Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair

Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca2+ activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca2+, AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing

Diffusion in low-dimensional lipid membranes

The diffusion behavior of biological components in cellular membranes is vital to the function of cells. By collapsing the complexity of planar 2D membranes down to one dimension, fundamental investigations of bimolecular behavior become possible in one dimension. Here we develop lipid nanolithography methods to produce membranes, under fluid, with widths as low as 6 nm but extending to microns in length. We find reduced lipid mobility, as the width is reduced below 50 nm, suggesting different lipid packing in the vicinity of boundaries. The insertion of a membrane protein, M2, into these systems, allowed characterization of protein diffusion using high-speed AFM to demonstrate the first membrane protein 1D random walk. These quasi-1D lipid bilayers are ideal for testing and understanding fundamental concepts about the roles of dimensionality and size on physical properties of membranes from energy transfer to lipid packing

Nanoporous Silicified Phospholipids and Application to Controlled Glycolic Acid Release

This work demonstrates the synthesis and characterization of novel nanoporous silicified phospholipid bilayers assembled inorganic powders. The materials are obtained by silicification process with silica precursor at the hydrophilic region of phospholipid bilayers. This process involves the co-assembly of a chemically active phospholipids bilayer within the ordered porosity of a silica matrix and holds promise as a novel application for controlled drug release or drug containers with a high level of specificity and throughput. The controlled release application of the synthesized materials was achieved to glycolic acid, and obtained a zero-order release pattern due to the nanoporosity

A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG•protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG•protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices

The antimicrobial effects of the alginate oligomer OligoG CF-5/20 are independent of direct bacterial cell membrane disruption

Concerns about acquisition of antibiotic resistance have led to increasing demand for new antimicrobial therapies. OligoG CF-5/20 is an alginate oligosaccharide previously shown to have antimicrobial and antibiotic potentiating activity. We investigated the structural modification of the bacterial cell wall by OligoG CF-5/20 and its effect on membrane permeability. Binding of OligoG CF-5/20 to the bacterial cell surface was demonstrated in Gram-negative bacteria. Permeability assays revealed that OligoG CF-5/20 had virtually no membrane-perturbing effects. Lipopolysaccharide (LPS) surface charge and aggregation were unaltered in the presence of OligoG CF-5/20. Small angle neutron scattering and circular dichroism spectroscopy showed no substantial change to the structure of LPS in the presence of OligoG CF-5/20, however, isothermal titration calorimetry demonstrated a weak calcium-mediated interaction. Metabolomic analysis confirmed no change in cellular metabolic response to a range of osmolytes when treated with OligoG CF-5/20. This data shows that, although weak interactions occur between LPS and OligoG CF-5/20 in the presence of calcium, the antimicrobial effects of OligoG CF-5/20 are not related to the induction of structural alterations in the LPS or cell permeability. These results suggest a novel mechanism of action that may avoid the common route in acquisition of resistance via LPS structural modification

Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

Single-molecule kinetics of pore assembly by the membrane attack complex

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion