The outer surface of cells presents a complex, nanostructured, yet fluid environment that controls the movement of signaling proteins. The lateral movement of many membrane biomolecules, including transmembrane or tethered proteins as well as lipids themselves, can be interpreted as being free and isotropic within compartments of the cell membrane measuring tens to hundreds of nanometers in scale1-6
. These compartments are delineated by semi-permeable barriers that arise from interactions between the plasma membrane, underlying cytoskeleton, and associated proteins6-8
. Fluctuations in these structures allow biomolecules to occasionally cross between compartments, allowing long-range, but comparatively slow, transport over the cell surface. More formally, transport along the membrane is an anomalous, non-Brownian process that can be characterized by two diffusion coefficients, one that describes short-range motion within an individual compartment and a second, smaller, effective
diffusion coefficient that is associated with long-range motion over many barriers. The extent to which these values differ is dependent on the spacing and properties of the barriers as well as the diffusing molecule. Emerging models suggest significant impacts of this behavior on cell signaling2, 9, 10
, but experimental systems for testing these hypotheses are not widely available. In this report, we capture this anomalous diffusion by nanopatterning supported lipid bilayers with barriers to lipid diffusion using a geometry that captures the semipermeable nature of those posed to be present in living cells. As is posed by models of these interactions, we aim to gain control over long-range diffusion, while maintaining local, isotropic diffusion associated with a membrane in the absence of such barriers. We demonstrate that these nanopatterned barriers give rise to different short- and long-range diffusion coefficients of lipids and membrane-associated proteins, and provide a quantitative model of this diffusion that suggests specific aspects of membrane structure at the sub-micrometer level.
The basic substrate supported lipid bilayer system consists of a phospholipid membrane in close association with an appropriate surface11, 12
. A thin, subnanometer-thick layer of water separates the bilayer and substrate13-15
, imparting lateral mobility to the membrane components. For reasons that are not well understood, bilayer formation is supported by a limited set of materials, notably silicon oxide-based substrates (glass, quartz, and silicon wafers, polydimethylsiloxane) and mica, which promote fusion of lipid vesicles into an extended, planar bilayer structure. Other materials such as metals, photoresists, or surface-adsorbed proteins are inert to vesicle fusion and provide a powerful method for directing the layout of supported lipid bilayers when micropatterned onto bilayer-compatible substrates16-18
. Here, we extend this micropatterning approach to nanoscale dimensions in order to capture the scales of membrane compartments observed in living cells.
Glass substrates were patterned with linear arrays of barriers () that are designed to restrict bilayer formation to the intervening regions of the surface. Barriers were spaced at either 125- or 250-nm center-to-center intervals (“spacing” in ), dimensions that are associated with the size of membrane compartments on living cells. For this study, the barriers are specified to be 50 nm in width, the narrowest that could reliably and controllably be fabricated using our processing equipment (as described below). To capture the semi-permeable properties of their cell-based counterparts, gaps of 30, 40, and 50 nm length were interspersed throughout the barriers. Diffusing membrane molecules thus periodically encounter a break in the barrier, capturing the basic mechanism of the anchored-protein “picket fence” model, in which transmembrane proteins attached to cytoskeleton disrupt both leaflets of the membrane. These gaps were spaced such that the periodicity of each pattern is 500 nm (“length”). Gaps were staggered between lines, as opposed to being aligned at the same location on each barrier. An important aspect of this barrier layout is that while compartments observed on cell membranes are essentially closed-perimeter patches, the barriers used here break up the surface into parallel stripes, a geometry chosen to simplify the measurement of short- and long-range diffusion coefficients. Specifically, molecular motion parallel to the barriers is unhindered; in the absence of any impact of the barriers on local membrane mobility, measurement of long-range diffusion coefficients in this direction will be numerically equivalent to their short-range counterparts. The impact of these barriers on short-range and long-range diffusive behavior can thus be estimated by simultaneously measuring long-range diffusion along and across the barriers. Importantly, these long-range diffusion coefficients can be measured using standard fluorescence recovery after photobleaching (FRAP) techniques, offering simplified analysis and longer timescale observations than contemporary single particle tracking approaches, which require specialized, high temporal- and spatial-resolution equipment to reveal these dynamics.
Figure 1 Nanopatterning of supported lipid bilayers. (A) A metal lift-off method was used to define parallel barriers on glass. These barriers are designed to disrupt supported lipid bilayers that will subsequently formed on these substrates. (B) Electron micrograph (more ...)
An electron-beam based lift-off process was used to create these barriers. Coverslips were spin-coated with a lower resist layer of 25kDa PMMA, an upper resist layer of 950kDa PMMA (together ~ 200 nm), and finally a charge dispersion layer of Aquasave (Mitsubishi Rayon Co., New York, NY). PMMA was patterned using an FEI XL-30 Scanning Electron Microscope fitted with a Nabity Pattern Generator and developed using standard methods. A 4 nm-thick layer (unless otherwise specified) of either Cr or Ti was then deposited using an electron beam evaporation system, and then the PMMA and overlying metal were finally removed in acetone. An electron micrograph of a typical surface is shown in , demonstrating the successful patterning of these substrates. Individual patterns covered an 80 μm × 80 μm region, each containing a specific combination of spacing and gap. Small unilamellar vesicles (SUVs) of egg phosphatidylcholine (egg PC, Avanti Polar Lipids) supplemented with 1 molar % Texas Red 1,2-dihexadecanoyl-sn
-glycero-3-phosphoethanolamine (TR-DHPE, Molecular Probes), at a concentration of 5 mg/ml in water, were prepared by extrusion through 50-nm pore polycarbonate membranes (Avanti Polar Lipids) using standard techniques19
. Supported lipid bilayers were formed by exposing the nanopatterned substrates to SUVs for 5 minutes, followed by extensive rinsing with PBS and then deionized water. Imaging of substrates was carried out in phosphate buffered saline (pH 7.4).