Isolation of pancreatic acinar cells
Acinar cells for secretion experiments, light microscopy, AFM, and EM were isolated using minor modification of a published procedure (Jena et al., 1991
). For each experiment, a male Sprague Dawley rat weighing 80–100 g was euthanized by CO2 inhalation. The pancreas was dissected and diced into 0.5 mm3 with a razor blade, mildly agitated for 10 min at 37°C in a siliconized glass tube with 5 ml of oxygenated buffer A (98 mM NaCl, 4.8 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 0.1% bovine serum albumin, 0.01% soybean trypsin inhibitor, 25 mM Hepes, pH 7.4) containing 1000 units of collagenase. The suspension of acini was filtered through a 224 µm Spectra-Mesh (Spectrum Laboratory Products, Rancho Dominguez, CA) polyethylene filter to remove large clumps of acini and undissociated tissue. The acini were washed six times, 50 ml per wash, with ice-cold buffer A. Isolated rat pancreatic acini and acinar cells were plated on Cell-Tak-coated (Collaborative Biomedical Products, Bedford, MA) glass cover slips. Two to three hours after plating, cells were imaged with the AFM before and during stimulation of secretion. Isolated acinar cells and hemiacinar preparations were used in the study because fusion of secretory vesicles at the PM in these cells occurs at the apical region facing the acinar lumen.
Pancreatic plasma membrane preparation
Rat pancreatic PM fractions were isolated using a modification of a published method (Rosenzweig et al., 1983). Male Sprague Dawley rats weighing 70–100 g were euthanized by CO2 inhalation. Pancreata were removed and placed in ice-cold phosphate-buffered saline (PBS), pH 7.5. Adipose tissue was removed, and the pancreata were diced into 0.5 mm3 pieces using a razor blade in a few drops of homogenization buffer A (1.25 M sucrose, 0.01% trypsin inhibitor, 25 mM Hepes, pH 6.5). The diced tissue was homogenized in 15% (w/v) ice-cold homogenization buffer A using four strokes at maximal speed of a motor-driven pestle (using a Wheaton Overhead Stirrer). A total of 1.5 ml of the homogenate was layered over a 125 µl cushion of 2M sucrose, and 500 µl of 0.3 M sucrose was layered onto the homogenate in Beckman centrifuge tubes. After centrifugation at 145,000 x g for 90 min in a Sorvall AH-650 rotor, the material banding between the 1.2 and 0.3 M sucrose interface was collected and the protein concentration (Bradford, 1976) was determined. For each experiment, fresh PM was prepared and used the same day in all AFM experiments.
Atomic force microscopy
Pits and fusion pores at the PM in live pancreatic acinar secreting cells in PBS, pH 7.5, were imaged with the AFM (BioScope III, Digital Instruments, Santa Barbara, CA) using both contact and "tapping" modes. All images presented in this manuscript were obtained in the tapping mode in fluid, using silicon nitride tips with a spring constant of 0.06 N/m, and an imaging force of Hz, with 512 lines per image, and constant image gains. Topographical dimensions of pits and fusion pores at the cell PM were analyzed using the software NanoScope IIIa version 4.43r8 supplied by Digital Instruments.
ImmunoAFM on live cells
Immunogold localization in live pancreatic acinar cells was assessed after 5 min stimulation of secretion with 10 µM of the secretagogue mastoparan. After stimulation of secretion, the live pancreatic acinar cells in buffer were exposed to a 1:200 dilution of 
-amylase-specific antibody (Biomeda, Foster City, CA) and 30 nm gold conjugated secondary antibody for 1 min and were washed in PBS before AFM imaging in PBS at room temperature. Pits and fusion pores within, at the apical end of live pancreatic acinar cells in PBS pH 7.5, were imaged by the AFM (BioScope III, Digital Instruments) using both contact and tapping mode. All images presented were obtained in the tapping mode in fluid, using silicon nitride tips as described previously.
ImmunoAFM on fixed cells
After stimulation of secretion with 10 µM mastoparan, the live pancreatic acinar cells were fixed for 30 min using ice-cold 2.5% paraformaldehyde (PFA) in PBS. Cells were then washed in PBS, followed by labeling with 1:200 dilution of -amylase-specific antibody (Biomeda) and 10 nm gold conjugated secondary antibody for 15 min, fixed, washed in PBS, and imaged in PBS with AFM at room temperature.
Isolation of zymogen granules
Zymogen granules (ZGs) were isolated by using a modification of the method of Jena et al. (1991). Male Sprague Dawley rats weighing 80–100 g were euthanized by CO2 inhalation for each ZG preparation. The pancreata were dissected and diced into 0.5-mm3 pieces. The diced pancreata were suspended in 15% (w/v) ice-cold homogenization buffer (0.3 M sucrose, 25 mM Hepes, pH 6.5, 1 mM benzamidine, 0.01% soybean trypsin inhibitor) and homogenized with a Teflon glass homogenizer. The resultant homogenate was centrifuged for 5 min at 300 x g at 4°C to obtain a supernatant fraction. One volume of the supernatant fraction was mixed with 2 volumes of a Percoll–sucrose–Hepes buffer (0.3 M sucrose, 25 mM Hepes, pH 6.5, 86% Percoll, 0.01% soybean trypsin inhibitor) and centrifuged for 30 min at 16,400 x g at 4°C. Pure ZGs were obtained as a loose white pellet at the bottom of the centrifuge tube.
Transmission electron microscopy
Isolated rat pancreatic acini and ZGs were fixed in 2.5% buffered PFA for 30 min, and the pellets were embedded in Unicryl resin and were sectioned at 40–70 nm. Thin sections were transferred to coated specimen transmission electron microscopy (TEM) grids, dried in the presence of uranyl acetate and methyl cellulose, and examined in a transmission electron microscope.
Immunoprecipitation and Western blot analysis
Immunoblot analysis was performed on pancreatic PM and total homogenate fractions. Protein in the fractions was estimated by the Bradford method (Bradford, 1976). Pancreatic fractions were boiled in Laemmli reducing sample preparation buffer (Laemmli, 1970) for 5 min, cooled, and used for SDS-PAGE. PM proteins were resolved in a 12.5% SDS-PAGE and electrotransferred to 0.2 µm nitrocellulose sheets for immunoblot analysis with a SNAP-23 specific antibody. The nitrocellulose was incubated for 1 h at room temperature in blocking buffer (5% non-fat milk in PBS containing 0.1% Triton X-100 and 0.02% NaN3), and immunoblotted for 2 h at room temperature with the SNAP-23 antibody (ABR, Golden, CO). The primary antibodies were used at a dilution of 1:10,000 in blocking buffer. The immunoblotted nitrocellulose sheets were washed in PBS containing 0.1% Triton X-100 and 0.02% NaN3 and were incubated for 1 h at room temperature in horseradish peroxidase-conjugated secondary antibody at a dilution of 1:2,000 in blocking buffer. The immunoblots were then washed in the PBS buffer, processed for enhanced chemiluminescence, and exposed to X-OMAT-AR film. To isolate the fusion complex for immunoblot analysis, SNAP-23 specific antibody conjugated to protein A-sepharose was used. One gram of total pancreatic homogenate solubilized in Triton/Lubrol solubilization buffer (0.5% Lubrol; 1 mM benzamidine; 5 mM ATP; 5 mM EDTA; 0.5% Triton X-100, in PBS) supplemented with protease inhibitor mix (Sigma, St. Louis, MO) was used. SNAP-23 antibody conjugated to the protein A-sepharose was incubated with the solubilized homogenate for 1 h at room temperature followed by washing with wash buffer (500 mM NaCl, 10 mM Tris, 2 mM EDTA, pH = 7.5). The immunoprecipitated sample attached to the immunosepharose beads was incubated in Laemmli sample preparation buffer, before 12.5% SDS-PAGE, electrotransfer to nitrocellulose, and immunoblot analysis using specific antibodies to actin (Sigma), fodrin (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin (Sigma), syntaxin 2, Ca2+-ß3, and Ca2+-1c (Alomone Labs, Jerusalem, Israel).
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