Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells
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...
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MDPI AG
2020-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/7/2643 |
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| author | Bingen G. Monasterio Noemi Jiménez-Rojo Aritz B. García-Arribas Howard Riezman Félix M. Goñi Alicia Alonso |
| author_facet | Bingen G. Monasterio Noemi Jiménez-Rojo Aritz B. García-Arribas Howard Riezman Félix M. Goñi Alicia Alonso |
| author_sort | Bingen G. Monasterio |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T20:32:49Z |
| format | Article |
| id | doaj.art-45b60b8cc8fc475ba27370806d25a899 |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T20:32:49Z |
| publishDate | 2020-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-45b60b8cc8fc475ba27370806d25a8992023-11-19T21:14:32ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-04-01217264310.3390/ijms21072643Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO CellsBingen G. Monasterio0Noemi Jiménez-Rojo1Aritz B. García-Arribas2Howard Riezman3Félix M. Goñi4Alicia Alonso5Instituto Biofisika (CSIC, UPV/EHU), Campus Universitario, 48940 Leioa, SpainNCCR Chemical Biology, Department of Biochemistry, University of Geneva, 1211 Geneva, SwitzerlandInstituto Biofisika (CSIC, UPV/EHU), Campus Universitario, 48940 Leioa, SpainNCCR Chemical Biology, Department of Biochemistry, University of Geneva, 1211 Geneva, SwitzerlandInstituto Biofisika (CSIC, UPV/EHU), Campus Universitario, 48940 Leioa, SpainInstituto Biofisika (CSIC, UPV/EHU), Campus Universitario, 48940 Leioa, SpainThis 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.https://www.mdpi.com/1422-0067/21/7/2643giant plasma membrane vesiclesblebsplasma membranegeneral polarizationAtomic Force Microscopy (AFM)lipidomics |
| spellingShingle | Bingen G. Monasterio Noemi Jiménez-Rojo Aritz B. García-Arribas Howard Riezman Félix M. Goñi Alicia Alonso Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells International Journal of Molecular Sciences giant plasma membrane vesicles blebs plasma membrane general polarization Atomic Force Microscopy (AFM) lipidomics |
| title | Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells |
| title_full | Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells |
| title_fullStr | Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells |
| title_full_unstemmed | Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells |
| title_short | Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells |
| title_sort | patches and blebs a comparative study of the composition and biophysical properties of two plasma membrane preparations from cho cells |
| topic | giant plasma membrane vesicles blebs plasma membrane general polarization Atomic Force Microscopy (AFM) lipidomics |
| url | https://www.mdpi.com/1422-0067/21/7/2643 |
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