Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Abstract

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

T Cells on Engineered Substrates: The Impact of TCR Clustering Is Enhanced by LFA-1 Engagement

“…The details of the fabrication process for making the patterned substrates was published previously ( 30 , 38 , 39 ). Briefly, hydrophilic glass coverslides (thickness = 170 microns, Assistant, Karl Hecht KG, Germany, 24 x 24 mm) were obtained by cleaning by ultrasonication in aqueous solution of a detergent (Hellmanex, Sigma, France), followed by a thorough rinsing in ultrapure water.…”

“…Finally, the layer of aluminum was removed by incubation in PBS + Sodium hydroxide (NaOH), pH ≥ 11 for at least 4 hours until complete dissolution at room temperature. At this stage the coverslide was covered with uniform nano-dots of functional BSA-Biotin surrounded by bare glass ( 39 ).…”

Ligand Nanocluster Array Enables Artificial-Intelligence-Based Detection of Hidden Features in T-Cell Architecture

“…33,34 Modification of T-cell membrane protein distribution, 35 and colocalisation of TCR-microclusters with patterned ligands of TCR, 36,37 was previously demonstrated. Ligand presentation as nano-scale pattern also impacts T cell spreading and activation, however, the interpretation remains complex [38][39][40] In order to explore the role of TCR µ-clusters in determining actin architecture, we exploit previous finding that clustering of TCR-ligands or antibodies against the TCR complex can induce clustering of TCRs themselves 8,36,37,41 to generate stable TCR µ-clusters arranged in an array. We leverage the ability of e-beam lithography to create flawless arrays of a repeated motif, to decipher the impact of presenting TCR-ligand-clustering on actin organization.…”

“…To explore the role of TCR μ-clusters in determining actin architecture, we exploit previous findings that clustering of TCR ligands or antibodies against the TCR complex can induce clustering of TCRs themselves ,,, to generate stable TCR μ-clusters arranged in an array. We leverage the ability of electron beam lithography to create flawless arrays of a repeated motif, to decipher the impact of presenting TCR ligand clustering on actin organization.…”