Paul A. Beales

Cytochrome c causes pore formation in cardiolipin-containing membranes

The release of cytochrome c from mitochondria is a key signaling mechanism in apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanisms remain unclear. Cytochrome c (cyt c) binds to and penetrates lipid structures containing the inner mitochondrial membrane lipid cardiolipin (CL), leading to protein conformational changes and increased peroxidase activity. We describe here a direct visualization of a fluorescent cyt c crossing synthetic, CL-containing membranes in the absence of other proteins. We observed strong binding of cyt c to CL in phospholipid vesicles and bursts of cyt c leakage across the membrane. Passive fluorescent markers such as carboxyfluorescein and a 10-kDa dextran polymer crossed the membrane simultaneously with cyt c, although larger dextrans did not. The data show that these bursts result from the opening of lipid pores formed by the cyt c–CL conjugate. Pore formation and cyt c leakage were significantly reduced in the presence of ATP. We suggest a model, consistent with these findings, in which the formation of toroidal lipid pores is driven by initial cyt c-induced negative spontaneous membrane curvature and subsequent protein unfolding interactions. Our results suggest that the CL–cyt c interaction may be sufficient to allow cyt c permeation of mitochondrial membranes and that cyt c may contribute to its own escape from mitochondria during apoptosis.

Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes.

We present a novel platform for investigating the composition-specific interactions of proteins (or other biologically relevant molecules) with model membranes composed of compositionally distinct domains. We focus on the interaction between a mitochondrial-specific lipid, cardiolipin (CL), and a peripheral membrane protein, cytochrome c (cyt c). We engineer vesicles with compositions such that they phase separate into coexisting liquid phases and the lipid of interest, CL, preferentially localizes into one of the domains (the liquid disordered (L(d)) phase). The presence of CL-rich and CL-depleted domains within the same vesicle provides a built-in control experiment to simultaneously observe the behavior of two membrane compositions under identical conditions. We find that cyt c binds strongly to CL-rich domains and observe fascinating morphological transitions within these regions of membrane. CL-rich domains start to form small buds and eventually fold up into a collapsed state. We also observe that cyt c can induce a strong attraction between the CL-rich domains of adjacent vesicles as demonstrated by the development of large osculating regions between these domains. Qualitatively similar behavior is observed when other polycationic proteins or polymers of a similar size and net charge are used instead of cyt c. We argue that these striking phenomena can be simply understood by consideration of colloidal forces between the protein and the membrane. We discuss the possible biological implications of our observations in relation to the structure and function of mitochondria.

Specific adhesion between DNA-functionalized “Janus” vesicles: size-limited clusters

Asymmetric building blocks afford assembly of more complex, sophisticated materials than their homogeneous counterparts. Phase separation of mixed membranes produces asymmetric surface textures in lipid vesicles. Membranes that demix into coexisting liquid phases ripen such that the vesicle domain morphology exhibits a Janus-like texture. DNA is commonly used in material science as a molecular glue. Hydrophobically modified DNA strands anchor to the membranes of vesicles such that the DNA is free to bind its complement. When DNA amphiphiles are anchored to phase separated vesicles, they thermodynamically partition between coexisting domains. This results in asymmetric surface distributions of adhesive functionalities. We enhance the partitioning of cholesteryl-anchored DNA into liquid ordered (Lo) domains of Janus-like vesicles by incorporating highly unsaturated lipids into membrane mixtures. We find that cardiolipin (CL) drives the strongest enrichment of DNA into Lo domains with apparent surface concentrations at least an order of magnitude greater than in coexisting liquid disordered domains. We also examine the partitioning of DNA with a lipid-like anchor in Janus-textured vesicles; the inclusion of CL also drives a very strong enhancement into Lo domains. The culmination of this work is the study of superstructures that form when populations of these Janus vesicles, functionalized by complementary DNA strands, are mixed. Unlike their homogeneous counterparts, which can form uncontrollably large clusters, size-limited multicompartmental architectures are observed. The DNA-rich Lo domains saturate in adhesion plaques of the clusters. This predominantly leaves DNA-depleted Lα phase accessible on the exterior surface of these structures, which does not favor binding of further vesicles.

Application of nucleic acid–lipid conjugates for the programmable organisation of liposomal modules

We present a critical review of recent work related to the assembly of multicompartment liposome clusters using nucleic acids as a specific recognition unit to link liposomal modules. The asymmetry in nucleic acid binding to its non-self complementary strand allows the controlled association of different compartmental modules into composite systems. These biomimetic multicompartment architectures could have future applications in chemical process control, drug delivery and synthetic biology. We assess the different methods of anchoring DNA to lipid membrane surfaces and discuss how lipid and DNA properties can be tuned to control the morphology and properties of liposome superstructures. We consider different methods for chemical communication between the contents of liposomal compartments within these clusters and assess the progress towards making this chemical mixing efficient, switchable and chemically specific. Finally, given the current state of the art, we assess the outlook for future developments towards functional modular networks of liposomes.

Partitioning of membrane-anchored DNA between coexisting lipid phases

The partitioning of different cholesterol-modified single-stranded DNA molecules (chol-DNAs) between the domains of phase-separated lipid vesicles is investigated by laser-scanning confocal fluorescence microscopy. All chol-DNAs studied preferentially localized into the fluid phase of giant vesicles in liquid-solid phase coexistence (1:1 DLPC:DPPC, 1:1 DLPC:DMPE). Partitioning behavior of chol-DNAs into liquid-liquid phase-separated vesicles (DOPC/DPPC/cholesterol) was found to be less straightforward. Single-cholesterol-anchored DNA molecules partitioned roughly equally between coexisting domains, whereas chol-DNAs with two cholesterol anchors were seen to be enriched in the liquid-ordered domains with apparent surface concentrations up to double that of the liquid-disordered phase. Quantitative analysis of the fluorescence intensity of DNA between the two phases also revealed a weaker dependence of the apparent partitioning on the initial lipid composition of the vesicles. We rationalize these observations by proposing a simple partitioning model based on the conformational entropy of insertion of a cholesterol anchor into each phase.

Protein crystallization: scaling of charge and salt concentration in lysozyme solutions

We studied the crystallization of lysozyme solutions by adding sodium chloride at pH = 4.5, 5.9 and 7.8. A universal crystallization boundary is found if data are scaled according to the salt concentration normalized by the square of the charge at the appropriate pH. Calculations show that this finding is consistent with recent attempts to rationalize protein crystallization using second virial coefficients.

Formation and dissolution of phospholipid domains with varying textures in hybrid lipo-polymersomes

The design of novel, soft membrane structures with chemical and physical properties that are tunable across a broad spectrum of parameter space is important for the development of new functional materials. Many potential applications of these novel membranes will likely require biocompatible interfaces, e.g., for functional reconstitution of integral proteins. Here we investigate the formation and control of compositional heterogeneities in hybrid lipo-polymersomes (HLPs) created by mixing the diblock copolymer poly(butadiene-b-ethylene oxide) with common natural lipids, e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-palmitoyl-oleoyl-sn-glycero-3-phosphocholine. Mixing and de-mixing of lipid-rich domains from the polymer-rich matrix of hybrid vesicles is controlled via thermally driven phase separation and by the inclusion of cholesterol. Domain size and morphology can be controlled by cooling rate and lipid composition, respectively. Macromolecular additives, e.g., cyclodextrins, enzymes and surfactants, can be used to remodel the hybrid membrane and its domains, resulting in domain dissolution, controlled release of contents or rupture of the hybrid vesicles. These composite membranes are promising materials for encapsulation-based technologies that require the combination of biocompatible membrane environments and enhanced structural stability, e.g., delivery and sensing applications or controlling (bio)chemical reactions within confinement.

PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties

Polybia-MP1 (MP1) is a bioactive host-defense peptide with known anticancer properties. Its activity is attributed to excess serine (phosphatidylserine (PS)) on the outer leaflet of cancer cells. Recently, higher quantities of phosphatidylethanolamine (PE) were also found at these cells’ surface. We investigate the interaction of MP1 with model membranes in the presence and absence of POPS (PS) and DOPE (PE) to understand the role of lipid composition in MP1’s anticancer characteristics. Indeed we find that PS lipids significantly enhance the bound concentration of peptide on the membrane by a factor of 7–8. However, through a combination of membrane permeability assays and imaging techniques we find that PE significantly increases the susceptibility of the membrane to disruption by these peptides and causes an order-of-magnitude increase in membrane permeability by facilitating the formation of larger transmembrane pores. Significantly, atomic-force microscopy imaging reveals differences in the pore formation mechanism with and without the presence of PE. Therefore, PS and PE lipids synergistically combine to enhance membrane poration by MP1, implying that the combined enrichment of both these lipids in the outer leaflet of cancer cells is highly significant for MP1’s anticancer action. These mechanistic insights could aid development of novel chemotherapeutics that target pathological changes in the lipid composition of cancerous cells.

DNA as Membrane-Bound Ligand-Receptor Pairs: Duplex Stability Is Tuned by Intermembrane Forces

We use membrane-anchored DNA as model adhesion receptors between lipid vesicles. By studying the thermal stability of DNA duplex formation, which tethers the vesicles into superstructures, we show that the melting temperature of a 10-base DNA sequence is dependent on the lipid composition of the tethered vesicles. We propose a simple model that describes how the intermembrane interactions tilt the free energy landscape for DNA binding. From our model, we estimate the area per DNA in the binding sites between vesicles and also the total area of the adhesion plaques. We find that vesicles containing a small proportion of cationic lipid that are modified with membrane-anchored DNA can be reversibly tethered by specific DNA interactions and that the DNA also induces a small attraction between these membranes, which stabilizes the DNA duplex. By increasing the equilibrium intermembrane distance on binding, we show that intermembrane interactions become negligible for the binding thermodynamics of the DNA and hence the thermal stability of vesicle aggregates becomes independent of lipid composition at large enough intervesicle separations. We discuss the implications of our findings with regards to cell adhesion and fusion receptors, and the programmable self-assembly of nano-structured materials by DNA hybridization.

β2-Microglobulin amyloid fibril-induced membrane disruption is enhanced by endosomal lipids and acidic pH.

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of β2-microglobulin (β2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which β2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of β2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that β2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between β2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of β2m amyloid-associated osteoarticular tissue destruction in DRA.