Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers

Kung, Li A.; Kam, Lance C.; Hovis, Jennifer S.; Boxer, Steven G.

doi:10.1021/la000653t

Cited by 192 publications

(185 citation statements)

References 25 publications

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“…23,24,27 For all stamping steps, fibronectin was labeled with monoamine-reactive Cy3.5 (Amersham) following the manufacturer's instructions and then mixed 1:3 with unlabeled protein for a total concentration of 100 μg/mL.…”

Section: Substrate Preparationmentioning

confidence: 99%

E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

et al. 2005

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Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 ± 0.3 μm 2 /s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

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Section: Substrate Preparationmentioning

confidence: 99%

E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

et al. 2005

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“…Fibronectin (Sigma) and BSA (Life Technologies, Rockville, MD) were labeled with cascade blue acetyl azide and Texas red X succinimidyl ester (Molecular Probes), respectively, in accordance with the manufacturer's instructions. Supported lipid bilayers on glass and on lithium niobate͞SiO 2 hybrid substrates were formed by vesicle fusion and patterned by microcontact printing protein grids, as described (10,22). The diffusion coefficient of fluorescent probes was determined as described (22,23).…”

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confidence: 99%

“…Supported lipid bilayers on glass and on lithium niobate͞SiO 2 hybrid substrates were formed by vesicle fusion and patterned by microcontact printing protein grids, as described (10,22). The diffusion coefficient of fluorescent probes was determined as described (22,23). Extraction and quantitative fluorescence of egg PC bilayers containing 1% Texas red was done as described (24).…”

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confidence: 99%

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Ajo‐Franklin

Kam

Boxer

2001

Proc. Natl. Acad. Sci. U.S.A.

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Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO 2 on lithium niobate (LiNbO3, n ‫؍‬ 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead͞ protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.

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“…Moreover, individual SLBs were completely confined within their protein boundaries. 47 To demonstrate this, the fluorescence recovery of a large bleach spot is shown in Figure 7. Taken together, the results in Figures 6 and 7 are strong evidence that highquality supported bilayers can be patterned on BSA-coated substrates that are selectively illuminated by deep UV radiation.…”

Section: Resultsmentioning

confidence: 99%

Multiplexing Ligand−Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

2008

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A method has been designed for patterning supported phospholipid bilayers (SLBs) on planar substrates and inside microfluidic channels. To do this, bovine serum albumin (BSA) monolayers were formed via adsorption at the liquid/solid interface. Next, this interfacial protein film was selectively patterned by using deep UV lithography. Subsequently, SLBs could be deposited in the patterned locations by vesicle fusion. By cycling through this process several times, spatially addressed bilayer arrays could be formed with intervening protein molecules serving as twodimensional corrals. By employing this method, phospholipid bilayers containing various concentrations of ganglioside GM1 were addressed along the length of individual microfluidic channels. Therefore, the binding of GM1 with pentameric cholera toxin B (CTB) subunits could be probed. A seven-channel microfluidic device was fabricated for this purpose. Each channel was simultaneously patterned with four chemically distinct SLBs containing 0, 0.2, 0.5, and 2.0 mol % GM1, respectively. Varying concentrations of CTB were then introduced into each of the channels. With the use of total internal reflection fluorescence microscopy, it was possible to simultaneously abstract multiple equilibrium dissociation constants as a function of ligand density for the CTB-GM1 system in a single shot.Multivalent ligand-receptor interactions are ubiquitous on cell surfaces. They have a wide variety of consequences including the modulation of equilibrium dissociation constants, high receptor selectivity, and receptor clustering. 1 Multivalency can also play a direct role in signal transduction processes. 2 Examination of the underlying thermodynamics of ligandreceptor binding may lead to a greater understanding of its biological role and could provide insight into biomedical applications involving inhibitory drug design. 1,3,4 Unfortunately, high-throughput, low-protein consumption assays are not presently well enough developed in this field to afford rapid, systematic studies of binding data at two-dimensionally fluid bilayer interfaces. 5 In previous work it has been demonstrated that microfluidic devices can be designed for measuring binding affinities in multivalent systems. [5][6][7][8][9][10][11][12][13][14] In our previous setup, ligands were incorporated into supported lipid bilayers (SLBs) coated on the walls and floors of polydimethylsiloxane/glass microchannels. Linear arrays of channels were then monitored by total internal reflection fluorescence microscopy (TIRFM) 15 to obtain equilibrium dissociation constants in one-shot assays. This could be done by using the same surface chemistry in each microchannel while varying the solution concentration of the aqueous proteins. Such a setup made these assays relatively rapid, afforded high accuracy, and used only a few microliters of protein solution. Nevertheless, such platforms could be markedly improved by measuring multiple binding constants with various membrane chemistries simultaneously. This could be achie...

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Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers

Cited by 192 publications

References 25 publications

E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Multiplexing Ligand−Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

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