Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells

Abstract

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

CHO/LY-B cell growth under limiting sphingolipid supply: correlation between lipid composition and biophysical properties of sphingolipid-restricted cell membranes+

“…PM preparations. PM preparations were obtained as described in Monasterio et al 37 . Briefly, GPMV formation was induced adding the GPMV formation reagent [freshly prepared 2 mM dithiothreitol, 25 mM paraformaldehyde in GPMV buffer (2 mM CaCl 2 , 10 mM HEPES, 150 mM NaCl, pH 7.4)] to T25 flasks with cells at confluence.…”

“…Several washing steps were performed to discard the released intracellular contents. Purification quality was checked using Di-4-ANEPPDHQ (λ ex = 465 nm, λ em = 635 nm) as a general fluorescent staining, together with organelle-specific fluorophores as described in Monasterio et al 37 . Images were taken in a Leica TCS SP5 II microscope (Leica Microsystems GmbH, Wetzlar, Germany) at room temperature with ImageJ software.…”

“…PM patch lipid extraction. PM patches were formed following the above mentioned protocol 37,42 . Chloroform:methanol (2:1) organic solvent was used to recover the lipid fraction of the attached PM patches.…”

“…Samples were left to equilibrate for 30 min at room temperature before performing five washing steps with non-CaCl 2 buffer in order to discard non-adsorbed vesicles and remove the remaining Ca 2+ cations 53 . Isolated PM patches for force spectroscopy were prepared as previously described 37,42 , this time using polylysine-coated mica slips instead of glass-bottom dishes. GPMV were first stained using Di-4-ANEPPQHD to allow detection on the mica slip.…”

“…SM synthases, which use Cer and phosphatidylcholine as substrates to produce SM and diacylglyceride, are used in the de novo synthesis pathway, sometimes also involved in reutilization of ceramide 30,36 from exogenous or endogenous sources. In a previous work 37 plasma membrane (PM) preparations from CHO cells and model membranes prepared from their lipid extracts had been characterized. In the present study we have applied those methods to the study of SL-synthesis deficient LY-B cells to determine the role of SL on cell growth, membrane physical properties and composition.…”