Osmotic Gradients Induce Bio-Reminiscent Morphological Transformations in Giant Unilamellar Vesicles

We report observations of large-scale, in-plane and out-of-plane membrane deformations in giant uni- and multilamellar vesicles composed of binary and ternary lipid mixtures in the presence of net transvesicular osmotic gradients. The lipid mixtures we examined consisted of binary mixtures of DOPC and DPPC lipids and ternary mixtures comprising POPC, sphingomyelin and cholesterol over a range of compositions – both of which produce co-existing phases for selected ranges of compositions at room temperature under thermodynamic equilibrium. In the presence of net osmotic gradients, we find that the in-plane phase separation potential of these mixtures is non-trivially altered and a variety of out-of-plane morphological remodeling events occur. The repertoire of membrane deformations we observe display striking resemblance to their biological counterparts in live cells encompassing vesiculation, membrane fission and fusion, tubulation and pearling, as well as expulsion of entrapped vesicles from multicompartmental giant unilamellar vesicles through large, self-healing transient pores. These observations suggest that the forces introduced by simple osmotic gradients across membrane boundaries could act as a trigger for shape-dependent membrane and vesicle trafficking activities. We speculate that such coupling of osmotic gradients with membrane properties might have provided lipid-mediated mechanisms to compensate for osmotic stress during the early evolution of membrane compartmentalization in the absence of osmoregulatory protein machinery

Reconstituting ring-rafts in bud-mimicking topography of model membranes.

During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding

Substrate Suppression of Thermal Roughness in Stacked Supported Bilayers

We have fabricated a stack of five 1,2-dipalmitoyl-sn-3-phosphatidylethanolamine (DPPE) bilayers supported on a polished silicon substrate in excess water. The density profile of these stacks normal to the substrate was obtained through analysis of x-ray reflectivity. Near the substrate, we find the layer roughness and repeat spacing are both significantly smaller than values found in bulk multilayer systems. The reduced spacing and roughness result from suppression of lateral fluctuations due to the flat substrate boundary. The layer spacing decrease then occurs due to reduced Helfrich repulsion.This work was partially supported by NSF Grants No. DMR-0706369 and No. DMR-0706665. Use of the Advanced Photon Sourcewas supported by theUSDepartment of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. SKS and ANP wish to acknowledge support from the Office of Basic Energy Sciences, US Department of Energy, via Grant No. DE-FG02- 04ER46173. We would also like to thank Suresh Narayanan for his support of the experimental work at Sector 8-ID

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes

Fas signaling induces raft coalescence that is blocked by cholesterol depletion in human RPE cells undergoing apoptosis

PURPOSE. To investigate whether the signaling events occurring in Fas-mediated apoptosis alter raft membrane formation in human RPE cells. METHODS. Formation of lipid rafts in cultured human retinal pigment epithelial cells (ARPE-19) was studied by confocal microscopy, with fluorescein-labeled cholera toxin subunit B binding protein (BODIPY)-labeled ganglioside GM1 lipid after Fas-L induction of apoptosis. Apoptosis was assessed by fluorescein-labeled annexin V detection of phosphatidylserine externalization and quadrant analysis with flow cytometry. Membrane rafts were localized into membrane vesicles by passing BODIPY-labeled GM1 RPE cells through a 2-m-pore polycarbonate membrane using an extruder device. The labeled fractions, containing vesicles enriched in GM1, were detected by flow cytometry and then analyzed for the presence of Fas protein. RESULTS. Differential punctate staining of membrane rafts was demonstrated in normal and FasL-induced apoptotic human ARPE-19 cells in culture by confocal microscopy, using cholera toxin B and GM1 labeling of extruded vesicles. The lipid raftassociated vesicles were derived by plasma membrane dissociation, via a newly developed whole-cell extrusion technique that produced 2-m vesicles with both GM1 lipid and Fas protein abundance enriched in a subpopulation of the membrane-derived vesicles. The amount of Fas protein in the vesicles containing raft domains markedly increased in FasL-treated cells. Treatment of human ARPE 19 cells with methyl ␤-cyclodextrin after FasL induction of apoptosis resulted in cellular cholesterol depletion and markedly reduced the incidence of Fas-receptor localization in GM1 rafts. CONCLUSIONS. Human ARPE-19 cells in culture contain membrane rafts with apoptotic signaling effectors uniformly distributed in the native state. The cells stimulated to undergo apoptosis appear to use membrane rafts in the death-signaling process by mobilization of rafts to localized regions of the membrane that are now enriched with apoptotic signaling effectors. Fas signaling induces apoptotic raft formation that results in polar condensation, or capping, of the rafts in the late stages of apoptosis. A novel extrusion technique is described that allows localization and enrichment of rafts into membrane vesicles, which can be assayed by flow cytometry. Cholesterol depletion, after Fas ligand activation of apoptosis, reduced raft formation in cells induced to undergo apoptosis. Therapeutic implications for the treatment of retinal disorders are discussed. (Invest Ophthalmol Vis Sci

Protein receptor-independent plasma membrane remodeling by HAMLET:a tumoricidal protein-lipid complex

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.Published versio