Elucidating membrane structure and protein behavior using giant plasma membrane vesicles, supplemental content
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To address the apical membrane of a particular vesicle, the confocal observation volume was first positioned in the center of the vesicle followed by an upward fluorescence intensity z-scan.
Receptor diffusion and ligand binding were measured on native vesicles immobilized on a poly-D-lysine coated 8-well coverglasses Nunc. Infrared trapping and fluorescence excitation laser light beams were separated from the emitted fluorescence light using a dielectric mirror Chroma, USA and appropriate filters.
For dual wavelength imaging a beam splitter Cairn Research was used. Phase separation in isolated vesicles was observed by the strong segregation of this fluorescent marker into the nonraft phase Fig. For determining ligand binding, the ACF of XAC-Atto was fitted as follows combining the three-dimensional diffusion of free ligands in solution and the two-dimensional diffusion of bound ligands: Maraviroc inhibits binding to the co-receptor CCR5 19and enfuvirtide inhibits fusion by blocking the required conformational change of gp41 GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.
J,K Fluorescence micrographs of the vesicle before J and after K perfusion with the NK1R agonist substance P showing enhanced concentration of arrestin-GFP at the plasma membrane; insets are the corresponding fluorescence profile cross-sections of the vesicles.
These vesicles offer a generic platform for bioanalysis of transmembrane signaling going beyond presently reached miniaturization and flexibility with many potential future applications: Such vesicles retain the functional integrity of the GPCR signaling cascade.
A Schematic drawing of the experimental design to understand the role of ordered lipid domains in HIV entry. For ligand binding experiments, vesicles were incubated for 30 min with various concentrations of XAC-Atto and KD was determined by plotting the apparent concentration of bound ligand to the total concentration of applied ligand.
This would provide a dynamic tomogram of the complex cellular signaling network and thus provide substantial new insight to the basics of cellular function. S2, C to E.
TMD length-dependent protein sorting between coexisting lipid domains has been addressed experimentally only recently by measuring partitioning of an oligomeric toxin perfringolysin O with multiple 35—40 TM segments in synthetic, phase-separated liposomes These PM vesicles separate into coexisting liquid phases 4 with different order 28which recruit membrane components in accordance with their predicted raft affinity, i.
Maraviroc reduced the targeting of HIV virions to phase boundaries Fig. The setup allowed acquisition of photon-count time traces and online correlation of the data. The selected rectangles are enlarged and shown below.
G Fluorescence micrograph of the vesicle obtained in F exhibiting fluorescence of the membrane expressed receptor. Excess area dependent scaling behavior of nano-sized membrane tethers.
Because ordered phases in lipid model systems consistently have been shown to be 0. These observations are made in giant plasma membrane vesicles GPMVswhich can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory.
Using this method, we readily observed striking differences between the mutants. A total of virions were analyzed for their distribution between the three compartments.
Integrins modulate TCR signaling by constraining actin flow at the immunological synapse. The partitioning of trLAT variants then was quantified by an established protocol 3031i.
Conclusion Taken together, we have demonstrated that individual plasma membrane vesicles function as minimized autonomous containers, capable of detecting external chemical signals and transducing them across the membrane to finally activate downstream signaling reactions inside the sub-femtoliter closed volume.
ACFs of freely diffusing molecules were fitted with a three-dimensional diffusion model including triplet formation: Erdinc Sezgin, Hermann-Josef Kaiser, Tobias Baumgart, Petra Schwille, Kai Simons, Ilya Levental Abstract The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts.
Membrane raft association is a determinant of plasma membrane localization
For example, a strong correlation exists between the TMD length of bitopic proteins and their organelle specificity 1920with longer TMDs targeting proteins to the PM and shorter TMDs found in the endoplasmic reticulum ERGolgi apparatus, and endocytic organelles.
Membrane morphology is actively transformed by covalent binding of the protein Atg8 to PE-lipids.
A movie version of this figure is available movie S3.
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