Aiwei Tian

Spotted vesicles, striped micelles and Janus assemblies induced by ligand binding

Selective binding of multivalent ligands within a mixture of polyvalent amphiphiles provides, in principle, a mechanism to drive domain formation in self-assemblies. Divalent cations are shown here to crossbridge polyanionic amphiphiles that thereby demix from neutral amphiphiles and form spots or rafts within vesicles as well as stripes within cylindrical micelles. Calcium and copper crossbridged domains of synthetic block copolymers or natural lipid (PIP2, phosphatidylinositol-4,5-bisphosphate) possess tunable sizes, shapes, and/or spacings that can last for years. Lateral segregation in these ‘responsive Janus assemblies’ couples weakly to curvature and proves restricted within phase diagrams to narrow regimes of pH and cation concentration that are centered near the characteristic binding constants for polyacid interactions. Remixing at high pH is surprising, but a theory for Strong Lateral Segregation (SLS) shows that counterion entropy dominates electrostatic crossbridges, thus illustrating the insights gained into ligand induced pattern formation within self-assemblies.

Sorting of Lipids and Proteins in Membrane Curvature Gradients

The sorting of lipids and proteins in cellular trafficking pathways is a process of central importance in maintaining compartmentalization in eukaryotic cells. However, the mechanisms behind these sorting phenomena are currently far from being understood. Among several mechanistic suggestions, membrane curvature has been invoked as a means to segregate lipids and proteins in cellular sorting centers. To assess this hypothesis, we investigate the sorting of lipid analog dye trace components between highly curved tubular membranes and essentially flat membranes of giant unilamellar vesicles. Our experimental findings indicate that intracellular lipid sorting, contrary to frequent assumptions, is unlikely to occur by lipids fitting into membrane regions of appropriate curvature. This observation is explained in the framework of statistical mechanical lattice models that show that entropy, rather than curvature energy, dominates lipid distribution in the absence of strongly preferential lateral intermolecular interactions. Combined with previous findings of curvature induced phase segregation, we conclude that lipid cooperativity is required to enable efficient sorting. In contrast to lipid analog dyes, the peripheral membrane binding protein Cholera toxin subunit B is effectively curvature-sorted. The sorting of Cholera toxin subunit B is rationalized by statistical models. We discuss the implications of our findings for intracellular sorting mechanisms.

Dynamic sorting of lipids and proteins in membrane tubes with a moving phase boundary

Cellular organelle membranes maintain their integrity, global shape, and composition despite vigorous exchange among compartments of lipids and proteins during trafficking and signaling. Organelle homeostasis involves dynamic molecular sorting mechanisms that are far from being understood. In contrast, equilibrium thermodynamics of membrane mixing and sorting, particularly the phase behavior of binary and ternary model membrane mixtures and its coupling to membrane mechanics, is relatively well characterized. Elucidating the continuous turnover of live cell membranes, however, calls for experimental and theoretical membrane models enabling manipulation and investigation of directional mass transport. Here we introduce the phenomenon of curvature-induced domain nucleation and growth in membrane mixtures with fluid phase coexistence. Membrane domains were consistently observed to nucleate precisely at the junction between a strongly curved cylindrical (tube) membrane and a pipette-aspirated giant unilamellar vesicle. This experimental geometry mimics intracellular sorting compartments, because they often show tubular-vesicular membrane regions. Nucleated domains at tube necks were observed to present diffusion barriers to the transport of lipids and proteins. We find that curvature-nucleated domains grow with characteristic parabolic time dependence that is strongly curvature-dependent. We derive an analytical model that reflects the observed growth dynamics. Numerically calculated membrane shapes furthermore allow us to elucidate mechanical details underlying curvature-dependent directed lipid transport. Our observations suggest a novel dynamic membrane sorting principle that may contribute to intracellular protein and lipid sorting and trafficking.

Bending Stiffness Depends on Curvature of Ternary Lipid Mixture Tubular Membranes

Lipid and protein sorting and trafficking in intracellular pathways maintain cellular function and contribute to organelle homeostasis. Biophysical aspects of membrane shape coupled to sorting have recently received increasing attention. Here we determine membrane tube bending stiffness through measurements of tube radii, and demonstrate that the stiffness of ternary lipid mixtures depends on membrane curvature for a large range of lipid compositions. This observation indicates amplification by curvature of cooperative lipid demixing. We show that curvature-induced demixing increases upon approaching the critical region of a ternary lipid mixture, with qualitative differences along two roughly orthogonal compositional trajectories. Adapting a thermodynamic theory earlier developed by M. Kozlov, we derive an expression that shows the renormalized bending stiffness of an amphiphile mixture membrane tube in contact with a flat reservoir to be a quadratic function of curvature. In this analytical model, the degree of sorting is determined by the ratio of two thermodynamic derivatives. These derivatives are individually interpreted as a driving force and a resistance to curvature sorting. We experimentally show this ratio to vary with composition, and compare the model to sorting by spontaneous curvature. Our results are likely to be relevant to the molecular sorting of membrane components in vivo.

Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion

Objective—The purpose of this study was to evaluate the role of membrane cholesterol on human neutrophil and HL-60 biomechanics, capture, rolling, and arrest to P-selectin– or IL-1–activated endothelium. Methods and Results—Methyl-β-cyclodextrin (MβCD) removed up to 73% and 45% of membrane cholesterol from HL-60 cells and neutrophils, whereas MβCD/cholesterol complexes resulted in maximum enrichment of 65% and 40%, respectively, above control levels. Cells were perfused at a venous wall shear rate of 100 s−1 over adherent P-selectin–coated 1-&mgr;m diameter beads, uncoated 10-&mgr;m diameter beads, P-selectin–coated surfaces, or activated endothelium. Elevated cholesterol enhanced capture efficiency to 1-&mgr;m beads and increased membrane tether growth rate by 1.5- to 2-fold, whereas cholesterol depletion greatly reduced tether formation. Elevated cholesterol levels increased tether lifetime by 17% in neutrophils and adhesion lifetime by 63% in HL-60 cells. Deformation of cholesterol-enriched neutrophils increased the contact time with 10-&mgr;m beads by 32% and the contact area by 7-fold. On both P-selectin surfaces and endothelial-cell monolayers, cholesterol-enriched neutrophils rolled more slowly, more stably, and were more likely to firmly arrest. Cholesterol depletion resulted in opposite effects. Conclusions—Increasing membrane cholesterol enhanced membrane tether formation and whole cell deformability, contributing to slower, more stable rolling on P-selectin and increased firm arrest on activated endothelium.

Polymersomes and Wormlike Micelles Made Fluorescent by Direct Modifications of Block Copolymer Amphiphiles

Wormlike micelles and vesicles prepared from diblock copolymers are attracting great interest for a number of technological applications. Although transmission electron microscopy has remained as the method of choice for assessing the morphologies, fluorescence microscopy has a number of advantages. We show here that when commercially available fluorophores are covalently attached to diblock copolymers, a number of their physicochemical characteristics can be investigated. This method becomes particularly useful for visualizing phase separation within polymer assemblies and assessing the dynamics of wormlike micelles in real time. Near-IR fluorophores can be covalently conjugated to polymers and this opens the possibility for deep-tissue fluorescence imaging of polymer assemblies in drug delivery applications.

Dynamic Domains in Polymersomes: Mixtures of Polyanionic and Neutral Diblocks Respond More Rapidly to Changes in Calcium than to pH

Chemical triggering of membrane domain dynamics is of broad relevance to cell signaling through lipid bilayers and might also be exploited in application of phase-separated vesicles. Here we describe the morphodynamics and remixing kinetics of spotted polymersomes made with mixtures of polyanionic and neutral amphiphiles plus calcium. Addition of the calcium chelator EDTA to vesicle dispersions produced a decrease in domain size within minutes, whereas increasing the pH with NaOH led to the viscous fingering of domains and decreased domain size over hours. Although the latter suggests that the charge of the polyanion contributes to domain formation, the remixing of more negative chains at high pH is surprising. Domain roughening at high pH is also accelerated by EDTA, which highlights the dominance of cross-bridging. Importantly, even though vesicles were perturbed only externally, the inner and outer leaflets remain coupled throughout, consistent with molecular dynamics simulations and suggestive of an order-disorder transition that underlies the remixing kinetics.

Mechanical stability of micropipet-aspirated giant vesicles with fluid phase coexistence.

Micropipet aspiration of phase-separated lipid bilayer vesicles can elucidate physicochemical aspects of membrane fluid phase coexistence. Recently, we investigated the composition dependence of line tension at the boundary between liquid-ordered and liquid-disordered phases of giant unilamellar vesicles obtained from ternary lipid mixtures using this approach. Here we examine mechanical equilibria and stability of dumbbell-shaped vesicles deformed by line tension. We present a relationship between the pipet aspiration pressure and the aspiration length in vesicles with two coexisting phases. Using a strikingly simple mechanical model for the free energy of the vesicle, we predict a relation that is in almost quantitative agreement with experiment. The model considers the vesicle free energy to be proportional to line tension and assumes that the vesicle volume, domain area fraction, and total area are conserved during aspiration. We also examine a mechanical instability encountered when releasing a vesicle from the pipet. We find that this releasing instability is observed within the framework of our model that predicts a change of the compressibility of a pipet-aspirated membrane cylinder from positive (i.e., stable) to negative (unstable) values, at the experimental instability. The model furthermore includes an aspiration instability that has also previously been experimentally described. Our method of studying micropipet-induced shape transitions in giant vesicles with fluid domains could be useful for investigating vesicle shape transitions modulated by bending stiffness and line tension.

Bending Stiffness and Curvature Coupling of Ternary Lipid Mixtures

There exists a wide range of curvature gradients within and between cellular organelles. Differences between membrane morphologies play important roles in cell homeostasis, for example, in the sorting and trafficking of membrane components, as well as in controlling the activities of membrane associated proteins. To better understand the mechanisms by which curvature regulates cellular functions, here, we investigate membrane curvature coupling to membrane composition and mechanical properties.We find that bending stiffness depends on membrane curvature of micro-scale homogeneous ternary lipid mixtures. Curvature gradients were generated by lipid tethers with controllable radius pulled from giant vesicles, and bending stiffness was obtained from tether radius and membrane tension measurements. As curvature increases, bending energy overcomes mixing entropy such that highly flexible lipid groups are sorted into the tube from the flat membrane. The sorting is enhanced as composition approaches the neighborhood of the mixing-demixing critical point, through two trajectories: parallel and perpendicular to the phase boundary. An expression that predicts bending stiffness to be a quadratic function of curvature in ternary mixture is derived, from which curvature sorting efficiency is obtained. We then interpret the sorting efficiency to be the ratio of a driving force for and a resistance to sorting. In addition, we estimate the bending stiffness of ternary mixtures at zero curvature, finding consistency with our measurements from the micropipette aspiration method.

Curvature Sorting of Lipids and Proteins in the Strong Segregation Limit: Curvature Mediated Domain Nucleation and Steady State Transport in Tubular Membranes with Phase Separation

Intracellular sorting centers, including the trans-Golgi network, the endoplasmic reticulum, and the endocytic recycling compartment, all contain membrane tube elements with cylindrical curvatures. The question of how curvature and sorting are coupled is central to the understanding of the function of organelle homeostasis, membrane trafficking, and intracellular sorting. Of particular interest, but underexplored, are non-equilibrium phenomena fundamentally linked to membrane transport.Here we present a straightforward and well controlled model system that allows us to characterize lipid transport at steady state: ternary lipid mixture tubular membranes pulled from phase-separated giant unilamellar vesicles (GUVs) by means of optical tweezers. The tubule composition, when initially formed from the liquid-ordered (Lo) phase region of the vesicle, is dependent on the velocity at which it is pulled: fast extraction velocities create tethers of the Lo phase, while slow extraction velocities can generate tethers that are liquid-disordered (Ld) phase. Thus, the speed with which highly curved tubules are formed may possibly serve as a control variable in living cells to adjust tubule composition. Furthermore, in tubules which are initially Lo phase, we find curvature-induced nucleation of Ld domains at the neck between tubule and vesicle. These Ld domains display characteristic, curvature dependent parabolic growth behavior that can be understood via a straightforward analytical mass transfer model that we derived from linear irreversible thermodynamics.We have also developed numerical schemes that capture shape transitions of tubular membranes on the basis of measured biophysical parameters in support of our experimental findings.