Technical Reference #214

214.

Surface-expressed Lamellar Body Membrane is Recycled to Lamellar Bodies S. Schaller-Bals; S.R. Bates; K. Notarfrancesco; J.Q. Tao; A.B. Fisher; H. Shuman, University of Pennsylvania Medical Center, American Journal of Physiology: Lung Cellular and Molecular Physiology, 279(214), (2000)
Link To Paper

Abstract:
Monoclonal antibody (MAb) 3C9 an antibody generated to the lamellar body of rat lung type II pneumocytes specifically labels the luminal face of the lamellar body membrane. To follow the retrieval of lamellar body membrane from the cell surface in these

Keywords:
endocytosis; membrane trafficking; organelle biogenesis; type II pneumocytes

Materials & Methods:
Production of MAb 3C9. MAb 3C9 was raised by immunizing BALB/c mice with lamellar body membrane fractions isolated from rat lungs. Mouse sera and hybridoma supernatants were screened by indirect immunofluorescence of isolated type II cells. After 24 h in culture MAb 3C9 labeled ringlike structures that were shown to be the limiting membrane of lamellar bodies in permeabilized freshly isolated type II cells (32). Labeling of MAb 3C9 and other markers. MAb 3C9 mouse IgG and human or rat iron-saturated transferrin (Sigma St. Louis MO) were covalently labeled with fluorescein-EX or rhodamine with the use of the FluoReporter or Texas Red-X protein labeling kits (Molecular Probes Eugene OR). Fluorescein- conjugated cationized ferritin from horse spleen was also purchased from Molecular Probes. Fluorescein-conjugated Chrom-Pure rat transferrin was from Jackson ImmunoResearch Laboratories (West Grove PA). MAb 3C9 and mouse IgG were conjugated with 10-nm colloidal gold (BBInternational Cardiff UK) by the addition of the minimum amount of MAb 3C9 or mouse IgG required to stabilize the gold in suspension. The antibody-gold conjugate was purified by centrifugation. Lipids and liposome preparation. NBD-PC {1-palmitoyl-2- [12-[(7-nitro-2–13-benzoxadiazol-4-yl)amino]dodecanoyl]phosphatidylcholine} dipalmitoylphosphatidylcholine (DPPC) egg phosphatidylcholine (PC) phosphatidylglycerol (PG) and cholesterol were obtained from Avanti Polar Lipids (Birmingham Al). NBD-PC is a fluorescent analog of PC (excitation maximum 463 nm and emission maximum 536 nm) previously used to follow lipid uptake in the lung and in cultured type II cells (5 23). The relative molar ratios of (NBD-PC)- DPPC-egg PC-PG-cholesterol used in the liposomes were 15:35:25:10:15. These ratios were chosen to reflect the lipid composition of the lung surfactant with NBD-PC lipid replacing the fraction of DPPC in the mixture that maximizes liposome fluorescence (23). Uniform unilamellar liposomes with a diameter of 100–200 nm were prepared by extrusion through polycarbonate membranes (14). In vivo experiments. Rhodamine-labeled MAb 3C9 or labeled mouse IgG (20 mg in 200 ml of 0.9% sodium chloride) was intratracheally instilled in an anesthetized rat. After 1 h on the respirator the rat was killed and the lung was perfused and fixed in 4% paraformaldehyde. A portion of the tissue was frozen and cryosectioned (8 mm) and another portion was embedded in Polybed 812 resin (Polysciences Warrington PA) for thinner sectioning. Some of the cryosections were also immunostained with a polyclonal antibody (NPROSP-C) against the amino terminus of the precursor peptide of the rat surfactant protein (SP) C which has previously been shown to be specific to type II cells of the lung and is found in lamellar bodies (3). Antibodies to the precursor peptide were used because it has been difficult to raise antibodies to the highly hydrophobic mature SP-C (3). Cell preparation. Type II cells were isolated from adult male Sprague-Dawley rat lungs according to the procedure of Dobbs et al. (8). Briefly the lungs were perfused via the pulmonary artery with solution II (0.9% saline and 0.1% glucose with 10 mM HEPES 5 mM KCl 2.5 mM sodium phosphate buffer 1.7 mM CaCl2 1.3 mM MgSO4 35 mg of penicillin and 50 mg of streptomycin in 500 ml). Lungs were then lavaged eight times through a tracheal cannula with solution I (0.9% saline and 0.1% glucose with 10 mM HEPES 5 mM KCl 2.5 mM sodium phosphate buffer 35 mg of EGTA 35 mg of penicillin and 50 mg of streptomycin in 500 ml) and two more times with warm solution II. For elastase digestion 10 ml of solution II containing 3 U/ml of elastase were instilled into the trachea. The elastase instillation was repeated twice more at 10-min intervals. The lung was minced with a McIlwain tissue chopper in the presence of a small volume of 100% FBS (ICN/Flow Laboratories ICN Biochemicals Costa Mesa CA) and then poured into 10 ml of solution II containing 4 mg of DNase (Sigma). Cells were separated by filtration through a sequence of nylon meshes (160 37 and 10 mm). The remaining macrophages were removed by plating the cell suspension onto a rat IgG (Sigma)-covered petri dish for 60 min. The unattached type II cells were harvested and centrifuged at 1000 rpm for 10 min then resuspended in MEM containing 10% FCS. The cells were plated onto glass coverslips (Fisher Scientific Pittsburgh PA) or glass-bottomed dishes (MatTek Ashland MA) at a density of 1–2 3 106/35-mm culture dish. Cells were cultured overnight in a humidified 37°C incubator supplemented with 5% CO2 in air. Purity of the type II cell preparation was .92% as determined by modified Papanicolau stain.

Microscopic Technique
Fluorescence Microscopy, Electron Microscopy

Cell Type(s)
Type II