Abstract
Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered (Lo) phase typical of the raft structure, namely “cholesterol-rich Lo phase”, with a phase fully missing in Cav-1 KO that we named “caveolin-induced Lo phase”. Furthermore, while the sole lipid component of both WT and KO-DRMs showed qualitatively similar isotherm configuration, the reinsertion of recombinant Cav-1 into WT-DRMs lipids restored the WT-DRM pattern. X-ray diffraction results confirmed that Cav-1 causes the formation of a “caveolin-induced Lo phase”, as suggested by Langmuir experiments, allowing us to speculate about a possible structural model. These results show that the unique molecular link between Cav-1 and cholesterol can spur functional order in a lipid bilayer strictly derived from biological sources.
Highlights
The initial discovery that important functional plasma membrane proteins, such as GPI anchored protein, caveolin-1 (Cav-1), and signal transduction proteins, could be isolated within restricted microdomains resistant to Triton X-100 solubilization was, since the beginning, connected to the hypothesis of the existence of lipid rafts in biological membranes [1,2]
We report Langmuir compression results, which displayed in WT-detergent resistant membranes (DRMs) an isotherm with a clear-cut feature representing a Lo phase fully missing in Cav-1 KO
Sucrose gradient fractions obtained from lung tissue treated with 1% TX 100 from WT and Cav-1 KO mice were analyzed by Western blot to determine the distribution of the caveolae marker Cav-1 and the lipid raft marker Flot-1 here used as KO DRM marker
Summary
The initial discovery that important functional plasma membrane proteins, such as GPI anchored protein, caveolin-1 (Cav-1), and signal transduction proteins, could be isolated within restricted microdomains resistant to Triton X-100 solubilization was, since the beginning, connected to the hypothesis of the existence of lipid rafts in biological membranes [1,2]. The original definition of lipid rafts as heterogeneous, highly dynamic, sterol, and sphingolipid-enriched domains that compartmentalize functional proteins [5], and possess a lipid structure that is equivalent to the liquid-ordered (Lo ) phase of model membranes [6], is nowadays insufficient to explain the many cellular roles attributed to lipid rafts [7,8]. In these last years there has been a revitalized interest in lipid rafts; despite recent advances in biochemical and microscopy approaches allowing light to be to shed on lipid organization and membrane heterogeneity in model membranes, the study of ordered membrane domains in vivo remains elusive. Caveolae are structurally recognizable invaginations of the plasma membrane, whose formation is dependent on
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