Name: ligand nano-cluster arrays in a supported lipid bilayer
Uploaded: 2017-04-23T14:00:00
Description: Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

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.

1. Cleaning Glass Cover-slides and Observation Chambers
<ol>
	<li>Arrange the glass cover-slides on a multi-slide tray made of an inert material like Polytetrafluoroethylene (PTFE).</li>
	<li>Immerse the tray with slides and the observation chamber in a surfactant solution (any product recommended for cleaning quartz cuvettes is suitable).</li>
	<li>Using an ultrasonic bath, ultra-sonicate in the surfactant solution for 30 min at room temperature (between 20 and 30 &#176;C).</li>
	<li>Rinse 5 times with ultrapure water...

<ol>
	<li>Alberts, B., Johnson, A., Lewis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Cell. , (2002).</li><li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cell+Receptor-Proximal+Signals+Are+Sustained+in+Peripheral+Microclusters+and+Terminated+in+the+Central+Supramolecular+Activation+Cluster.">T Cell Receptor-Proximal Signals Are Sustained in Peripheral Microclusters and Terminated in the Central Supramolecular Activation Cluster.</a> Immunity. 25 (1), 117-127 (2006).</li><li>Kaizuka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+segregating+T+cell+receptor+and+adhesion+molecules+during+immunological+synapse+formation+in+Jurkat+T+cells.">Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells.</a> Proc Natl Acad Sci USA. 104 (51), 20296-20301 (2007).</li><li>Dustin, M. L., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+signaling+clusters+in+the+immune+synapse.">Receptor signaling clusters in the immune synapse.</a> Annu Rev Biophys. 41, 543-556 (2012).</li><li>Huppa, J. B., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell-antigen+recognition+and+the+immunological+synapse.">T-cell-antigen recognition and the immunological synapse.</a> Nat Rev Immunol. 3 (12), 973-983 (2003).</li><li>Grakoui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Immunological+Synapse:+A+Molecular+Machine+Controlling+T+Cell+Activation.">The Immunological Synapse: A Molecular Machine Controlling T Cell Activation.</a> Science. 285, 221-228 (1999).</li><li>Dillard, P., Varma, R., Sengupta, K., Limozin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-mediated+friction+determines+morphodynamics+of+spreading+T+cells.">Ligand-mediated friction determines morphodynamics of spreading T cells.</a> Biophys J. 107 (11), 2629-2638 (2014).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+viral+antigen+processing+generates+peptide-specific+MHC+class+I+cell-surface+clusters.">Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.</a> Proc Natl Acad Sci U S A. 109 (38), 15407-15412 (2012).</li><li>Pi, F., Dillard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-Tunable+Organic+Nanodot+Arrays:+A+Versatile+Platform+for+Manipulating+and+Imaging+Cells.">Size-Tunable Organic Nanodot Arrays: A Versatile Platform for Manipulating and Imaging Cells.</a> Nano Lett. 15 (8), 5178-5184 (2015).</li><li>Deeg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+activation+is+determined+by+the+number+of+presented+antigens.">T cell activation is determined by the number of presented antigens.</a> Nano Lett. 13 (11), 5619-5626 (2013).</li><li>Delcassian, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+ligand+spacing+influences+receptor+triggering+in+T+cells+and+NK+cells.">Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.</a> Nano Lett. 13 (11), 5608-5614 (2013).</li><li>Matic, J., Deeg, J., Scheffold, A., Goldstein, I., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+tuning+and+efficient+T+cell+activation+with+stimulatory+aCD3+nanoarrays.">Fine tuning and efficient T cell activation with stimulatory aCD3 nanoarrays.</a> Nano Lett. 13 (11), 5090-5097 (2013).</li><li>Dillard, P., Pi, F., Lellouch, A. C., Limozin, L., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-clustering+of+ligands+on+surrogate+antigen+presenting+cells+modulates+T+cell+membrane+adhesion+and+organization.">Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization.</a> Integr Biol. 8 (3), 287-301 (2016).</li><li>Pi, F., Dillard, P., Limozin, L., Charrier, A., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometric+protein-patch+arrays+on+glass+and+polydimethylsiloxane+for+cell+adhesion+studies.">Nanometric protein-patch arrays on glass and polydimethylsiloxane for cell adhesion studies.</a> Nano lett. 13 (7), 3372-3378 (2013).</li><li>Fenz, S. F., Merkel, R., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+and+intermembrane+distance:+case+study+of+avidin+and+E-cadherin+mediated+adhesion.">Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.</a> Langmuir. 25 (2), 1074-1085 (2009).</li><li>Sengupta, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+tissue+surfaces+by+supported+membrane+coupled+ultra-thin+layer+of+hyaluronic+acid.">Mimicking tissue surfaces by supported membrane coupled ultra-thin layer of hyaluronic acid.</a> Langmuir. 19 (5), 1775-1781 (2003).</li><li>Taylor, Z. R., Keay, J. C., Sanchez, E. S., Johnson, M. B., Schmidtke, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independently+controlling+protein+dot+size+and+spacing+in+particle+lithography.">Independently controlling protein dot size and spacing in particle lithography.</a> Langmuir. 28 (25), 9656-9663 (2012).</li><li>Massou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+ordered+topographical+and+chemical+nano-features+from+anodic+alumina+templates.">Large scale ordered topographical and chemical nano-features from anodic alumina templates.</a> Appl. Surf Sci. 256 (2), 395-398 (2009).</li><li>Selhuber-Unkel, C., Lopez-Garcia, M., Kessler, H., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperativity+in+adhesion+cluster+formation+during+initial+cell+adhesion.">Cooperativity in adhesion cluster formation during initial cell adhesion.</a> Biophys J. 95 (11), 5424-5431 (2008).</li><li>Arnold, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cell+polarization+and+migration+by+a+gradient+of+nanoscale+variations+in+adhesive+ligand+spacing.">Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing.</a> Nano Lett. 8 (7), 2063-2069 (2008).</li><li>Cavalcanti-Adam, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+spreading+and+focal+adhesion+dynamics+are+regulated+by+spacing+of+integrin+ligands.">Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.</a> Biophys J. 92 (8), 2964-2974 (2007).</li><li>Schvartzman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanolithographic+Control+of+the+Spatial+Organization+of+Cellular+Adhesion+Receptors+at+the+Single-Molecule+Level.">Nanolithographic Control of the Spatial Organization of Cellular Adhesion Receptors at the Single-Molecule Level.</a> Nano Lett. 11 (3), 1306-1312 (2011).</li><li>Mossman, K., Groves, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+supported+membranes+as+tools+for+quantitative+studies+of+the+immunological+synapse.">Micropatterned supported membranes as tools for quantitative studies of the immunological synapse.</a> Chem.Soc.Rev. 36 (1), 46-54 (2007).</li><li>Furlan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatase+CD45+both+positively+and+negatively+regulates+T+cell+receptor+phosphorylation+in+reconstituted+membrane+protein+clusters.">Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.</a> J Biol Chem. 289 (41), 28514-28525 (2014).</li><li>Hsu, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+mobility+modulates+immunological+synapse+formation+and+T+cell+activation.">Ligand mobility modulates immunological synapse formation and T cell activation.</a> PloS One. 7 (2), e32398 (2012).</li><li>Yu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+integrin+binding+to+Arg-Gly-Asp+peptide+activates+actin+polymerization+and+contractile+movement+that+stimulates+outward+translocation.">Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation.</a> Proc Natl Acad Sci U S A. 108 (51), 20585-20590 (2011).</li></ol>

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...

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 &#8212; 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 &#8212; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass coverslips</td>
			<td>Assistent, Karl Hecht KG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Observation chamber</td>
			<td>Home made</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline surfactant concentrate (Hellmanex)</td>
			<td>Hella Analytics</td>
			<td>9-307-011-4-507</td>
		</tr>
		<tr>
			<td>Ultra-sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Labbox</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallizer&#160;</td>
			<td>Shott</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidine</td>
			<td>Thermo Fischer Scientifique</td>
			<td>84607</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Sigma-aldrich</td>
			<td>P3813</td>
		</tr>
		<tr>
			<td>Water MQ&#160;</td>
			<td>ELGA, Veolia France</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica beads</td>
			<td>Corpuscular Inc</td>
			<td>147114-10</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma-aldrich</td>
			<td>A3648</td>
		</tr>
		<tr>
			<td>BSA-Biotin</td>
			<td>Sigma-aldrich</td>
			<td>A8549</td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
		</tr>
		<tr>
			<td>Dansyl-PE</td>
			<td>Avanti Polar Lipids</td>
			<td>810330C</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-aldrich</td>
			<td>650471</td>
		</tr>
		<tr>
			<td>Gastight syringe&#160;</td>
			<td>Dominique Dustcher , France</td>
			<td>74453</td>
		</tr>
		<tr>
			<td>Film balance</td>
			<td>NIMA</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss, Germany</td>
			<td>TIRF-III system</td>
		</tr>
		<tr>
			<td>Aluminium Target&#160;</td>
			<td>Kurt J. Lesker Compagny, USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Radio Frequency Magnetron sputtering Syst&#232;me&#160;</td>
			<td>modified SMC 600 tool by ALCATEL , France</td>
			<td></td>
		</tr>
	</tbody>
</table>

ligand nano-cluster arrays in a supported lipid bilayer

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.

The fluorescence images were analyzed to measure the spacing and size of the dots. Typical spacing was found to be 1,900 &#177; 80 nm and typical dot-size was 600 &#177; 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 i...

Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

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.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

The overall goal of this technique is to fabricate an array of protein nano-clusters in a supported lipid bilayer, which is compatible with sensitive surface microscopy techniques for cell biology applications. This method can help answer key questions in the field of cell biophysics, in particular, how the nano-clustering of ligands influences T cell activation and adhesion. The main advantage of this technique is that it is easily reproducible in standard biophysical lab and it is compatible with advanced surface sensitive microscopy technique, such as TIRF and RICM.Though this technique is designed to provide insight into immunology, it can be applied to other cell types with potential applications in cell biology, oncology and tissue engineering. To begin this procedure, clean the glass cover slides and observation chambers as outlined in the text protocol. Then, deposit 70 microliters of 2%silica bead suspension drop by drop onto a cover slide held at a 15 degree incline.Flip the glass 90 degrees every 15 seconds for one minute while the suspensions dries. It is critical to create a closely-packed monolayer of beads and to avoid the formation of multilayers and oil clusters. That's why the concentration and the volume of the bead solution as well as the hydrophilicity of the slide need to be optimized.After the liquid has evaporated, place the slide inside a radio frequency magnatron sputtering device onto a rotating table situated 105 millimeters away from an aluminum silicon target. After this, use a turbo-molecular pump to decrease the pressure in the deposition chamber to 2.6 times 10 to the negative fourth pascals. Introduce a pure argon atmosphere with a flux of 10 standard cubic centimeters per minute and a pressure of 0.8 pascals.Next, switch on the radio frequency power generator. After the plasma is stabilized, sputter for two minutes while keeping the shutter closed, to remove possible impurities from the target surface. Open the shutter, and allow the sputtering to continue for 60 minutes to deposit a 200-nanometer thick layer of aluminum on the slide.Then cut the flow of argon. Close the gate valve to isolate the turbo-molecular pump from the deposition chamber. After this, vent the chamber with clean nitrogen to reach ambient pressure.Recover the aluminum-coated slide from the chamber. Immerse the recovered slide in ultra-pure water at room temperature and ultra-sonicate at 50 watts and 50 hertz for 30 seconds. Next, deposit 0.5 milliters of APTES at the bottom of a desiccator.Place the glass slide on a ceramic grid. Place the grid into the desiccator. Connect the desiccator to a membrane pump.Then run the pump at maximum power for 30 minutes to generate a low vacuum. Close the valve of the desiccator, and switch off the pump. Heat to 50 degrees celsius for one hour.After this open the desiccator and collect the slide. Place the collected slide on a PTFE support. Deposit 2 milliliters of 25 micrograms per milliliter biotin dissolved in PBS.Allow the slide to rest for 30 minutes at room temperature. Then rinse 10 times with PBS. Incubate the slide in a solution of sodium hydroxide and PBS at room temperature overnight.After this, rinse the slide 10 times with ultra-pure water. After cleaning the Langmuir trough, place PTFE trays in the trough's PTFE enclosure. Then fill it with ultra-pure water.Set the measured pressure to zero millinewtons per meter. Using a gas-tight glass metal syringe, deposit 30 microliters of 1 milligram per millimeter DOPC and chloroform solution on the water's surface. Close the PTFE barrier until the desired pressure of 27 millinewtons per meter is reached to compress the lipid monolayer.Next, using a motorized clamp, dip the prepared glass slide into the PTFE enclosure. Hold the slide in the clamp perpendicular to the air-water interface. Raise the slide through the interface at a rate of 15 millimeters per minute while maintaining a constant pressure of 27 millinewtons per meter.Then, place the glass slide horizontally on the surface of the water above a PTFE tray. Next, using metal tweezers, push the slide down into the PTFE tray, immersing it in the ultra-pure water. Use the tweezers to transfer the PTFE tray containing the slide into a crystallizer filled with ultra-pure water.Transfer the coated slide underwater into an observation chamber. After this, close the chamber, ensuring that approximately 1 milliliter of water is trapped inside. Another delicate step is the assembly of the sample chamber underwater.And one should absolutely avoid contact with the air of the deposited bilayers, so to ensure this use a large volume of water and ensure that the whole assembly process can occur underwater. Remove the assembled chamber from the water, check to ensure that the chamber is watertight and free from leaks. Add 500 microliters of PBS, then remove 500 microliters of liquid from the chamber.Repeat this process 10 times to fully replace the one milliliter of ultra-pure water in the chamber with PBS. Next, introduce 100 microgram per milliliter of bovine serum albumen to the observation chamber. Incubate at room temperature for 30 minutes.After this, rinse the bilayer by removing and adding 500 microliters from the chamber, 10 times. Then, functionalize with ligands, deposit cells, and observe the proteolipidic nanopattern and SLB fluidity as outlined in the text protocol. In this protocol, a bead mask is used to create a secondary metal mask by controlled deposition of aluminum.Then, the bead mask is removed, leaving a layer of aluminum with holes. Next, an organo-amino siline, called APTES, is deposited in the vapor phase, followed by deposition of BSA-Biotin in aqueous phase. Then, aluminum is removed, revealing nanometric protein patches.Finally the inter-dot space is back-filled with a supported lipid bilayer. Epiflourescence images are then taken of the nano-dots and supported lipid bilayer. A composite image shows perfect complementarity with the lipid bilayer deposited uniquely around the protein dots, but not on them.In an epiflourescence image of a freshly-deposited supported lipid bilayer, the protein dots appear as dark spots in a bright sea of lipids. However, after continuous photo bleaching for 50 seconds, the image shows a halo inside the region delimited by the field diaphragm, indicating that the lipids are mobile. Analysis of the average intensity profile along the edge of the field diaphragm, and the decay of this intensity over time during the bleaching process, reveals that the diffusion constant is five micrometers squared per second.This technique can be done in two days if it is performed properly. Its's important to maintain very clean conditions during aluminum deposition, and to carefully calibrate the deposition curve. Following this procedure, the patterned slide can be further functionalized.For example, by adding another biomolecule in the bilayer. We use this to mimic the antigen plant-eating cells, in order to study the T cell activation. So after its development, this technique should interest diverse audiences.For example, tissue engineers may want to use it as scaffold to grow artificial tissues, and oncologists may use this as a platform for posing fundamental questions about addition of cancer cells. After watching this video you should have a good understanding of how to fabricate an area of protein nano-clusters in supported lipid bilayer, which is compatible with an advanced microscopy technique. While we use this to study T cells, we believe that this substrate have the potential to become the platform of choice to study the interaction of any kind of cells with controlled pattern proteolipidic membrane.

ligand-nano-cluster-arrays-in-a-supported-lipid-bilayer

Research

JoVE Journal

Bioengineering

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...