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Method Article

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

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DOI:

10.3791/55060

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April 23rd, 2017

 ,  ,  ,  , 
1Aix-Marseille Université, CNRS, UMR 7325, CINaM, 2State Key Laboratory of Food Science and Technology, School of Food Science of Jiangnan University

In This Article

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 receptors (TCRs) on the surface of the T cell form micron scale clusters that serve as signaling platforms 2,3,4, and are eventually centralized to form a larger central supramolecular cluster (cSMAC) 5,6,7. Recently, it was shown that on the APC side, the ligands of the TCR are also clustered 8.

In the context of T cell-APC interaction, the deployment of hybrid systems, where the APC is mimicked by an artificial surface functionalized with relevant proteins, has greatly advanced our understanding of the synaptic interface 2,3,4,5,6,7. In this context, it is highly relevant to design APC mimetic surfaces that capture one or more aspects of the target cell. For example, if ligands are grafted on supported lipid bilayers, they can diffuse in the plane of the bilayer, mimic the situation on the APC surface and at the same time allow the formation of the cSMAC 6,7. Similarly, the clusters on the APC have been mimicked by creating islands of ligands in a sea of polymers 9,10,11,12,13,14. However, these two features have so far not been combined.

Here we describe a novel technique to create nano-dots of anti-CD3 (an antibody that targets the TCR complex) surrounded by a lipid bilayer with diffusing lipids. The bilayer is deposited using Langmuir-Blodgett/Langmuir-Schaefer technique 7,15,16 and if desired, could be functionalized with a specific protein — for example, the ligand of the T cell integrin (called ICAM1). In addition, the anti-CD3 protein dots could be replaced with another antibody or CAM. While we have chosen the proteins for future use as platform for T cell adhesion studies, the strategy detailed here can be adapted for any protein and even DNA.

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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 with ultrapure water.
  7. Repeat steps 1.5 and 1.6 twice.
  8. Store in water at room temperature (between 20 and 30 °C) for up to one week.

2. Fabrication of Protein Nano-dots

  1. Deposition of beads
    1. Deposit the suspension of silica beads (2% v/v, 70 µL) drop by drop on a cover-slide (24 x 24 mm, thickness 170 µm) held at an inclination of 15 degrees.
    2. Let the suspension spread for about 1 min while flipping the glass by 90° every 15 s.
    3. Allow the liquid to evaporate under ambient conditions.
    4. Store in a clean glass or PTFE chamber at ambient conditions for up to 2 days.
  2. Deposition of aluminum
    1. Place the slides (prepared in section 2.1) inside a radio frequency (RF) magnetron sputtering equipment 8, on a rotating table at a distance of about 105 mm from an aluminum (99%) : silicon (1%) target.
    2. Pump down the deposition chamber to 2.6 x 10-4 Pa using a turbo-molecular pump. This step is important for the removal of possible gaseous impurities.
    3. Introduce pure Argon atmosphere (5N, 99.999%) with a flux of 10 s.c.c.m. at a pressure of 0.8 Pa (6.6 mTorr).
      NOTE: The incoming argon flux is controlled by a mass flow controller and the process pressure is monitored using a pressure gauge. The argon gas flow is continuous and is controlled by a balance between the inlet and outlet opening valves.
    4. Switch on the RF power generator.
      NOTE: Here, a typical range of 400-600 W radiofrequency power at 13.56 MHz frequency was used. The RF generator is used with a matching network in a capacitive plasma coupling mode. The reflected power is monitored and minimized using the network adaptation.
    5. After the plasma is stabilized, sputter for 2 min keeping the shutter closed to remove possible impurities from the surface of the target.
    6. Open the shutter and allow the sputtering to continue for 60 min to deposit aluminum on the glass slide to a thickness of 200 nm.
    7. Cut the flow of argon, close the gate valve to isolate the turbomolecular pump from the deposition chamber and vent the chamber with clean nitrogen to obtain the room pressure. Recover the aluminum-coated slides. Store for up to a month in a clean and hermetically sealed glass or PTFE box under ambient conditions.
  3. Vapor deposition of Organosilane
    1. Immerse the aluminum-coated slide prepared in step 2.2.6 in ultrapure water at room temperature and ultra-sonicate for 30 s in an ultrasonic bath (50 W, 50/60 Hz, between 20 and 30 °C).
    2. Deposit 0.5 mL of (3-aminopropyl)-triethoxysilane (APTES) at the bottom of a desiccator.
      CAUTION: APTES is an organosilane, which evaporates easily and is toxic. APTES should be handled only under a flow-hood and with gloves.
    3. Put the glass slides (prepared in 2.3.1) on a ceramic grid and place inside the desiccator.
    4. Connect the desiccator to a membrane pump and run at maximum power for 30 min to generate a low vacuum.
    5. Close the valve of the desiccator and switch off the pump.
    6. Heat the desiccator to about 50 °C for 1 h.
    7. Open the desiccator and collect the slides.
    8. Transfer to another clean desiccator for storage for up to 48 h at room temperature (20 to 30 °C).
  4. Deposition of first layer of protein - bovine serum albumin labelled with biotin (BSA-biotin)
    1. Place one of the slides prepared in section 2.3 on a PTFE support.
    2. Deposit 1 mL of 25 µg/mL BSA-biotin dissolved in Phosphate Buffered Saline (PBS). Leave for 30 min at room temperature (20 to 30 °C).
    3. Rinse 10 times with PBS. Store the sample for up to 24 h at 4 °C away from light source.
  5. Removal of aluminum mask
    1. Incubate the slides in a solution of NaOH in PBS (prepared by dropwise addition of 1 M NaOH to about 100 mL PBS to obtain pH≈12) over-night at room temperature (between 20 and 30 °C).
      CAUTION. NaOH is corrosive and should be handled using gloves.
    2. Rinse 10 times in ultrapure water.

3. Deposition of Supported Lipid Bilayer (SLB)

  1. Cleaning the Langmuir trough
    1. Remove the water in the PTFE enclosure of the Langmuir trough using a pump.
    2. Clean with a lint-free non-woven disposable towel soaked in chloroform.
      CAUTION. Chloroform is toxic and should be manipulated in a well-ventilated area, with a mask (or under a flow-hood) and with appropriate gloves.
    3. Clean 4 times with lukewarm ultrapure water (40 to 50 °C).
    4. Clean at least 6 times with cold ultrapure water.
  2. Deposition of the first lipid layer
    1. Place PTFE trays in the PTFE enclosure of the Langmuir trough and then fill it with ultrapure water.
    2. Use the control software of the Langmuir apparatus to set the measured pressure to 0 mN/m.
    3. Use a gastight glass/metal syringe to deposit 30 µL of a 1 mg/mL lipid solution (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in chloroform) on the surface of the water. The chloroform evaporates and the lipid molecules spontaneously form a monolayer.
    4. Use the control software to compress the lipid monolayer by closing the PTFE barrier till the desired pressure (27 mN/m for DOPC) is reached.
    5. Use the control software to dip the glass slide prepared in the section 2.5.2 into the PTFE enclosure using a motorized clamp.
    6. Hold the slide, in the clamp, perpendicular to the air-water interface. Use the control software to raise it slowly (15 mm/min) through the interface, while maintaining a constant pressure at 27 mN/m.
    7. Place in a dry environment for either immediate use or for storage up to 24 h at room temperature (between 20 and 30 °C).
  3. Deposition of the second lipid layer
    1. Maintain the surface pressure in the Langmuir trough at the desired value of 27 mN/m. Compress using the control software of the Langmuir apparatus and/or add a small amount of lipid to achieve the desired pressure.
    2. Place the glass slides carrying the lipid monolayer, prepared in section 3.2.6, horizontally on the surface of the water. Ensure that each slide is floating above a PTFE tray.
    3. Push the glass slides down, one slide at a time, into its corresponding PTFE tray using PTFE or metal tweezers such that they are immersed in the water. Avoid tilting the slides while pushing.
    4. Use PTFE or metal tweezers to transfer the PTFE trays containing the slides into a crystallizer filled with ultrapure water making sure that the slides are not exposed to air.
    5. Use PTFE or metal tweezers to transfer, while working underwater, a bilayer coated glass slide into an observation chamber.
      NOTE: The observation chamber is placed under water inside the crystallizer. The observation chamber is custom made and consists of a PTFE ring with a rubber gasket and steel casings, which can be assembled underwater to make a water-tight chamber whose bottom surface is made from the glass slide previously coated with the lipid bilayer.
    6. Close the chamber while continuing to work under water and ensuring that about 1 mL of water is trapped inside the chamber.
    7. Take the assembled chamber out of the water. Make sure that it is water-tight and leakage free.
    8. Replace the 1 mL of ultrapure water present in the observation chamber with PBS by adding and removing 500 µL of PBS 10 times. Ensure that the chamber is never devoid of liquid.
  4. SLB blocking step
    1. Introduce 100 µg/mL of bovine serum albumin (BSA) in the observation chamber containing the slide prepared in section 3.3.6 and incubate for 30 min at room temperature (between 20 and 30 °C).
    2. Rinse the bilayer by removing and adding 500 µL of PBS 10 times. The bilayer can be kept 24 h at 4 °C.

4. Functionalization with Ligands

  1. Add fluorescent or non-fluorescent neutravidin (NAV, a deglycosylated version of avidin not charged at pH 7) at 2 µg/mL into the chamber containing the slide prepared in section 3.4.2 for 30 min at room temperature (between 20 and 30 °C).
  2. Rinse by adding and removing 500 µL of PBS 10 times.
  3. Add biotinylated anti-CD3 at 2 µg/mL to the chamber containing the slide prepared in section 3.4.2. For double functionalization of the SLB and the dots, add Fc-ICAM1 His-tag at 5 µg/mL (here, the bilayer is made of DOPC + 1% of nitrilotriacetic acid (NTA) lipids). Leave for 30 min at room temperature (between 20 and 30 °C).
  4. Rinse by adding and removing 500 µL of PBS 10 times
  5. Replace the PBS with cell medium (PBS + 0.1% BSA) by adding and removing 500 µL of the cell medium 10 times.
  6. Leave 200 µL of cell medium in the chamber with the slide.
  7. Place the chamber for 10 min at 37 °C before adding cells.

5. Cell Deposition (see reference 7 for details)

  1. Carefully deposit 400 µL of the cell suspension into the chamber. Incubate the cells for 30 min at 37 °C.
  2. Fix the cell with 2% of paraformaldehyde (PFA) for 15 min at 37 °C. Replace the PFA with PBS by repeated removal and addition of 500 µL of PBS.

6. Observation

  1. Proteo-lipidic Nano-pattern and SLB fluidity
    1. Use a fluorescence microscope to image the protein pattern in epi-fluorescence mode using appropriate illumination wavelength (639 nm) and filter cubes (e.g. here, EX TBP 483+564+642; BS TFT 506+582+659; EM TBP 526+601+688).
      NOTE: The intensity of the signal can be quantified to estimate the amount of protein inside and outside the dot. Use an appropriate illumination wavelength (330 nm) and filter cube to image the bilayer (BP 365/12, FT 395, LP 397), which appears bright with dark holes.
    2. Use continuous photo bleaching (CPB) technique13,15 to measure the diffusion constant of tracer lipids in the bilayer. This observation is preferably made just after the SLB deposition.
      NOTE: In order to quantify the diffusion constant, two parameters are measured during CPB process: the specific bleaching time of the dye (tauT) and the decay length of the fluorescent profile of the halo formed around the bleached area (tauD). The first is obtained by plotting the time evolution of the fluorescence intensity at the center of the illuminated field during the CPB process. The second is obtained by plotting the intensity profile at the edge of the illuminated zone (averaged over 12 lines and 5 images after steady-state is reached). The curves are fitted to obtain tauT and tauD. The diffusion constant is given by tauD2/ tauT (D=Equation for decay rate ratio, τ₂/τ₁, in transient absorption analysis.).

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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...

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Laurent Limozin, Pierre Dillard and Astrid Wahl for continuing fruitful discussions about cellular applications. We also thank Frederic Bedu from PLANETE cleanroom facility for his help with SEM observations. This work was partially funded by the European Research Council via grant No. 307104 FP/2007-2013/ERC.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Glass coverslipsAssistent, Karl Hecht KG 
Observation chamberHome made
Alkaline surfactant concentrate (Hellmanex)Hella Analytics9-307-011-4-507
Ultra-sonicatorThermoFisher
DesiccatorLabbox
Crystallizer Shott
NeutravidineThermo Fischer Scientifique84607
PBS Sigma-aldrichP3813
Water MQ ELGA, Veolia France
Silica beadsCorpuscular Inc147114-10
APTESSigma-aldrichA3648
BSA-BiotinSigma-aldrichA8549
DOPCAvanti Polar Lipids850375C
Dansyl-PEAvanti Polar Lipids810330C
ChloroformSigma-aldrich650471
Gastight syringe Dominique Dustcher , France74453
Film balanceNIMAMedium
MicroscopeZeiss, GermanyTIRF-III system
Aluminium Target Kurt J. Lesker Compagny, USA
Radio Frequency Magnetron sputtering Système modified SMC 600 tool by ALCATEL , France

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Tags

Protein Nano clustersLigand ArraysBead Mask TechniqueAluminum DepositionAPTES FunctionalizationBSA Biotin CoatingTIRF MicroscopyRICM MicroscopyLipid Bilayer Fluidity
Ligand Nano-cluster Arrays in a Supported Lipid Bilayer
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