Photoswitchable Endocytosis of Biomolecular Condensates in Giant Vesicles.

Interactions between membranes and biomolecular condensates can give rise to complex phenomena such as wetting transitions, mutual remodeling, and endocytosis. In this study, light-triggered manipulation of condensate engulfment is demonstrated using giant vesicles containing photoswitchable lipids. UV irradiation increases the membrane area, which can be stored in nanotubes. When in contact with a condensate droplet, the UV light triggers rapid condensate endocytosis, which can be reverted by blue light. The affinity of the protein-rich condensates to the membrane and the reversibility of the engulfment processes is quantified from confocal microscopy images. The degree of photo-induced engulfment, whether partial or complete, depends on the vesicle excess area and the relative sizes of vesicles and condensates. Theoretical estimates suggest that utilizing the light-induced excess area to increase the vesicle-condensate adhesion interface is energetically more favorable than the energy gain from folding the membrane into invaginations and tubes. The overall findings demonstrate that membrane-condensate interactions can be easily and quickly modulated via light, providing a versatile system for building platforms to control cellular events and design intelligent drug delivery systems for cell repair.

Photomanipulation of Minimal Synthetic Cells: Area Increase, Softening, and Interleaflet Coupling of Membrane Models Doped with Azobenzene-Lipid Photoswitches.

Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), a holistic picture of light-triggered changes in membrane kinetics, morphology, and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers, and molecular dynamics simulations is provided. Light-induced membrane area increases as high as ≈25% and a ten-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than palmitoyl-oleoyl phosphatidylcholine (POPC) bilayers but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions results in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models, and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders.

Kinetic relaxation of giant vesicles validates diffusional softening in a binary lipid mixture.

The stiffness of biological membranes determines the work required by cellular machinery to form and dismantle vesicles and other lipidic shapes. Model membrane stiffness can be determined from the equilibrium distribution of giant unilamellar vesicle surface undulations observable by phase contrast microscopy. With two or more components, lateral fluctuations of composition will couple to surface undulations depending on the curvature sensitivity of the constituent lipids. The result is a broader distribution of undulations whose complete relaxation is partially determined by lipid diffusion. In this work, kinetic analysis of the undulations of giant unilamellar vesicles made of phosphatidylcholine-phosphatidylethanolamine mixtures validates the molecular mechanism by which the membrane is made 25% softer than a single-component one. The mechanism is relevant to biological membranes, which have diverse and curvature-sensitive lipids.

Assessing membrane material properties from the response of giant unilamellar vesicles to electric fields.

Knowledge of the material properties of membranes is crucial to understanding cell viability and physiology. A number of methods have been developed to probe membranes in vitro, utilizing the response of minimal biomimetic membrane models to an external perturbation. In this review, we focus on techniques employing giant unilamellar vesicles (GUVs), model membrane systems, often referred to as minimal artificial cells because of the potential they offer to mimick certain cellular features. When exposed to electric fields, GUV deformation, dynamic response and poration can be used to deduce properties such as bending rigidity, pore edge tension, membrane capacitance, surface shear viscosity, excess area and membrane stability. We present a succinct overview of these techniques, which require only simple instrumentation, available in many labs, as well as reasonably facile experimental implementation and analysis.

Zwitterionic Dendrimersomes: A Closer Xenobiotic Mimic of Cell Membranes.

Building functional mimics of cell membranes is an important task toward the development of synthetic cells. So far, lipid and amphiphilic block copolymers are the most widely used amphiphiles with the bilayers by the former lacking stability while membranes by the latter are typically characterized by very slow dynamics. Herein, a new type of Janus dendrimer containing a zwitterionic phosphocholine hydrophilic headgroup (JDPC ) and a 3,5-substituted dihydrobenzoate-based hydrophobic dendron is introduced. JDPC self-assembles in water into zwitterionic dendrimersomes (z-DSs) that faithfully recapitulate the cell membrane in thickness, flexibility, and fluidity, while being resilient to harsh conditions and displaying faster pore closing dynamics in the event of membrane rupture. This enables the fabrication of hybrid DSs with components of natural membranes, including pore-forming peptides, structure-directing lipids, and glycans to create raft-like domains or onion vesicles. Moreover, z-DSs can be used to create active synthetic cells with life-like features that mimic vesicle fusion and motility as well as environmental sensing. Despite their fully synthetic nature, z-DSs are minimal cell mimics that can integrate and interact with living matter with the programmability to imitate life-like features and beyond.

GM1 asymmetry in the membrane stabilizes pores.

Cell membranes are highly asymmetric and their stability against poration is crucial for survival. We investigated the influence of membrane asymmetry on electroporation of giant unilamellar vesicles with membranes doped with GM1, a ganglioside asymmetrically enriched in the outer leaflet of neuronal cell membranes. Compared with symmetric membranes, the lifetimes of micronsized pores are about an order of magnitude longer suggesting that pores are stabilized by GM1. Internal membrane nanotubes caused by the GM1 asymmetry, obstruct and additionally slow down pore closure, effectively reducing pore edge tension and leading to leaky membranes. Our results point to the drastic effects this ganglioside can have on pore resealing in biotechnology applications based on poration as well as on membrane repair processes.

Characterization of DAG Binding to TRPC Channels by Target-Dependent cis-trans Isomerization of OptoDArG.

Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel-lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable cis conformation to the trans conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand-protein interactions. Cis photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal cis-trans isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by cis PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), cis OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of cis OptoDArG-induced currents in the dark was sensitive to mutations in the L2 lipid coordination site of TRPC channels. We conclude that the binding of cis OptoDArG to TRPC channels promotes transition of cis OptoDArG to the trans conformation. This process is suggested to provide valuable information on DAG-ion channel interactions and may enable highly selective photopharmacological interventions.

Glycopolymer Based LbL Multilayer Thin Films with Embedded Liposomes.

Layer-by-layer (LbL) self-assembly emerged as an efficient technique for fabricating coating systems for, e.g., drug delivery systems with great versatility and control. In this work, protecting group free and aqueous-based syntheses of bioinspired glycopolymer electrolytes aredescribed. Thin films of the glycopolymers are fabricated by LbL self-assembly and function as scaffolds for liposomes, which potentially can encapsulate active substances. The adsorbed mass, pH stability, and integrity of glycopolymer coatings as well as the embedded liposomes are investigated via whispering gallery mode (WGM) technology and quartz crystal microbalance with dissipation (QCM-D) monitoring , which enable label-free characterization. Glycopolymer thin films, with and without liposomes, are stable in the physiological pH range. QCM-D measurements verify the integrity of lipid vesicles. Thus, the fabrication of glycopolymer-based surface coatings with embedded and intact liposomes is presented.

Perfluorinated Ligands Induce Meridional Metal Stereochemistry to Generate M8L12, M10L15, and M12L18 Prisms.

Meridional (mer) coordination promotes the generation of larger and lower-symmetry prismatic metallosupramolecular structures, in contrast with the facial (fac) coordination common to smaller and higher-symmetry polyhedra. Here, we describe a general route to the selective formation of large metallosupramolecular prisms that contain exclusively mer-coordinated metal vertices. The use of 2-formylpyridine subcomponents that contain perfluorophenylene substituents at their 5-positions resulted in stereoselective formation of the iron(II) complexes from these subcomponents. Only mer vertices were observed, as opposed to the statistical fac/mer mixture otherwise generated. This mer-selective self-assembly could be used to prepare tetragonal (M8L12), pentagonal (M10L15), and hexagonal (M12L18) prisms by taking advantage of the subtle selectivities imposed by the different anilines and counterions employed. The equilibrium between the tetragonal and pentagonal prism followed a linear free-energy relationship, with the ratio between structures correlating with the Hammett σp(+) parameter of the incorporated aniline. The contrasting preferences of the fluorinated and nonfluorinated ligands to generate prisms and tetrahedra, respectively, were quantified energetically, with the destabilization increasing linearly for each "incorrect ligand" incorporated into either structure.