Planar Supported Lipid Bilayers Research Articles

Sum-frequency vibrational spectroscopy, a tutorial: Applications for the study of lipid membrane structure and dynamics.

Planar supported lipid bilayers (PSLBs) are an ideal model for the study of lipid membrane structures and dynamics when using sum-frequency vibrational spectroscopy (SFVS). In this paper, we describe the construction of asymmetric PSLBs and the basic SFVS theory needed to understand and make measurements on these membranes. Several examples are presented, including the determination of phospholipid orientation and measuring phospholipid transmembrane translocation (flip-flop).

Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge. These changes hint at additional molecular targets for interactions between virus and cell, such as the cell membrane or proteoglycans on the cell surface. Herein, bottom-up assembled synthetic SARS-CoV-2 miniviruses (MiniVs), with a lipid composition similar to that of infectious particles, are implemented to study and compare the binding properties of Omicron and Alpha variants. Toward this end, a systematic functional screening is performed to study the binding ability of Omicron and Alpha S proteins to ACE2-functionalized and nonfunctionalized planar supported lipid bilayers. Moreover, giant unilamellar vesicles are used as a cell membrane model to perform competitive interaction assays of the two variants. Finally, two cell lines with and without presentation of the ACE2 receptor are used to confirm the binding properties of the Omicron and Alpha MiniVs to the cellular membrane. Altogether, the results reveal a significantly higher affinity of Omicron S toward both the lipid membrane and ACE2 receptor. The research presented here highlights the advantages of creating and using bottom-up assembled SARS-CoV-2 viruses to understand the impact of changes in the affinity of S for ACE2 in infection studies.

Gold-Supported Lipid Membranes Formed by Redox-Triggered Vesicle Fusion on Binary Self-Assembled Monolayers: Ion-Pairing Association and Surface Hydrophilicity.

The assembly of biomimetic, planar supported lipid bilayers (SLBs) by the popular vesicle fusion method, which relies on the spontaneous adsorption and rupture of small unilamellar vesicles from aqueous solution on a solid surface, typically works with a limited range of support materials and lipid systems. We previously reported a conceptual advance in the formation of SLBs from vesicles in the gel or fluid phase using the interfacial ion-pairing association of charged phospholipid headgroups with electrochemically generated cationic ferroceniums bound to a self-assembled monolayer (SAM) chemisorbed to gold. This redox-driven approach lays down a single bilayer membrane on the SAM-modified gold surface at room temperature within minutes and is compatible with both anionic and zwitterionic phospholipids. The present work explores the effects of the surface ferrocene concentration and hydrophobicity/hydrophilicity on the formation of continuous SLBs of dialkyl phosphatidylserine, dialkyl phosphatidylglycerol, and dialkyl phosphatidylcholine using binary SAMs of ferrocenylundecanethiolate (FcC11S) and dodecanethiolate (CH3C11S) or hydroxylundecanethiolate (HOC11S) comprising different surface mole fractions of ferrocene (χFcsurf). An increase in the surface hydrophilicity and surface free energy of the FcC11S/HOC11S SAM mitigates the decrease in the attractive ion-pairing interactions resulting from a reduced χFcsurf. SLBs of ≳80% area coverage form on the FcC11S/HOC11S SAM for all the phospholipid types down to χFcsurf of at least 0.2, composition yielding a water contact angle (θW) of 44 ± 4°. By contrast, a greater number of ion-pairing interactions is required on the hydrophobic FcC11S/CH3C11S surface to drive the vesicle fusion process; bilayers or bilayer patches form at χFcsurf ≳ 0.6 (θW = 97 ± 3°). These findings will aid in tailoring the surface chemistry of redox-active modified surfaces to widen the conditions that yield supported lipid membranes.

Bioinspired Nanoplatforms Based on Graphene Oxide and Neurotrophin-Mimicking Peptides.

Neurotrophins (NTs), which are crucial for the functioning of the nervous system, are also known to regulate vascularization. Graphene-based materials may drive neural growth and differentiation, and, thus, have great potential in regenerative medicine. In this work, we scrutinized the nano-biointerface between the cell membrane and hybrids made of neurotrophin-mimicking peptides and graphene oxide (GO) assemblies (pep-GO), to exploit their potential in theranostics (i.e., therapy and imaging/diagnostics) for targeting neurodegenerative diseases (ND) as well as angiogenesis. The pep-GO systems were assembled via spontaneous physisorption onto GO nanosheets of the peptide sequences BDNF(1-12), NT3(1-13), and NGF(1-14), mimicking the brain-derived neurotrophic factor (BDNF), the neurotrophin 3 (NT3), and the nerve growth factor (NGF), respectively. The interaction of pep-GO nanoplatforms at the biointerface with artificial cell membranes was scrutinized both in 3D and 2D by utilizing model phospholipids self-assembled as small unilamellar vesicles (SUVs) or planar-supported lipid bilayers (SLBs), respectively. The experimental studies were paralleled via molecular dynamics (MD) computational analyses. Proof-of-work in vitro cellular experiments with undifferentiated neuroblastoma (SH-SY5Y), neuron-like, differentiated neuroblastoma (dSH-SY5Y), and human umbilical vein endothelial cells (HUVECs) were carried out to shed light on the capability of the pep-GO nanoplatforms to stimulate the neurite outgrowth as well as tubulogenesis and cell migration.

Propranolol induces large-scale remodeling of lipid bilayers: tubules, patches, and holes.

Herein, we report fluorescence microscopy analysis of the interaction between propranolol (PPN), a beta-adrenergic blocking agent, and planar supported lipid bilayers (SLBs), as model membranes. The results indicate that PPN can remarkably promote largescale remodeling in SLBs with various lipid compositions. It was found that PPN insertion induces the formation of long microtubules that can retract into hemispherical caps on the surface of the bilayer. These transformations are dynamic, partially reversible, and dependent upon the drug concentration. Quantitative analysis revealed a three-step model for PPN-lipid bilayer interaction, with the first step involving interfacial electrostatic adsorption, the second step centered on hydrophobic insertion, and the third step associated with membrane disruption and hole formation. By introducing cholesterol, phosphoethanolamine, phosphatidylglycerol, and phosphatidylserine lipids into the phosphocholine SLBs, it was illustrated that both the chemistry of the lipid headgroups and the packing of lipid acyl chains can substantially affect the particular steps in the interactions between PPN and lipid bilayers. Our findings may help to elucidate the possible mechanisms of PPN interaction with lipid membranes, the toxic behavior and overdosage scenarios of beta-blockers, and provide valuable information for drug development and modification.

Inhibitory Effect of Lanthanides on Native Lipid Flip-Flop

The influence of ytterbium ions (Yb3+), a commonly used paramagnetic NMR chemical shift reagent, on the physical properties and flip-flop kinetics of dipalmitoylphosphatidylcholine (DPPC) planar supported lipid bilayers (PSLBs) was investigated. Langmuir isotherm studies revealed that Yb3+ interacts strongly with the phosphate headgroup of DPPC, evidenced by the increases in shear and compression moduli. Using sum-frequency vibrational spectroscopy, changes in the acyl chain ordering and phase transition temperature were also observed, consistent with Yb3+ interacting with the phosphate headgroup of DPPC. The changes in the physical properties of the membrane were also observed to be concentration dependent, with more pronounced modification observed at low (50 μM) Yb3+ concentrations compared to 6.5 mM Tb3+, suggesting a cross-linking mechanism between adjacent DPPC lipids. Additionally, the changes in membrane packing and phase transition temperatures in the presence of Tris buffer suggested that a putative Yb(Tris)3+ complex forms that coordinates to the PC headgroup. The kinetics of DPPC flip-flop in the gel and liquid crystalline (lc) phases were substantially inhibited in the presence of Yb3+, regardless of the Yb3+ concentration. Analysis of the flip-flop kinetics under the framework of transition state theory revealed that the free energy barrier to flip-flop in both the gel and lc phases was substantial increased over a pure DPPC membrane. In the gel phase, the trend in the free energy barrier appeared to follow the trend in the shear moduli, suggesting that the Yb3+-DPPC headgroup interaction was driving the increase in the activation free energy barrier. In the lc phase, activation free energies of DPPC flip-flop in the presence of 50 μM or 6.5 mM Yb3+ were found to mirror the free energies of TEMPO-DPPC flip-flop, leading to the conclusion that the strong interaction between Yb3+ and the PC headgroup was essentially manifested as a headgroup charge modification. These studies illustrate that the presence of the lanthanide Yb3+ results in significant modification to the lipid membrane physical properties and, more importantly, results in a pronounced inhibition of native lipid flip-flop.

Snf7 spirals sense and alter membrane curvature

Endosomal Sorting Complex Required for Transport III (ESCRT-III) is a conserved protein system involved in many cellular processes resulting in membrane deformation and scission, topologically away from the cytoplasm. However, little is known about the transition of the planar membrane-associated protein assembly into a 3D structure. High-speed atomic force microscopy (HS-AFM) provided insights into assembly, structural dynamics and turnover of Snf7, the major ESCRT-III component, on planar supported lipid bilayers. Here, we develop HS-AFM experiments that remove the constraints of membrane planarity, crowdedness, and support rigidity. On non-planar membranes, Snf7 monomers are curvature insensitive, but Snf7-spirals selectively adapt their conformation to membrane geometry. In a non-crowded system, Snf7-spirals reach a critical radius, and remodel to minimize internal stress. On non-rigid supports, Snf7-spirals compact and buckle, deforming the underlying bilayer. These experiments provide direct evidence that Snf7 is sufficient to mediate topological transitions, in agreement with the loaded spiral spring model.

Nanomechanical Properties of Artificial Lipid Bilayers Composed of Fluid and Polymerizable Lipids.

Polymerization enhances the stability of a planar supported lipid bilayer (PSLB) but it also changes its chemical and mechanical properties, attenuates lipid diffusion, and may affect the activity of integral membrane proteins. Mixed bilayers composed of fluid lipids and poly(lipids) may provide an appropriate combination of polymeric stability coupled with the fluidity and elasticity needed to maintain the bioactivity of reconstituted receptors. Previously (Langmuir, 2019, 35, 12483-12491) we showed that binary mixtures of the polymerizable lipid bis-SorbPC and the fluid lipid DPhPC form phase-segregated PSLBs composed of nanoscale fluid and poly(lipid) domains. Here we used atomic force microscopy (AFM) to compare the nanoscale mechanical properties of these binary PSLBs with single-component PSLBs. The elastic (Young's) modulus, area compressibility modulus, and bending modulus of bis-SorbPC PSLBs increased upon polymerization. Before polymerization, breakthrough events at forces below 5 nN were observed, but after polymerization, the AFM tip could not penetrate the PSLB up to an applied force of 20 nN. These results are attributed to the polymeric network in poly(bis-SorbPC), which increases the bilayer stiffness and resists compression and bending. In binary DPhPC/poly(bis-SorbPC) PSLBs, the DPhPC domains are less stiff, more compressible, and are less resistant to rupture and bending compared to pure DPhPC bilayers. These differences are attributed to bis-SorbPC monomers and oligomers present in DPhPC domains that disrupt the packing of DPhPC molecules. In contrast, the poly(bis-SorbPC) domains are stiffer and less compressible relative to pure PSLBs; this difference is attributed to DPhPC filling the nm-scale pores in the polymerized domains that are created during bis-SorbPC polymerization. Thus, incomplete phase segregation increases the stability of poly(bis-SorbPC) but has the opposite, detrimental effect for DPhPC. Overall, these results provide guidance for the design of partially polymerized bilayers for technological uses.

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane.

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D- decreasing from ≃ to ≃) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

Temporal Analysis of T-Cell Receptor-Imposed Forces Via Quantitative Single Molecule Fret Measurements

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and dynamical behavior are still poorly defined. We here report a FRET-based sensor equipped with a TCR-reactive single chain antibody fragment, which was tethered to planar supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5pN and moderate force loading rates on the TCR of 1.5pN per second. When facing fluid SLBs instead, T-cells still exerted tensile forces yet with threefold reduced magnitude and only prior to but not upon activation. Our findings not only provide first truly molecular information on TCR-imposed forces within the immunological synapse, they also recalibrate their significance in antigen recognition.

Effect of tetracaine on dynamic reorganization of lipid membranes

To understand the intrinsic influence of a drug on lipid membranes is of critical importance in pharmacological science. Herein, we report fluorescence microscopy analysis of the interaction between the local anesthetic tetracaine (TTC) and planar supported lipid bilayers (SLBs), as model membranes. Our results show that TTC increases lipid chain mobility, destabilizes the SLBs and remarkably induces membrane disruption and solubilization. Upon TTC binding, a local curvature change in the bilayer was observed, which led to the subsequent formation of up to 20-μm-long flexible lipid tubules as well as the formation of micron-size holes. Quantitative analysis revealed that membrane solubilization process can be divided into two distinct different stages as a function of TTC concentration. In the first stage (<800 μM), the bilayer disruption profiles fit well to a Langmuir isotherm, while in the second stage (800 μM–25 mM), TTC solubilizes the membrane in a detergent-like manner. Notably, the onset of membrane solubilization occurred below the critical micelle concentration (cmc) of TTC, indicating a local accumulation of the drug in the membrane. Additionally, cholesterol increases the insertion of TTC into the membrane and thus promotes the solubilization effect of TTC on lipid bilayers. These findings may help to elucidate the possible mechanisms of TTC interaction with lipid membranes, the dose dependent toxicity attributed to local anesthetics, as well as provide valuable information for drug development and modification.

Getting CD19 Into Shape: Expression of Natively Folded "Difficult-to- Express" CD19 for Staining and Stimulation of CAR-T Cells.

The transmembrane protein CD19 is exclusively expressed on normal and malignant B cells and therefore constitutes the target of approved CAR-T cell-based cancer immunotherapies. Current efforts to assess CAR-T cell functionality in a quantitative fashion both in vitro and in vivo are hampered by the limited availability of the properly folded recombinant extracellular domain of CD19 (CD19-ECD) considered as “difficult-to-express” (DTE) protein. Here, we successfully expressed a novel fusion construct consisting of the full-length extracellular domain of CD19 and domain 2 of human serum albumin (CD19-AD2), which was integrated into the Rosa26 bacterial artificial chromosome vector backbone for generation of a recombinant CHO-K1 production cell line. Product titers could be further boosted using valproic acid as a chemical chaperone. Purified monomeric CD19-AD2 proved stable as shown by non-reduced SDS-PAGE and SEC-MALS measurements. Moreover, flow cytometric analysis revealed specific binding of CD19-AD2 to CD19-CAR-T cells. Finally, we demonstrate biological activity of our CD19-AD2 fusion construct as we succeeded in stimulating CD19-CAR-T cells effectively with the use of CD19-AD2-decorated planar supported lipid bilayers.

ESCRT-III Assembles Simultaneously and Without Preference on Supported Lipid Bilayers of Varying Curvatures

Endosomal Sorting Complex Required for Transport III proteins (ESCRT-III) are downstream effective components of an evolutionarily conserved system of proteins that is involved in crucial processes in eukaryotic cells resulting in membrane deformation and scission. Snf7, the major component of this system, must be able to polymerize efficiently on lipid bilayers displaying a variety of curvatures to account for differences in function and intracellular localization. High-speed atomic force microscopy (HS-AFM), a method distinguished by its high spatial and temporal resolution in ambient conditions, has provided insights into the assembly, structural arrangement and turnover of Snf7 assemblies on planar supported lipid bilayers. We now established novel non-planar HS-AFM supports, and demonstrate that Snf7 assembles concurrently and without preference on supported lipid bilayer patches of different curvatures, saturating the sample surface in minutes. By analyzing the dimensions and topographical properties of the spirals, we find that the non-planar substrate introduces constraints into the size distribution of Snf7 assemblies, and that assembly topology is an emergent property of the ensemble (the spiral). Furthermore, we show that there is no significant difference in Snf7 assembly stability in relation to surface curvature. This implies that Snf7 assembly into three-dimensional spirals on curved lipid bilayer patches does not constitute an unfavorable conformation, and that Snf7 assemblies are able to form rapidly on lipid bilayers regardless of their shape - a requirement that is crucial for efficient Snf7 recruitment to budding sites in various physiological conditions and functions.

Defects in Self-Assembled Monolayers on Nanoparticles Prompt Phospholipid Extraction and Bilayer-Curvature-Dependent Deformations

Metal nanoparticles (NPs) functionalized with self-assembled monolayers (SAMs) of long alkanethiol ligands are subject to defects in the SAM structure due to the interplay between alkyl chain packing and the free volume available in space per ligand. We find via dissipative particle dynamics (DPD) simulations that hydrophobic contact between protruding acyl chains of phospholipids from lipid vesicles and exposed alkyl chains in SAM defects prompts NP insertion and that defects become sites for phospholipid extraction. Experiments show that AuNPs coated with cationic (11-mercaptoundecyl)trimethylammonium bromide, analogous to the models used for the simulations, attach to and acquire lipids from planar supported lipid bilayers, while AuNPs coated with anionic 11-mercaptoundecanoic acid do not. Phospholipid extraction and the structure of the ligands inserted in bilayers collectively contribute to bilayer thinning at the site of NP insertion and bilayer-curvature-dependent deformability, revealing how these typical engineered SAM-coated NPs interface with lipid-bilayer systems with potential biological consequences.

Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells.

Planar supported lipid bilayers (PSLB) presenting T cell receptor (TCR) ligands and ICAM-1 induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but incorporation of other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. We demonstrate selective enrichment of CD40L and ICOS in SE in response to addition of CD40 and ICOSL, respectively, to SLB presenting TCR ligands and ICAM-1. SE are enriched in tetraspanins, BST-2, TCR signaling and ESCRT proteins. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2. CD40L+ SE retain the capacity to induce dendritic cell maturation and cytokine production.

Nanodomain Formation in Planar Supported Lipid Bilayers Composed of Fluid and Polymerized Dienoyl Lipids.

Polymerization of synthetic phospholipid monomers has been widely used to enhance the stability of lipid membranes in applications such as membrane-based biosensing, where the inherent instability of fluid-phase lipid bilayers can be problematic. However, lipid polymerization typically decreases membrane fluidity, which may be required to maintain the activity of reconstituted integral proteins and peptides. Prior work has shown that a bilayer composed of binary mixtures of poly(lipid) and fluid lipid exhibits enhanced stability and supports the function of incorporated biomolecules. This work examines the structural basis of these findings using planar supported lipid bilayers (PSLBs) composed of binary mixtures of a polymerizable lipid, 1,2-bis[10-(2',4'-hexadienoloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC), and a nonpolymerizable lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). Fluorescence recovery after photobleaching (FRAP) measurements showed that long-range lateral diffusion was minimally affected when the poly(lipid) mole ratio was ≤0.7. Atomic force microscopy, used to examine phase segregation in these PSLBs, showed that DPhPC forms a continuous lipid matrix that is 0.2-0.4 nm thicker than the island-like poly(bis-SorbPC) domains, with lateral dimensions of ≤200 nm. The nanoscale phase segregation allows for long-range lateral diffusion of lipid probes in the DPhPC matrix. The combination of fluidity and stability in these materials should make them useful in membrane-based biosensing applications.

Determination of the Rate-Limiting Step in Fatty Acid Transport.

The generally accepted model of free fatty acid (FA) transport through cellular membranes occurs in three steps, adsorption of the FA onto the membrane, translocation across the membrane ("flip-flop"), and subsequent desorption of the FA into the cytosol. There still exists some dispute as to the identity of the rate-limiting step of FA transport. In the present study, sum-frequency vibrational spectroscopy (SFVS) was used to directly measure the rate of stearic acid (SA) flip-flop in planar supported lipid bilayers (PSLBs) comprised of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The impact of SA on the physical properties of binary mixtures of SA and DSPC was investigated via Π-A isotherms from which the excess free energies of mixing and compression moduli were calculated. The manner in which these physical changes influenced the rates of SA and DSPC flip-flop was subsequently examined using SFVS. The rates of SA and DSPC flip-flop revealed that SA flip-flops independently of DSPC and on much faster time scales than its phospholipid counterpart. SFVS was also used to probe the rate of protein-unassisted SA desorption from hybrid supported lipid bilayers (HSLBs), allowing for the first decoupled measurement of the rates of desorption and flip-flop. These results provide strong evidence for desorption being the rate-limiting step in FA transport through the membrane in the absence of proteins.

Single-Molecule Analyses Reveal Rhomboid Proteins Are Strict and Functional Monomers in the Membrane

Intramembrane proteases hydrolyze peptide bonds within the membrane as a regulatory paradigm that is conserved across all forms of cellular life. Many of these enzymes are thought to be oligomeric, and that their resulting quaternary interactions form the basis of their regulation. However, technical limitations have precluded directly determining the oligomeric state of intramembrane proteases in any membrane. Using single-molecule photobleaching, we determined the quaternary structure of 10 different rhomboid proteins (the largest superfamily of intramembrane proteases) and six unrelated control proteins in parallel detergent micelle, planar supported lipid bilayer, and whole-cell systems. Bacterial, parasitic, insect, and human rhomboid proteases and inactive rhomboid pseudoproteases all proved to be monomeric in all membrane conditions but dimeric in detergent micelles. These analyses establish that rhomboid proteins are, as a strict family rule, structurally and functionally monomeric by nature and that rhomboid dimers are unphysiological.

Confocal-Raman Microscopy Characterization of Supported Phospholipid Bilayers Deposited on the Interior Surfaces of Chromatographic Silica.

A common approach to exploring the structure and dynamics of biological membranes is through the deposition of model lipid bilayers on planar supports by Langmuir-trough or vesicle-fusion methods. Planar-supported lipid bilayers have been shown to exhibit structure and properties similar to those of lipid-vesicle membranes and are suitable for biosensing applications. Investigations using these planar-membrane models are limited to high-sensitivity methods capable of detecting a small population of molecules at the interface between a planar support and aqueous solution. In this work, we present evidence that supported-lipid bilayers can be deposited by vesicle fusion onto the interior surfaces throughout the wide-pore network of chromatographic silica particles. The thickness of a 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) film and headgroup spacing are consistent with a single bilayer of DMPC deposited onto the pore surfaces. The high specific surface area of these materials generates phospholipid concentrations easily detected by confocal-Raman microscopy within an individual particle, which allows the structure of these supported bilayers to be investigated. Raman spectra of porous-silica-supported DMPC bilayers are equivalent to spectra of DMPC vesicle membranes, both above and below their melting phase transitions, suggesting comparable phospholipid organization and bilayer structure. These porous-silica-supported model membranes could share benefits that planar-supported lipid bilayers bring to biosensing applications, but in a material that overcomes the limited surface area of a planar support. To test this concept, the potential of these porous-silica-supported lipid bilayers as high-surface-area platforms for label-free Raman-scattering-based protein biosensing is demonstrated with detection of concanavalin A selectively binding to a lipid-immobilized mannose target.

Molecular AFM imaging of Hsp70-1A association with dipalmitoyl phosphatidylserine reveals membrane blebbing in the presence of cholesterol.

Hsp70-1A-the major stress-inducible member of the HSP70 chaperone family-is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5μg/ml (7nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.