Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Summary

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

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.

Introduction

Cell adhesion takes place through specialized cell adhesion molecules (CAMs), proteins present on the cell membrane that are capable of binding to their counterpart on the extra cellular matrix or on another cell. On adhered cells, most adhesion molecules including the ubiquitous integrin and cadherin, are found in the form of clusters ¹. The interaction of T lymphocytes (T cells) with antigen presenting cells (APCs) provides a particularly striking illustration of the importance of receptor clusters formed at the interface between the two cells — often called an immunological synapse. Upon forming the first contact with the APC, T cell r....

Protocol

1. Cleaning Glass Cover-slides and Observation Chambers

1. Arrange the glass cover-slides on a multi-slide tray made of an inert material like Polytetrafluoroethylene (PTFE).
2. Immerse the tray with slides and the observation chamber in a surfactant solution (any product recommended for cleaning quartz cuvettes is suitable).
3. Using an ultrasonic bath, ultra-sonicate in the surfactant solution for 30 min at room temperature (between 20 and 30 °C).
4. Rinse 5 times with ultrapure water (18.2 MΩ.cm, 0.059 µS/cm).
5. Ultra-sonicate in surfactant solution for 30 min at room temperature (between 20 and 30 °C).
6. Rinse 10 times....

Results

The fluorescence images were analyzed to measure the spacing and size of the dots. Typical spacing was found to be 1,900 ± 80 nm and typical dot-size was 600 ± 100 nm (Figure 1g). The spacing is set by the size of the beads used for the mask. The dot-size is set by the bead-size as well as deposition conditions. The SLB is deposited uniquely around the protein dots and not on them (Figure 2), with perfect complementarity between the holes seen in SLB imag.......

Discussion

The critical steps within the protocol described above are related to the formation of the protein nano-dots or the back-filling of the space around the dots by a supported lipid bilayer. The first critical step with respect to protein nano-dots is the preparation of the bead-mask. The cleaning of the cover-slide is critical. The slides need to be either cleaned with a detergent solution that is recommended for cleaning quartz cuvettes, or with oxygen plasma. Other cleaning techniques like immersion in ethanol or iso-pro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass coverslips	Assistent, Karl Hecht KG
Observation chamber	Home made
Alkaline surfactant concentrate (Hellmanex)	Hella Analytics	9-307-011-4-507
Ultra-sonicator	ThermoFisher
Desiccator	Labbox
Crystallizer	Shott
Neutravidine	Thermo Fischer Scientifique	84607
PBS	Sigma-aldrich	P3813
Water MQ	ELGA, Veolia France
Silica beads	Corpuscular Inc	147114-10
APTES	Sigma-aldrich	A3648
BSA-Biotin	Sigma-aldrich	A8549
DOPC	Avanti Polar Lipids	850375C
Dansyl-PE	Avanti Polar Lipids	810330C
Chloroform	Sigma-aldrich	650471
Gastight syringe	Dominique Dustcher , France	74453
Film balance	NIMA	Medium
Microscope	Zeiss, Germany	TIRF-III system
Aluminium Target	Kurt J. Lesker Compagny, USA
Radio Frequency Magnetron sputtering Système	modified SMC 600 tool by ALCATEL , France