Near-Critical Fluctuations and Cytoskeleton-Assisted Phase Separation Lead to Subdiffusion in Cell Membranes

Ehrig, Jens; Petrov, Eugene P.; Schwille, Petra

doi:10.1016/j.bpj.2010.11.002

Cited by 104 publications

(118 citation statements)

References 63 publications

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“…Moreover, increase of cholesterol submicrometric domains upon acute uncoupling of membrane:spectrin anchorage at 4.1R complexes indicates restriction by protein:based fences [54,55]. These three lines of evidence suggest that cholesterol submicrometric domains are restricted by membrane:spectrin anchorage, in perfect agreement with the picket-fence network which allows confined diffusion of lipids and proteins in mesoscale domains [54,56] while preventing macroscopic domain formation [57,58].…”

Section: Biogenesis Of Cholesterol Submicrometric Domains: Restrictiosupporting

confidence: 52%

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

Carquin

Conrard

Pollet

et al. 2015

Cell. Mol. Life Sci.

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Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

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Section: Biogenesis Of Cholesterol Submicrometric Domains: Restrictiosupporting

confidence: 52%

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

Carquin

Conrard

Pollet

et al. 2015

Cell. Mol. Life Sci.

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“…Formation of Pore-Suspending Membranes Composed of DOPC/Sphingomyelin/Cholesterol/Gb 3 We made use of phase-separated pore-spanning membranes, and we show-as a proof-of-principle-that these bilayers are suitable mimics of the eukaryotic plasma membrane including the underlying cytoskeleton. Pore-spanning membranes were achieved by spreading GUVs on micrometer-sized functionalized highly ordered pore-arrays with pore diameters of 0.8, 1.2 and 2 mm (see Figure 1 D).…”

Section: Resultsmentioning

confidence: 99%

“…Monte Carlo simulations provide evidence that the membrane-cytoskeleton interaction strongly affects phase separation in lipid bilayers. [3] It was demonstrated that the cytoskeleton inhibits large-scale phase separation, which might explain why micrometer-scaled domains are observed in giant unilamellar vesicle membranes [4] as a result of a minimization of the line tension occurring at the border of different lipid compositions [5] but not in plasma membranes of living cells. [6] Thus, to create a more realistic model of the plasma membrane, not only the membrane composition has to be properly adjusted but also the underlying cytoskeleton has to be taken into account.…”

Section: Introductionmentioning

confidence: 99%

Creating and Modulating Microdomains in Pore‐Spanning Membranes

et al. 2011

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The architecture of the plasma membrane is not only determined by the lipid and protein composition, but is also influenced by its attachment to the underlying cytoskeleton. Herein, we show that microscopic phase separation of "raft-like" lipid mixtures in pore-spanning bilayers is strongly determined by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb(3) were prepared on ordered pore arrays in silicon with pore diameters of 0.8, 1.2 and 2 μm, respectively, by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol-bearing cholesterol derivative rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liquid-ordered- (l(o)) and liquid-disordered (l(d)) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb(3) (40:35:20:5) membranes. The size of the l(o)-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl-β-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb(3)-doped membranes increased the l(o)-phase considerably and even induced l(o)-phase domains in non-phase separated bilayers composed of DOPC/sphingomyelin/cholesterol/Gb(3) (65:10:20:5).

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“…The effects of immobile clustered obstacles on diffusion have been investigated in several theoretical studies (44,57,58,71). It was shown by Saxton (43) that a percolation threshold, c p , exists when the occupied area fraction of obstacles, c, exceeds 0.332.…”

Section: Existence Of Racks Of Rhodopsin Dimersmentioning

confidence: 99%

Explicit Spatiotemporal Simulation of Receptor-G Protein Coupling in Rod Cell Disk Membranes

Schöneberg

Heck

Hofmann

et al. 2014

Biophysical Journal

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Dim-light vision is mediated by retinal rod cells. Rhodopsin (R), a G-protein-coupled receptor, switches to its active form (R(∗)) in response to absorbing a single photon and activates multiple copies of the G-protein transducin (G) that trigger further downstream reactions of the phototransduction cascade. The classical assumption is that R and G are uniformly distributed and freely diffusing on disk membranes. Recent experimental findings have challenged this view by showing specific R architectures, including RG precomplexes, nonuniform R density, specific R arrangements, and immobile fractions of R. Here, we derive a physical model that describes the first steps of the photoactivation cascade in spatiotemporal detail and single-molecule resolution. The model was implemented in the ReaDDy software for particle-based reaction-diffusion simulations. Detailed kinetic in vitro experiments are used to parametrize the reaction rates and diffusion constants of R and G. Particle diffusion and G activation are then studied under different conditions of R-R interaction. It is found that the classical free-diffusion model is consistent with the available kinetic data. The existence of precomplexes between inactive R and G is only consistent with the data if these precomplexes are weak, with much larger dissociation rates than suggested elsewhere. Microarchitectures of R, such as dimer racks, would effectively immobilize R but have little impact on the diffusivity of G and on the overall amplification of the cascade at the level of the G protein.

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Near-Critical Fluctuations and Cytoskeleton-Assisted Phase Separation Lead to Subdiffusion in Cell Membranes

Cited by 104 publications

References 63 publications

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

Creating and Modulating Microdomains in Pore‐Spanning Membranes

Explicit Spatiotemporal Simulation of Receptor-G Protein Coupling in Rod Cell Disk Membranes

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