeformed and deformed states. Seed points for regions of interest were utilized and then fit to the cell by hand drawn pixel intensity limitations. The frequency distribution of fluorescent pixel intensities was analyzed and intensity measurements are reported as mean and SD values. Magnetic Twisting Cytometry Ferromagnetic (Fe 3 O 4 ) microbeads (4.5 µm diameter, magnetic moment = 4 Am 2 /kg) were coated with a synthetic RGD peptide (RGD Peptite; Telios Pharmaceuticals Inc., San Diego, CA)

4 E4 at a concentration of 50 µg/ml (E6). The RGD-coated beads were dispersed in serum free medium and added to each well at 20 µg/well (1 2 beads/cell) for 20 minutes. Unbound beads were washed away before magnetic twisting cytometry measurements. Cell wells were placed into the magnetic twisting cytometer and kept at 37 C or cooled to 4 C as dictated by the experimental protocol. A 10-second 1,000-Gauss homogeneous magnetic pulse was then applied to magnetize the beads in the horizontal direction. A fluxgate magnetometer (Foerster, Reutlingen, Germany) was used to measure the remnant magnetic field of the beads in the horizontal direction (B). A magnetic twisting field, the strength of which defines local stress, was applied in the vertical direction to twist the beads upward. The consequent decline in B, which is proportional to angular bead rotation, was used to compute apparent cell stiffness (E7). Statistical Analysis Throughout the results section we specify the meaning of n, the number of observations, as either the number of cells that were analyzed, the number of wells from which measurements were derived, or the number of experiments that were carried out on different study occasions. Statistical comparisons between experimental conditions were made using the Student t tests for paired observations (Microsoft Excel, Redmond, WA). Also, analysis of variance was used to determine strain effect on percent wounded cells across different strain amplitude, strain rate, and temperature (SYSTAT, SPSS Science, Chicago, IL). Statistical significance was assumed at p < 0.05 with respect to a two-tailed probability distribution. For stretch analyses of lipid trafficking, each cell served as its own static control. Fluorescent intensity measurements and cell injury percentages are presented as means and standard deviations. Quantification of lipid trafficking was also compared against a nondeformed time control cell population that was exposed to the same environmental conditions, including laser light exposure time.

5 E5 References E1. Dobbs LG. Isolation and culture of alveolar type II cells. Am J Physiol: Lung Cell Mol Physiol 1990;258:L134-L147. E2. Stroetz RW, Vlahakis NE, Walters BJ, Schroeder MA, Hubmayr RD. Validation of a new live cell strain system: characterization of plasma membrane stress failure. J Appl Physiol 2001;90: E3. Vlahakis NE, Schroeder MA, Pagano RE, Hubmayr RD. Deformation-induced lipid trafficking in alveolar epithelial cells. Am J Physiol-Lung 2001;280:L938-L946. E4. Leppimaki P, Kronqvist R, Slotte JP. The rate of sphingomyelin synthesis de novo is influenced by the level of cholesterol in cultured human skin fibroblasts. Biochem J 1998;335: E5. Hanson DP, Robb RA, Aharon S, Augustine KE, Cameron BM, Camp JJ, Karwoski RA, Larson AG, Stacy MC, Workman EL. New software toolkits for comprehensive visualization and analysis of three-dimensional multimodal biomedical images. J Digital Imaging 1997;10: E6. Berrios JC, Schroeder MA, Hubmayr RD. Mechanical properties of alveolar epithelial cells in culture. J Appl Physiol 2001;91: E7. Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton. Science 1993;260: