8 research outputs found
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 ââprotein-centricâ 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 ââreceptor independentâ 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
Pulsatile Gating of Giant Vesicles Containing Macromolecular Crowding Agents Induced by Colligative Nonideality
The ability of large
macromolecules to exhibit nontrivial deviations
in colligative properties of their aqueous solutions is well-appreciated
in polymer physics. Here, we show that this colligative nonideality
subjects giant lipid vesicles containing inert macromolecular crowding
agents to osmotic pressure differentials when bathed in small-molecule
osmolytes at comparable concentrations. The ensuing influx of water
across the semipermeable membrane induces characteristic swell-burst
cycles: here, cyclical and damped oscillations in size, tension, and
membrane phase separation occur <i>en route</i> to equilibration.
Mediated by synchronized formation of transient pores, these cycles
orchestrate pulsewise ejection of macromolecules from the vesicular
interior reducing the osmotic differential in a stepwise manner. These
experimental findings are fully corroborated by a theoretical model
derived by explicitly incorporating the contributions of the solution
viscosity, solute diffusivity, and the colligative nonideality of
the osmotic pressure in a previously reported continuum description.
Simulations based on this model account for the differences in the
details of the noncolligatively induced swell-burst cycles, including
numbers and periods of the repeating cycles, as well as pore lifetimes.
Taken together, our observations recapitulate behaviors of vesicles
and red blood cells experiencing sudden osmotic shocks due to large
(hundreds of osmolars) differences in the concentrations of small
molecule osmolytes and link intravesicular macromolecular crowding
with membrane remodeling. They further suggest that any tendency for
spontaneous overcrowding in single giant vesicles is opposed by osmotic
stresses and requires independent specific interactions, such as associative
chemical interactions or those between the crowders and the membrane
boundary
Kinetic control of shape deformations and membrane phase separation inside giant vesicles
A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.Nanyang Technological UniversityW.-C.S., D.L.G. and A.N.P. acknowledge funding from the National Science Foundation (DMR-1810540). J.C.S.H. and A.N.P. acknowledge funding and support from the Singapore Centre for Environmental Life Sciences Engineering and the Institute for Digital Molecular Analytics and Science, Nanyang Technological University. C.D.K. and A.T.R. were supported by the US Department of Energy, Office of Science, Basic Energy Sciences under award no. DE-SC0008633
Recurrent dynamics of rupture transitions of giant lipid vesicles at solid surfaces
Single giant unilamellar vesicles (GUVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophobic surfaces, the GUVs rupture via a recurrent, bouncing ball rhythm. During each contact, the GUVs, rendered tense by the substrate interactions, porate, and spread a molecularly transformed motif of a monomolecular layer on the hydrophobic surface from the point of contact in a symmetric manner. The competition from pore closure, however, limits the spreading and produces a daughter vesicle, which re-engages with the substrate. At solid hydrophilic surfaces, by contrast, GUVs rupture via a distinctly different recurrent burst-heal dynamics; during burst, single pores nucleate at the contact boundary of the adhering vesicles, facilitating asymmetric spreading and producing a "heart"-shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. In both cases, the pattern of burst-reseal events repeats multiple times, splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise, not because of the elastic properties of the solid surface, but because the competition between membrane spreading and pore healing, prompted by the surface-energy-dependent adhesion, determine the course of the topological transition
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Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide
The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of âŒ100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity
Mixing, Diffusion, and Percolation in Binary Supported Membranes Containing Mixtures of Lipids and Amphiphilic Block Copolymers
Substrate-mediated
fusion of small polymersomes, derived from mixtures
of lipids and amphiphilic block copolymers, produces hybrid, supported
planar bilayers at hydrophilic surfaces, monolayers at hydrophobic
surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces,
directly responding to local measures of (and variations in) surface
free energy. Despite the large thickness mismatch in their hydrophobic
cores, the hybrid membranes do not exhibit microscopic phase separation,
reflecting irreversible adsorption and limited lateral reorganization
of the polymer component. With increasing fluid-phase lipid fraction,
these hybrid, supported membranes undergo a fluidity transition, producing
a fully percolating fluid lipid phase beyond a critical area fraction,
which matches the percolation threshold for the immobile point obstacles.
This then suggests that polymer-lipid hybrid membranes might be useful
models for studying obstructed diffusion, such as occurs in lipid
membranes containing proteins
Mixing, Diffusion, and Percolation in Binary Supported Membranes Containing Mixtures of Lipids and Amphiphilic Block Copolymers
Substrate-mediated
fusion of small polymersomes, derived from mixtures
of lipids and amphiphilic block copolymers, produces hybrid, supported
planar bilayers at hydrophilic surfaces, monolayers at hydrophobic
surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces,
directly responding to local measures of (and variations in) surface
free energy. Despite the large thickness mismatch in their hydrophobic
cores, the hybrid membranes do not exhibit microscopic phase separation,
reflecting irreversible adsorption and limited lateral reorganization
of the polymer component. With increasing fluid-phase lipid fraction,
these hybrid, supported membranes undergo a fluidity transition, producing
a fully percolating fluid lipid phase beyond a critical area fraction,
which matches the percolation threshold for the immobile point obstacles.
This then suggests that polymer-lipid hybrid membranes might be useful
models for studying obstructed diffusion, such as occurs in lipid
membranes containing proteins
Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide
The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of âŒ100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity