Wrapping anisotropic microgel particles in lipid membranes: Effects of particle shape and membrane rigidity.

Cellular engulfment and uptake of macromolecular assemblies or nanoparticles via endocytosis can be associated to both healthy and disease-related biological processes as well as delivery of drug nanoparticles and potential nanotoxicity of pollutants. Depending on the physical and chemical properties of the system, the adsorbed particles may remain at the membrane surface, become wrapped by the membrane, or translocate across the membrane through an endocytosis-like process. In this paper, we address the question of how the wrapping of colloidal particles by lipid membranes can be controlled by the shape of the particles, the particle-membrane adhesion energy, the membrane phase behavior, and the membrane-bending rigidity. We use a model system composed of soft core-shell microgel particles with spherical and ellipsoidal shapes, together with phospholipid membranes with varying composition. Confocal microscopy data clearly demonstrate how tuning of these basic properties of particles and membranes can be used to direct wrapping and membrane deformation and the organization of the particles at the membrane. The deep-wrapped states are more favorable for ellipsoidal than for spherical microgel particles of similar volume. Theoretical calculations for fixed adhesion strength predict the opposite behavior-wrapping becomes more difficult with increasing aspect ratio. The comparison with the experiments implies that the microgel adhesion strength must increase with increasing particle stretching. Considering the versatility offered by microgels systems to be synthesized with different shapes, functionalizations, and mechanical properties, the present findings further inspire future studies involving nanoparticle-membrane interactions relevant for the design of novel biomaterials and therapeutic applications.

Molecular Biomechanics Controls Protein Mixing and Segregation in Adherent Membranes.

Cells interact with their environment by forming complex structures involving a multitude of proteins within assemblies in the plasma membrane. Despite the omnipresence of these assemblies, a number of questions about the correlations between the organisation of domains and the biomechanical properties of the involved proteins, namely their length, flexibility and affinity, as well as about the coupling to the elastic, fluctuating membrane, remain open. Here we address these issues by developing an effective Kinetic Monte Carlo simulation to model membrane adhesion. We apply this model to a typical experiment in which a cell binds to a functionalized solid supported bilayer and use two ligand-receptor pairs to study these couplings. We find that differences in affinity and length of proteins forming adhesive contacts result in several characteristic features in the calculated phase diagrams. One such feature is mixed states occurring even with proteins with length differences of 10 nm. Another feature are stable nanodomains with segregated proteins appearing on time scales of cell experiments, and for biologically relevant parameters. Furthermore, we show that macroscopic ring-like patterns can spontaneously form as a consequence of emergent protein fluxes. The capacity to form domains is captured by an order parameter that is founded on the virial coefficients for the membrane mediated interactions between bonds, which allow us to collapse all the data. These findings show that taking into account the role of the membrane allows us to recover a number of experimentally observed patterns. This is an important perspective in the context of explicit biological systems, which can now be studied in significant detail.

Hepatitis C virus p7 mediates membrane-to-membrane adhesion.

Viroporin p7 of the hepatitis C virus (HCV) acts as an ion channel for pH equilibration to stabilize HCV particles; most studies of p7 have focused on this role. However, pH equilibration by p7 via its ion channel activity does not fully explain the importance of p7 in HCV particle production. Indeed, several researchers have suggested p7 to have an unidentified ion channel-independent function. Here, we show that p7 has a novel role as a lipid raft adhesion factor, which is independent of its ion channel activity. We found that p7 targets not only the liquid-disordered (Ld) phase, but also the negatively-charged liquid-ordered (Lo) phase that can be represented as a lipid raft. p7 clusters at the phase boundary of the neutral Ld phase and the negatively-charged Lo phase. Interestingly, p7 targeting the Lo phase facilitates membrane-to-membrane adhesion, and this activity is not inhibited by p7 ion channel inhibitors. Our results demonstrated that HCV p7 has dual roles as a viroporin and as a lipid raft adhesion factor. This ion channel-independent function of p7 might be an attractive target for development of anti-HCV compounds.

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

Adhesive Nanoparticles as Local Probes of Membrane Curvature.

Biological and biomimetic membranes display complex shapes with nonuniform curvature. Because the interaction of adhesive nanoparticles with such membranes depends on the local membrane curvature, different segments of the same membrane can differ in their engulfment behavior. For a single vesicle in contact with many nanoparticles, we predict ten distinct engulfment patterns as well as morphological transitions between these patterns, which are directly accessible to experiment.

Lattice-based mesoscale simulations and mean-field theory of cell membrane adhesion.

Adhesion of cell membranes involves multi-scale phenomena, ranging from specific molecular binding at Angstrom scale all the way up to membrane deformations and phase separation at micrometer scale. Consequently, theory and simulations of cell membrane adhesion require multi-scale modeling and suitable approximations that capture the essential physics of these phenomena. Here, we present a mesoscale model for membrane adhesion which we have employed in a series of our recent studies. This model quantifies, in particular, how nanoscale lipid clusters physically affect and respond to the intercellular receptor-ligand binding that mediates membrane adhesion. The goal of this Chapter is to present all details and subtleties of the mean-field theory and Monte Carlo simulations of this mesoscale model, which can be used to further explore physical phenomena related to cell membrane adhesion.

Membrane-Mediated Cooperative Interactions of CD47 and SIRPα.

The specific binding of the ubiquitous 'marker of self' protein CD47 to the SIRPα protein anchored in the macrophage plasma membrane results in the inhibition of the engulfment of 'self' cells by macrophages and thus constitutes a key checkpoint of our innate immune system. Consequently, the CD47-SIRPα protein complex has been recognized as a potential therapeutic target in cancer and inflammation. Here, we introduce a lattice-based mesoscale model for the biomimetic system studied recently in fluorescence microscopy experiments where GFP-tagged CD47 proteins on giant plasma membrane vesicles bind to SIRPα proteins immobilized on a surface. Computer simulations of the lattice-based mesoscale model allow us to study the biomimetic system on multiple length scales, ranging from single nanometers to several micrometers and simultaneously keep track of single CD47-SIRPα binding and unbinding events. Our simulations not only reproduce data from the fluorescence microscopy experiments but also are consistent with results of several other experiments, which validates our numerical approach. In addition, our simulations yield quantitative predictions on the magnitude and range of effective, membrane-mediated attraction between CD47-SIRPα complexes. Such detailed information on CD47-SIRPα interactions cannot be obtained currently from experiments alone. Our simulation results thus extend the present understanding of cooperative effects in CD47-SIRPα interactions and may have an influence on the advancement of new cancer treatments.

Structural bases for the Charcot-Marie-Tooth disease induced by single amino acid substitutions of myelin protein zero.

Myelin protein zero (MPZ or P0) is a transmembrane protein which functions to glue membranes in peripheral myelin. Inter-membrane adhesion is mediated by homophilic interactions between the extracellular domains (ECDs) of MPZ. Single amino acid substitutions in an ECD cause demyelinating neuropathy, Charcot-Marie-Tooth disease (CMT), with unknown mechanisms. In this study, by using a novel assay system "nanomyelin," we revealed that a stacked-rings-like ECD-8-mer is responsible for membrane adhesion. Two inter-ECD interactions, cis and head-to-head, are essential to constituting the 8-mer and to gluing the membranes. This result was reinforced by the observation that the CMT-related N87H substitution at the cis interface abolished membrane-adhesion activity. In contrast, the CMT-related D32G and E68V variants retained membrane-stacking activity, whereas their thermal stability was lower than that of the WT. Reduced thermal stability may lead to impairment of the long-term stability of ECD and the layered membranes of myelin.

Interplay of Trans- and Cis-Interactions of Glycolipids in Membrane Adhesion.

Glycolipids mediate stable membrane adhesion of potential biological relevance. In this article, we investigate the trans- and cis-interactions of glycolipids in molecular dynamics simulations and relate these interactions to the glycolipid-induced average separations of membranes obtained from neutron scattering experiments. We find that the cis-interactions between glycolipids in the same membrane leaflet tend to strengthen the trans-interactions between glycolipids in apposing leaflets. The trans-interactions of the glycolipids in our simulations require local membrane separations that are significantly smaller than the average membrane separations in the neutron scattering experiments, which indicates an important role of membrane shape fluctuations in glycolipid trans-binding. Simulations at the experimentally measured average membrane separations provide a molecular picture of the interplay between glycolipid attraction and steric repulsion of the fluctuating membranes probed in the experiments.

Enhanced Cytotoxic Activity of Mitochondrial Mechanical Effectors in Human Lung Carcinoma H520 Cells: Pharmaceutical Implications for Cancer Therapy.

Cancer cell mitochondria represent an attractive target for oncological treatment as they have unique hallmarks that differ from their healthy counterparts, as the presence of a stronger membrane potential that can be exploited to specifically accumulate cytotoxic cationic molecules. Here, we explore the selective cytotoxic effect of 10-N-nonyl acridine orange (NAO) on human lung carcinoma H520 cells and compare them with healthy human lung primary fibroblasts. NAO is a lipophilic and positively charged molecule that promotes mitochondrial membrane adhesion that eventually leads to apoptosis when incubated at high micromolar concentration. We found an enhanced cytotoxicity of NAO in H520 cancer cells. By means Fluorescence lifetime imaging microscopy (FLIM) we also confirmed the formation of H-dimeric aggregates originating from opposing adjacent membranes that interfere with the mitochondrial membrane structure. Based on our results, we suggest the mitochondrial membrane as a potential target in cancer therapy to mechanically control the cell proliferation of cancer cells.

Neurolysis with Amniotic Membrane Nerve Wrapping for Treatment of Secondary Wartenberg Syndrome: A Preliminary Report.

BACKGROUND: Entrapment of the superficial sensory branch of the radial nerve (SRN) commonly results in debilitating pain of the dorsoradial wrist. Symptom relief following SRN neurolysis is often incomplete or temporary due to recurrent perineural scarring. METHODS: We performed a retrospective review with prospective follow-up of all patients with SRN neuropathy who were treated with neurolysis and nerve wrapping using an amnion-based allograft adhesion barrier over a one-year interval. Measured outcomes included pain rated by Visual Analog Scale (VAS) and Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) functional outcome scores. RESULTS: Three females satisfied inclusion. At mean follow-up of 28.9 months, all three patients exhibited improved pain (mean VAS change -4.7 ± 0.6), function (mean QuickDASH change -40 ± 5), and subjective satisfaction. No adverse events or reactions to the implanted tissue occurred. CONCLUSIONS: SRN entrapment neuropathy was safely and effectively treated with neurolysis and amnion nerve wrapping in this small series. Use of this technique for perineural scar prevention warrants additional study in larger groups of patients and in other upper extremity entrapment neuropathies.

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

Evaluating adhesion reduction efficacy of type I/III collagen membrane and collagen-GAG resorbable matrix in primary flexor tendon repair in a chicken model.

BACKGROUND: Reduction of peritendinous adhesions after injury and repair has been the subject of extensive prior investigation. The application of a circumferential barrier at the repair site may limit the quantity of peritendinous adhesions while preserving the tendon's innate ability to heal. The authors compare the effectiveness of a type I/III collagen membrane and a collagen-glycosaminoglycan (GAG) resorbable matrix in reducing tendon adhesions in an experimental chicken model of a "zone II" tendon laceration and repair. METHODS: In Leghorn chickens, flexor tendons were sharply divided using a scalpel and underwent repair in a standard fashion (54 total repairs). The sites were treated with a type I/III collagen membrane, collagen-GAG resorbable matrix, or saline in a randomized fashion. After 3 weeks, qualitative and semiquantitative histological analysis was performed to evaluate the "extent of peritendinous adhesions" and "nature of tendon healing." The data was evaluated with chi-square analysis and unpaired Student's t test. RESULTS: For both collagen materials, there was a statistically significant improvement in the degree of both extent of peritendinous adhesions and nature of tendon healing relative to the control group. There was no significant difference seen between the two materials. There was one tendon rupture observed in each treatment group. Surgical handling characteristics were subjectively favored for type I/III collagen membrane over the collagen-GAG resorbable matrix. CONCLUSION: The ideal method of reducing clinically significant tendon adhesions after injury remains elusive. Both materials in this study demonstrate promise in reducing tendon adhesions after flexor tendon repair without impeding tendon healing in this model.