Spherical Supported Lipid Bilayers Research Articles

Assembling the Bacillus subtilis Spore Coat Basement Layer on Spherical Supported Lipid Bilayers.

Micro- and nanoparticles are often designed by mimicking naturally occurring structures. Bacterial spores are dormant cells elaborated by some Gram-positive bacteria during poor growth conditions to protect their genetic material from harsh environmental stresses. In Bacillus subtilis, this protection is, in part, conferred by a proteinaceous shell, the "coat", which is composed of ~80 different proteins. The basement layer of the coat contains two unusual proteins, which we have recently reconstituted around silica beads to generate synthetic spore-like particles termed "SSHELs". Here, we describe the protocol for generating SSHEL particles, and describe the procedure to covalently link molecules of interest (in this case an anti-HER2 affibody) to SSHEL surfaces. SSHELs therefore represent a versatile platform for the display of ligands or antigens for the site-specific delivery of cargo or vaccines.

Enrichment of membrane curvature-sensing proteins from Escherichia coli using spherical supported lipid bilayers

Bacteria display dynamically organized curved membrane structures, especially during cell division. The importance of membrane curvature-sensing (MCS) proteins for the recognition and regulation of biological membrane morphologies has predominately been investigated in eukaryotic cells. Recently, a technique for screening MCS proteins from solutions that contain peripheral membrane proteins was developed, and MCS protein candidates were identified from mammalian cells. The technique uses differently sized spherical supported lipid bilayers (SSLBs), which consist of spherical SiO2 particles covered with a lipid bilayer. To discriminate between proteins possessing the MCS property, SSLBs with the same surface area were used in a comparative sedimentation assay with shotgun proteome analysis. In this study, to prove that the technique could be applied to other samples, MCS proteins in Escherichia coli were investigated. Through a comparative proteomic study, 35 and 47 proteins were enriched as candidate MCS proteins preferentially bound to SSLBs of 100nm and 1000nm, respectively. Among the identified MCS candidate proteins, FtsZ and SecA were further examined for their MCS properties using the two SSLB sizes, which revealed a high binding affinity for the low membrane curvature (large SSLB). This is the first study to explore MCS proteins in prokaryotic cells and the MCS property of the SecA protein. The results demonstrate a method to enrich MCS proteins that could be utilized to better elucidate membrane dynamics and protein function expression on curved membrane structures in prokaryotic cells.

Proteomic Exploration of Membrane Curvature Sensors Using a Series of Spherical Supported Lipid Bilayers.

Membrane curvature-sensing (MCS) proteins recognize and regulate the morphologies of biological membranes. As these proteins lack characteristic sequence motifs in their primary structure, they are not instantly recognizable by genomic databases. Overcoming this technological challenge toward the agile identification of new proteins can promote the elucidation of membrane morphological regulation. Here, for the selective identification of MCS proteins, comparative proteomic analysis was performed using different sizes of the spherical supported lipid bilayer (SSLB), which consists of spherical SiO2 particles covered with a lipid bilayer. Because of the presence of SiO2 core, the curvature of the surrounding membrane is well-controlled and stable even on a micron scale. To prove this concept, known membrane curvature-sensing protein domains, Bin/Amphiphysin/Rvs (BAR) and Epsin N-terminal homology (ENTH), were evaluated by performing a binding assay using SSLBs, and the preferential binding to the highly curved membrane was confirmed. Peripheral membrane proteins obtained from normal human dermal fibroblast (NHDF) and human breast cancer (MDA-MB-231) cells were used in shotgun proteomic analysis, and 786 and 949 proteins were identified from SSLBs as lipid membrane binders, respectively. Statistical quantitative analyses of proteins detected from each SSLB with a different size revealed 118 candidate proteins, including 23 proteins unique to MDA-MB-231 cells, as membrane curvature sensors, including some previously reported curvature sensors. Functional clustering analysis based on the KEGG orthology database revealed that the protein-binding property to specific high or low membrane curvature correlated with their functions. Further investigation of candidate proteins will lead to the identification of new MCS proteins as well as cancer biomarkers.

Nanogap dielectrophoresis combined with buffer exchange for detecting protein binding to trapped bioparticles

We demonstrate a gold nanogap electrode platform that can rapidly create a linear array of biological particles by low-voltage dielectrophoresis (DEP). We further combine microfluidic buffer exchange to introduce protein molecules in high-conductivity solutions while trapping and immobilizing particles. The nanogap between the gold electrodes enables low operating voltages that prevent unwanted Joule heating in high-conductivity buffers. This platform is used to trap bioparticles composed of lipid membranes such as spherical supported lipid bilayers and brain-derived myelin particles, followed by detection of protein binding to specific membrane-bound receptors. We use bioparticles with different sizes, physicochemical properties, and origins to demonstrate a platform that can be used to study a variety of biomolecular interactions. The low-power linear DEP trap combined with microfluidic buffer exchange has potential to enable a portable biosensing platform to rapidly concentrate rare biological particles and perform on-chip binding assays with improved detection limits in physiological buffers.

Investigating the order parameters of saturated lipid molecules under various curvature conditions on spherical supported lipid bilayers.

The conformations and motions of lipid molecules under different membrane curvatures have important implications for transmembrane protein function, binding events, and overall membrane organization. This work reports on the local order parameters of saturated lipid molecules, as measured by (13)C NMR relaxation, under several curvature conditions to probe structural changes as a function of lipid bilayer curvature. Different curvature conditions are created by depositing phosphatidylcholine membranes on spherical beads of various diameters. The findings reveal that the order parameters are not a continuous function of the membrane curvature. While small (30 nm) and large (110 nm) diameter bilayers exhibit similar order parameters, bilayers with curvatures of 60-80 nm diameter show a consistently increased order parameter along the entire lipid molecule, indicating a higher packing density and lateral tension. Order parameters for curvatures between 60 and 80 nm also show molecular evidence for interdigitation.

Formation of biomembrane microarrays with a squeegee-based assembly method.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

High-density arrays of submicron spherical supported lipid bilayers.

Lipid bilayer membranes found in nature are heterogeneous mixtures of lipids and proteins. Model systems, such as supported lipid bilayers (SLBs), are often employed to simplify experimental systems while mimicking the properties of natural lipid bilayers. Here, we demonstrate a new method to form SLB arrays by first forming spherical supported lipid bilayers (SSLBs) on submicrometer-diameter SiO(2) beads. The SSLBs are then arrayed into microwells using a simple physical assembly method that requires no chemical modification of the substrate nor modification of the lipid membrane with recognition moieties. The resulting arrays have submicrometer SSLBs with 3 μm periodicity where >75% of the microwells are occupied by an individual SSLB. Because the arrays have high density, fluorescence from >1000 discrete SSLBs can be acquired with a single image capture. We show that 2-component random arrays can be formed, and we also use the arrays to determine the equilibrium dissociation constant for cholera toxin binding to ganglioside GM1. SSLB arrays are robust and are stable for at least one week in buffer.

Kinetics of Myosin-I-Membrane Detachment Under Load

Myo1c is a single-headed motor that links cell membranes to the underlying actin cytoskeleton. Actin binding occurs via the motor domain, while the tail domain interacts with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) through a putative pleckstrin homology (PH) domain. In this study, we measured the strength and lifetime of the myo1c attachment to spherical supported lipid bilayers (SSL) composed of 1 μm diameter silica or polystyrene beads coated with various mole fractions of PI(4,5)P2, dioleoyl-phosphatidylcholine (DOPC), and dioleoyl-phosphatidylserine (DOPS). The SSLs trapped by a laser beam were brought into contact with immobilized spherical (2 μm) silica pedestals sparsely labeled with the myo1c tail domain. Upon repeated contact and retraction cycles, binding events and subsequent rupture forces were measured. The most probable rupture forces (< 10 pN) from membranes containing 2% PI(4,5)P2 were found to be largely independent of the loading rate (pN/s). Nevertheless, the frequency of single myo1c molecules interacting with membranes containing 2% PI(4,5)P2 - 98% DOPC was considerably higher than with membranes that contained 20 - 80% DOPS. Lifetime measurements of myo1c attachment to 2% PI(4,5)P2 - 98% DOPC membranes under constant pulling forces (1 - 3 pN) were fitted to Bell's equation and the extrapolated duration of the bond at zero force was found to be ∼ 25 ms. Although phosphoinositide binding is crucial for the proper cellular targeting of myo1c, our results suggest that it is unlikely that this connection is a suitable anchor for force generation.