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- ItemSuperelasticity of Plasma- and Synthetic Membranes Resulting from Coupling of Membrane Asymmetry, Curvature, and Lipid Sorting(Weinheim : Wiley-VCH, 2021) Steinkühler, Jan; Fonda, Piermarco; Bhatia, Tripta; Zhao, Ziliang; Leomil, Fernanda S. C.; Lipowsky, Reinhard; Dimova, RumianaBiological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the apparent initial PM area. Isolated lipids self-organize into membranes, but are prone to rupture at small (<2–4%) area strains, which limits progress for synthetic reconstitution of cellular features. Here, it is shown that by preserving PM structure and composition during isolation from cells, vesicles with cell-like elasticity can be obtained. It is found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These act as lipid reservoirs and are recruited by mechanical tension applied to the outer vesicle membrane. Both in experiment and theory, it is shown that a “superelastic” response emerges from the interplay of lipid domains and membrane curvature. This finding allows for bottom-up engineering of synthetic biomaterials that appear one magnitude softer and with threefold larger deformability than conventional lipid vesicles. These results open a path toward designing superelastic synthetic cells possessing the inherent mechanics of biological cells.
- ItemLabel‐Free Imaging of Cholesterol Assemblies Reveals Hidden Nanomechanics of Breast Cancer Cells(Hoboken, NJ : Wiley, 2020) Dumitru, Andra C.; Mohammed, Danahe; Maja, Mauriane; Yang, Jinsung; Verstraeten, Sandrine; del Campo, Aranzazu; Mingeot-Leclercq, Marie-Paule; Tyteca, Donatienne; Alsteens, DavidTumor cells present profound alterations in their composition, structural organization, and functional properties. A landmark of cancer cells is an overall altered mechanical phenotype, which so far are linked to changes in their cytoskeletal regulation and organization. Evidence exists that the plasma membrane (PM) of cancer cells also shows drastic changes in its composition and organization. However, biomechanical characterization of PM remains limited mainly due to the difficulties encountered to investigate it in a quantitative and label‐free manner. Here, the biomechanical properties of PM of a series of MCF10 cell lines, used as a model of breast cancer progression, are investigated. Notably, a strong correlation between the cell PM elasticity and oncogenesis is observed. The altered membrane composition under cancer progression, as emphasized by the PM‐associated cholesterol levels, leads to a stiffening of the PM that is uncoupled from the elastic cytoskeletal properties. Conversely, cholesterol depletion of metastatic cells leads to a softening of their PM, restoring biomechanical properties similar to benign cells. As novel therapies based on targeting membrane lipids in cancer cells represent a promising approach in the field of anticancer drug development, this method contributes to deciphering the functional link between PM lipid content and disease.
- ItemNanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging(Washington, DC : ACS Publ., 2018-6-12) Schneider, Falk; Waithe, Dominic; Galiani, Silvia; Bernardino de la Serna, Jorge; Sezgin, Erdinc; Eggeling, ChristianThe diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.
- ItemMechanical properties of plasma membrane vesicles correlate with lipid order, viscosity and cell density(Berlin : Nature Publishing, 2019) Steinkühler, Jan; Sezgin, Erdinc; Urbančič, Iztok; Eggeling, Christian; Dimova, RumianaRegulation of plasma membrane curvature and composition governs essential cellular processes. The material property of bending rigidity describes the energetic cost of membrane deformations and depends on the plasma membrane molecular composition. Because of compositional fluctuations and active processes, it is challenging to measure it in intact cells. Here, we study the plasma membrane using giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. We show that the bending rigidity of plasma membranes under varied conditions is correlated to readout from environment-sensitive dyes, which are indicative of membrane order and microviscosity. This correlation holds across different cell lines, upon cholesterol depletion or enrichment of the plasma membrane, and variations in cell density. Thus, polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Additionally, our results allow for identifying synthetic membranes with a few well defined lipids as optimal plasma membrane mimetics.
- ItemNanoscale dynamics of cholesterol in the cell membrane(Bethesda, Md. : ASBMB Publications, 2019) Pinkwart, Kerstin; Schneider, Falk; Lukoseviciute, Martyna; Sauka-Spengler, Tatjana; Lyman, Edward; Eggeling, Christian; Sezgin, ErdincCholesterol constitutes ~30-40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane. © 2019 Pinkwart et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.