Micropatterned supported membranes as tools for quantitative studies of the immunological synapse

In living cells, membrane receptors transduce ligand binding into signals that initiate proliferation, specialization, and secretion of signaling molecules. Spatial organization of such receptors regulates signaling in several key immune cell interactions. In the most extensively studied of these, a T cell recognizes membrane-bound antigen presented by another cell, and forms a complex junction called the "immunological synapse" (IS). The importance of spatial organization at the IS and the quantification of its effect on signaling remain controversial topics. Researchers have successfully investigated the IS using lipid bilayers supported on solid substrates as model antigen-presenting membranes. Recent technical developments have enabled micron- and nanometre-scale patterning of supported lipid bilayers (SLBs) and their application to immune cell studies with provocative results, including spatial mutation of the IS. In this tutorial review, we introduce the IS; discuss SLB techniques, including micropatterning; and discuss various methods used to perturb and quantify the IS.     

Label‐Free Measurements of the Diffusivity of Molecules in Lipid Membranes

An important and characteristic property of a cell membrane is the lateral mobility of protein molecules in the lipid bilayer. This has conventionally been measured by labeling the molecules with fluorescent markers and monitoring their mobility by different fluorescence‐based techniques. However, adding the label to the studied molecule may affect the system, so it is an assumption in almost all experiments that the measured mobility of the biomolecule with its label is the same as that of the unlabeled molecule. However, this assumption is rarely tested due to a lack of suitable methods. In this work, a new technique to perform label‐free diffusivity measurements is developed and used to measure the effect of the label for two common protein–lipid systems: 1) streptavidin (SA) coupled to a supported lipid bilayer (SLB) through biotinylated lipids and 2) the extracellular part of the T‐cell adhesion protein CD2, coupled to an SLB through histidine tags to nickel‐chelating lipids. A measurable (≈12 %) decrease in diffusivity is found for both labeled proteins, even though the molecular mass of the label is almost 100 times smaller than those of the proteins (≈50 kDa). The results illustrate the importance of being able to study different biophysical properties of cell membranes and their mimics without relying on fluorescent labels, especially if fluorescent labeling is difficult or is expected to affect the nature of the intermolecular interactions being studied.     

Structure and dynamics of crystalline protein layers bound to supported lipid bilayers

We study proteins at the surface of bilayer membranes using streptavidin and avidin bound to biotinylated lipids in a supported lipid bilayer (SLB) at the solid-liquid interface. Using X-ray reflectivity and simultaneous fluorescence microscopy, we characterize the structure and fluidity of protein layers with varied relative surface coverages of crystalline and noncrystalline protein. With continuous bleaching, we measure a 10-15% decrease in the fluidity of the SLB after the full protein layer is formed. We propose that this reduction in lipid mobility is due to a small fraction (0.04) of immobilized lipids bound to the protein layer that create obstacles to membrane diffusion. Our X-ray reflectivity data show a 40 A thick layer of protein, and we resolve an 8 A layer separating the protein layer from the bilayer. We suggest that the separation provided by this water layer allows the underlying lipid bilayer to retain its fluidity and stability.     

Single cell 3-D platform to study ligand mobility in cell-cell contact

Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have yet to be combined and applied in a single in vitro platform to address, e.g., cell adhesion in a setting mimicking the three-dimensional environment of neighboring cells in a reductionist way. To study the effect of the lateral mobility of cell adhesive ligands in three dimensions we present and characterize a platform, which enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Soluble E-cadherin extracellular domains coupled through an optimized streptavidin-antibody linkage to lipids in a supported lipid bilayer (SPB) were presented on the microwell walls as either laterally mobile or immobile ligands. The fluidity was controlled through a small change in temperature by choosing phospholipids for the SPB with a lipid phase transition temperature around 30 °C. The platform thus enabled the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligands presented on the same cell membrane mimetic surface. Chinese hamster ovary (CHO) cells engineered to express E-cadherin that were cultured on the platform demonstrated that enhanced cadherin lateral mobility significantly decreased the formation of actin bundles and resulted in more diffuse actin organization, while constraining the cell shape to that of the microwell. This example highlights the potential to use in vitro cell culture platforms to mimic direct cell-cell interaction in a controlled environment that nevertheless captures the dynamic nature of the native cell environment.     

Issues of ligand accessibility and mobility in initial cell attachment

The influence of lateral ligand mobility on cell attachment and receptor clustering has previously been explored for membrane-anchored molecules involved in cell-cell adhesion. In this study, we considered instead a cell binding motif from the extracellular matrix. Even though the lateral mobility of extracellular matrix ligands in membranes does not occur in vivo, we believe it is of interest for cell engineering in vitro. As is the case for cell-cell adhesion molecules, lateral mobility of extracellular matrix ligands could influence cell attachment and, subsequently, cell behavior in cell culture. In this paper, the accessibility and functionality of extracellular matrix ligands presented at surfaces were evaluated for the conditions of laterally mobile versus non-mobile ligands by studying ligand-antibody binding events and early cell attachment as a function of ligand concentration. We compare the initial attachment of rat-derived adult hippocampal progenitor (AHP) cells on laterally mobile, supported phospholipid bilayer membranes to non-mobile, poly-L-lysine-grafted-poly(ethylene glycol) (PLL-g-PEG) polymer films functionalized with a range of laminin-derived IKVAV-containing peptide densities. To this end, synthesis of a new PLL-g-PEG/PEG-IKVAV polymer is described. The characterization of available IKVAV peptides on both surface presentations schemes was explored by studying the mass uptake of anti-IKVAV antibodies using a combination of the surface-sensitive techniques quartz crystal microbalance with dissipation monitoring, surface plasmon resonance spectroscopy, and optical waveguide lightmode spectroscopy. IKVAV-containing peptides presented on laterally mobile, supported phospholipid bilayers and non-mobile PLL-g-PEG were recognized by the anti-IKVAV antibody in a dose-dependent manner, indicating that the amount of available IKVAV ligands increases proportionally with ligand density over the concentrations tested. Attachment of AHP cells to IKVAV-functionalized PLL-g-PEG and supported phospholipid bilayers followed a sigmoidal dependence on peptide concentration, with a critical concentration of approximately 3 pmol/cm2 IKVAV ligands required to support initial AHP cell attachment for both surface modifications. There appeared to be little influence of IKVAV peptide mobility on the initial attachment of AHP cells. Although the spread in the cell attachment data was larger for the PLL-g-PEG surface modification, this was reduced when observed after 24 h, indicating that the cells might need longer times to establish attachment strengths equivalent to those observed on peptide-functionalized supported lipid bilayers. The present study is a step toward understanding the influence of extracellular-matrix-derived ligand mobility on cell fate. Further analysis should focus on the systematic tuning of lateral ligand diffusion, as well as a comparison between the response of non-spreading cells (i.e., AHPs), versus spreading cells (i.e., fibroblasts).     

Lipopeptides incorporated into supported phospholipid monolayers have high specific activity at low incorporation levels

The ability to present cell adhesion molecule (CAM) ligands in controlled amounts on a culture surface would greatly facilitate the control of cell growth and differentiation. Supported lipid monolayer/bilayer systems have previously been developed that allow for presentation of CAM ligands for cell interaction; however, these systems have employed peptide loadings much higher than those used in poly(ethylene glycol) (PEG)-based immobilization systems. We report the development of synthetic methods that can be used for the efficient and versatile creation of many linear and cyclic lipid-linked peptide moieties. Using RGD-based peptides for the alpha5beta1 integrin as a model system, we have demonstrated that these lipopeptides support efficient cell binding and spreading at CAM ligand loadings as low as 0.1 mol %, which is well below that previously reported for supported lipid systems. Engineered lipopeptide-based surfaces offer unique presentation options not possible with other immobilization systems, and the high activity at low loadings we have shown here may be extremely useful in presenting multiple CAM ligands for studying cell growth, differentiation, and signaling.     

Double cushions preserve transmembrane protein mobility in supported bilayer systems

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)-conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 x 10(-8) cm2/s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 x 10(-8) cm2/s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.     

Coupled membrane fluctuations and protein mobility in supported intermembrane junctions

Junctions between lipid membranes make possible cell-free explorations of physical mechanisms that can contribute to protein and lipid organization at a variety of biophysical interfaces. Recent studies of mobile antibodies sandwiched between lipid bilayer membranes have shown that strong intermembrane adhesion and protein mobility alone are sufficient to drive inert proteins into micron-scale patterns of dense and sparse zones. Though the length scale of these patterns was suspected to be related to membrane rigidity, a quantitative understanding has so far been unavailable. We introduce data showing radially structured protein patterns that also demonstrate micron-scale organization. We then provide a simple model that relates the spectrum of membrane fluctuations to the observed protein distributions; in brief, only membrane modes that are slow enough to couple to the protein mobility drive intermembrane protein patterns.     

Stability of DNA-tethered lipid membranes with mobile tethers

We recently introduced two approaches for tethering planar lipid bilayers as membrane patches to either a supported lipid bilayer or DNA-functionalized surface using DNA hybridization (Chung, M.; Lowe, R. D.; Chan, Y-H. M.; Ganesan, P. V.; Boxer, S. G. J. Struct. Biol.2009, 168, 190-9). When mobile DNA tethers are used, the tethered bilayer patches become unstable, while they are stable if the tethers are fixed on the surface. Because the mobile tethers between a patch and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions, we have investigated the sources of instability, focusing on membrane composition. The most stable patches were made with a mixture of saturated lipids and cholesterol, suggesting an important role for membrane stiffness. Other factors such as the effect of tether length, lateral mobility, and patch membrane edge were also investigated. On the basis of these results, a model for the mechanism of patch destruction is developed.     

Incorporation of alpha-hemolysin in different tethered bilayer lipid membrane architectures

Tethered bilayer lipid membranes are stable solid supported model membrane systems. They can be used to investigate the incorporation and function of membrane proteins. In order to study ion translocation mediated via incorporated proteins, insulating membranes are necessary. The architecture of the membrane can have an important effect on both the electrical properties of the lipid bilayer as well as on the possibility to functionally host proteins. Alpha-hemolysin pores have been functionally incorporated into a tethered bilayer lipid membrane coupled to a gold electrode. The protein incorporation has been monitored optically and electrically and the influence of the molecular structure of the anchor lipids on the insertion properties has been investigated.     

Interkingdom signaling: integration, conformation, and orientation of N-acyl-L-homoserine lactones in supported lipid bilayers

N-Acyl-L-homoserine lactones (AHLs) are small cell-to-cell signaling molecules involved in the regulation of population density and local gene expression in microbial communities. Recent evidence shows that contact of this signaling system, usually referred to as quorum sensing, to living eukaryotes results in interactions of AHL with host cells in a process termed "interkingdom signaling". So far details of this process and the binding site of the AHLs remain unknown; both an intracellular and a membrane-bound receptor seem possible, the first of which requires passage through the cell membrane. Here, we used sum-frequency-generation (SFG) spectroscopy to investigate the integration, conformation, orientation, and translocation of deuterated N-acyl-L-homoserine lactones (AHL-d(n)) with varying chain length (8, 12, and 14 C atoms) in lipid bilayers consisting of a 1:1 mixture of POPC:POPG supported on SiO(2) substrates (prepared by vesicle fusion). We found that all AHL-d(n) derivatives are well-ordered within the supported lipid bilayer (SLB) in a preferentially all-trans conformation of the deuterated alkyl chain and integrated into the upper leaflet of the SLB with the methyl terminal groups pointing downward. For the bilayer system described above, no flip-flop of AHL-d(n) from the upper leaflet to the lower one could be observed. Spectral assignments and interpretations were further supported by Fourier transform infrared and Raman spectroscopy.     

Binding of Lipid Vesicles to Protein-Coated Solid Polymer Surfaces: A Model for Cell Adhesion to Artificial Biocompatible Materials

The adhesion of lipid vesicles (liposomes) having controlled chemical and physical structure to polymer supported human serum albumin (HSA) thin layers was investigated by a spectrofluorimetric technique. The vesicle lipid bilayer was labeled with a small amount of an apolar fluorescent probe (diphenylexathriene) and the vesicle suspension was set in contact with the protein film. After washing and drying, the adhering vesicles containing sample was dissolved in chloroform and the homogeneous solution was analyzed by standard spectrofluorimetric techniques. Different parameters of the lipid bilayer, suspending solution, and protein film were varied and their influence on the liposome binding was investigated. Concerning the lipid bilayer, we studied the effect of liposome surface charge by using different mixtures of neutral (dipalmitoyl-phosphatidylcholine) and charged (dipalmitoyl-phosphatidic acid) phospholipids and the fluid or gel nature of the lipid bilayer (switched on and off by temperature variation). Variations of the local environment involve Ca(2+) and H(+) changes in the millimolar range as well as different hydrodynamical flows (in the range 0.1-10 cm/s). Preliminary measurements using different protein layers were also performed. Results show: (a) negligible adhesion without the protein layer, (b) the presence of a maximum for the liposome adhesion vs ion concentration (depending on the liposome composition and kind of the adsorbed ions), (c) a much stronger adhesion for vesicles in the fluid phase (overcoming the entropy-driven desorption increase with temperature), and (d) a dramatic lowering of the adhesion capability under hydrodynamic flow. Points a-c have been interpreted on the basis of a simple mechanoelectrical model. Copyright 2000 Academic Press.     

Supported lipid bilayers at skeletonized surfaces for the study of transmembrane proteins

Skeletonized zirconium phosphonate surfaces are used to support planar lipid bilayers and are shown to be viable substrates for studying transmembrane proteins. The skeletonized surfaces provide space between the bilayer and the solid support to enable protein insertion and avoid denaturation. The skeletonized zirconium octadecylphosphonate surfaces were prepared using Langmuir-Blodgett techniques by mixing octadecanol with octadecylphosphonic acid. After zirconation of the transferred monolayer, rinsing the coating with organic solvent removes the octadecanol, leaving holes in the film ranging from ～50 to ～500 nm in diameter, depending on the octadecanol content. Upon subsequent deposition of a lipid bilayer, either by vesicle fusion or by Langmuir-Blodgett/Langmuir-Schaefer techniques, the lipid assemblies span the holes providing reservoirs beneath the bilayer. The viability of the supported bilayers as model membranes for transmembrane proteins was demonstrated by examining two approaches for incorporating the proteins. The BK channel protein inserts directly into a preformed bilayer on the skeletonized surface, in contrast to a bilayer on a nonskeletonized film, for which the protein associates only weakly. As a second approach, the integrin α(5)β(1) was reconstituted in lipid vesicles, and its inclusion in supported bilayers on the skeletonized surface was achieved by vesicle fusion. The integrin retains its ability to recognize the extracellular matrix protein fibronectin when supported on the skeletonized film, again in contrast to the response if the bilayer is supported on a nonskeletonized film.     

Bilayer lipid membrane formation on a chemically modified S-layer lattice

The present paper describes the generation of a biomimetic model lipid membrane on bacterial surface (S-)layer which covered the entire surface of various sensors. The S-layer lattice allows one to be independent from the underlying solid material and provides a biological surface and anchoring structure for lipid membranes. S-layer proteins were chemically modified via binding of two amine-terminated phospholipids. Subsequently, a bimolecular lipid membrane anchored to the previously generated viscoelastic lipid monolayer was generated by the rapid solvent exchange technique. Characterization of the intermediate (monolayer) and final membrane structures (bilayer) was performed by imaging, surface-sensitive, and electrochemical techniques. This bilayer lipid membrane generated on an S-layer lattice revealed a thickness of ～6 nm and constitutes a stable supported model membrane system with highly isolating properties showing a membrane resistance of 8.5 MΩ × cm(2).     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

Bilayer hydrophobic thickness and integral membrane protein function

The influence of the lipid environment on the function of membrane proteins is increasingly recognized as crucial. Nevertheless, the molecular mechanisms underlying protein-lipid interactions remain obscure. Membrane lipid composition has a regulatory effect on membrane protein activity, and for a number of membrane proteins a clear correlation was found between protein activity and properties of the membrane bilayer such as fluidity. Membrane thickness is an important property of a lipid bilayer. It is expected that hydrophobic thickness match the hydrophobic thickness of transmembrane segments of integral membrane proteins. Any mismatch between the hydrophobic thicknesses of the lipid bilayer and the protein would lead to some modification in either the structure of the protein or the structure of the bilayer, or both. Consequent rearrangements may result in changes in protein activity. Here we review the behavior of several transmembrane proteins whose activity is altered by hydrophobic core thickness.     

二维平面细胞微图案化技术及其生物学应用

可以控制细胞粘附形状、大小的方法统称为细胞图案化技术.这些方法结合微纳米制备、表面化学、电化学和光化学等手段可以动态控制细胞的粘附、迁移、分化及其相互作用,为细胞生物学研究提供了一个新平台.本文介绍了二维平面细胞图案化的各种方法,并对其优缺点进行了总结,评述了细胞图案化技术在细胞生物学基础研究、组织工程以及基于细胞的生物传感器领域的应用.     

F-Actin reassembly during focal adhesion impacts single cell mechanics and nanoscale membrane structure

Focal adhesions play an important role in cell spreading,migration,and overall mechanical integrity.The relationship of cell structural and mechanical properties was investigated in the context of focal adhesion processes.Combined atomic force microscopy(AFM) and laser scanning confocal microscopy(LSCM) was utilized to measure single cell mechanics,in correlation with cellular morphology and membrane structures at a nanometer scale.Characteristic stages of focal adhesion were verified via confocal fluorescent studies,which confirmed three representative F-actin assemblies,actin dot,filaments network,and long and aligned fibrous bundles at cytoskeleton.Force-deformation profiles of living cells were measured at the single cell level,and displayed as a function of height deformation,relative height deformation and relative volume deformation.As focal adhesion progresses,single cell compression profiles indicate that both membrane and cytoskeleton stiffen,while spreading increases especially from focal complex to focal adhesion.Correspondingly,AFM imaging reveals morphological geometries of spherical cap,spreading with polygon boundaries,and elongated or polarized spreading.Membrane features are dominated by protrusions of 41-207 nm tall,short rods with 1-6 μm in length and 10.2-80.0 nm in height,and long fibrous features of 31-246 nm tall,respectively.The protrusion is attributed to local membrane folding,and the rod and fibrous features are consistent with bilayer decorating over the F-actin assemblies.Taken collectively,the reassembly of F-actin during focal adhesion formation is most likely responsible for the changes in cellular mechanics,spreading morphology,and membrane structural features.     

Groove-spanning behavior of lipid membranes on microfabricated silicon substrates

We report on a spreading behavior of phospholipid membranes that arise from a lump of phospholipid (a lipid source) on topographically patterned substrates immersed in an aqueous solution. Microgrooves with well-defined shapes were prepared on Si111 surfaces by anisotropic etching in an alkaline solution. A spreading front that consists of membrane lobes and a single lipid bilayer was observed on the patterned silicon substrates by utilizing fluorescence interference contrast (FLIC) microscopy. FLIC images indicate that the membrane lobes span the microgrooves, while the underlying single lipid bilayer spread along the surface of the microgrooves. In fact, fluorescent polystyrene nanoparticles could be encapsulated in the microgrooves that were completely covered with the membrane lobes. The groove-spanning behavior of membrane lobes is discussed in terms of a balance between adhesion and bending energies of lipid bilayers.     

Phospholipid morphologies on photochemically patterned silane monolayers

We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.