Name: ligand nano-cluster arrays in a supported lipid bilayer
Uploaded: 2017-04-23T14:00:00.000+00:00
Duration: 10 min 34 s
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 (18.2 M&#8486;.cm, 0.059 &#181;S/cm).</li>
	<li>Ultra-sonicate in surfactant solution for 30 min at room temperature (between 20 and 30 &#176;C).</li>
	<li>Rinse 10 times with ultrapure water.</li>
	<li>Repeat steps 1.5 and 1.6 twice.</li>
	<li>Store in water at room temperature (between 20 and 30 &#176;C) for up to one week.</li>
</ol>
2. Fabrication of Protein Nano-dots
<ol>
	<li>Deposition of beads
	<ol>
		<li>Deposit the suspension of silica beads (2% v/v, 70 &#181;L) drop by drop on a cover-slide (24 x 24 mm, thickness 170 &#181;m) held at an inclination of 15 degrees.</li>
		<li>Let the suspension spread for about 1 min while flipping the glass by 90&#176; every 15 s.</li>
		<li>Allow the liquid to evaporate under ambient conditions.</li>
		<li>Store in a clean glass or PTFE chamber at ambient conditions for up to 2 days.</li>
	</ol>
	</li>
	<li>Deposition of aluminum
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>After the plasma is stabilized, sputter for 2 min keeping the shutter closed to remove possible impurities from the surface of the target.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Vapor deposition of Organosilane
	<ol>
		<li>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 &#176;C).</li>
		<li>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.</li>
		<li>Put the glass slides (prepared in 2.3.1) on a ceramic grid and place inside the desiccator.</li>
		<li>Connect the desiccator to a membrane pump and run at maximum power for 30 min to generate a low vacuum.</li>
		<li>Close the valve of the desiccator and switch off the pump.</li>
		<li>Heat the desiccator to about 50 &#176;C for 1 h.</li>
		<li>Open the desiccator and collect the slides.</li>
		<li>Transfer to another clean desiccator for storage for up to 48 h at room temperature (20 to 30 &#176;C).</li>
	</ol>
	</li>
	<li>Deposition of first layer of protein - bovine serum albumin labelled with biotin (BSA-biotin)
	<ol>
		<li>Place one of the slides prepared in section 2.3 on a PTFE support.</li>
		<li>Deposit 1 mL of 25 &#181;g/mL BSA-biotin dissolved in Phosphate Buffered Saline (PBS). Leave for 30 min at room temperature (20 to 30 &#176;C).</li>
		<li>Rinse 10 times with PBS. Store the sample for up to 24 h at 4 &#176;C away from light source.</li>
	</ol>
	</li>
	<li>Removal of aluminum mask
	<ol>
		<li>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&#8776;12) over-night at room temperature (between 20 and 30 &#176;C). 
		CAUTION. NaOH is corrosive and should be handled using gloves.</li>
		<li>Rinse 10 times in ultrapure water.</li>
	</ol>
	</li>
</ol>
3. Deposition of Supported Lipid Bilayer (SLB)
<ol>
	<li>Cleaning the Langmuir trough
	<ol>
		<li>Remove the water in the PTFE enclosure of the Langmuir trough using a pump.</li>
		<li>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.</li>
		<li>Clean 4 times with lukewarm ultrapure water (40 to 50 &#176;C).</li>
		<li>Clean at least 6 times with cold ultrapure water.</li>
	</ol>
	</li>
	<li>Deposition of the first lipid layer
	<ol>
		<li>Place PTFE trays in the PTFE enclosure of the Langmuir trough and then fill it with ultrapure water.</li>
		<li>Use the control software of the Langmuir apparatus to set the measured pressure to 0 mN/m.</li>
		<li>Use a gastight glass/metal syringe to deposit 30 &#181;L 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.</li>
		<li>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.</li>
		<li>Use the control software to dip the glass slide prepared in the section 2.5.2 into the PTFE enclosure using a motorized clamp.</li>
		<li>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.</li>
		<li>Place in a dry environment for either immediate use or for storage up to 24 h at room temperature (between 20 and 30 &#176;C).</li>
	</ol>
	</li>
	<li>Deposition of the second lipid layer
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Close the chamber while continuing to work under water and ensuring that about 1 mL of water is trapped inside the chamber.</li>
		<li>Take the assembled chamber out of the water. Make sure that it is water-tight and leakage free.</li>
		<li>Replace the 1 mL of ultrapure water present in the observation chamber with PBS by adding and removing 500 &#181;L of PBS 10 times. Ensure that the chamber is never devoid of liquid.</li>
	</ol>
	</li>
	<li>SLB blocking step
	<ol>
		<li>Introduce 100 &#181;g/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 &#176;C).</li>
		<li>Rinse the bilayer by removing and adding 500 &#181;L of PBS 10 times. The bilayer can be kept 24 h at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Functionalization with Ligands
<ol>
	<li>Add fluorescent or non-fluorescent neutravidin (NAV, a deglycosylated version of avidin not charged at pH 7) at 2 &#181;g/mL into the chamber containing the slide prepared in section 3.4.2 for 30 min at room temperature (between 20 and 30 &#176;C).</li>
	<li>Rinse by adding and removing 500 &#181;L of PBS 10 times.</li>
	<li>Add biotinylated anti-CD3 at 2 &#181;g/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 &#181;g/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 &#176;C).</li>
	<li>Rinse by adding and removing 500 &#181;L of PBS 10 times</li>
	<li>Replace the PBS with cell medium (PBS + 0.1% BSA) by adding and removing 500 &#181;L of the cell medium 10 times.</li>
	<li>Leave 200 &#181;L of cell medium in the chamber with the slide.</li>
	<li>Place the chamber for 10 min at 37 &#176;C before adding cells.</li>
</ol>
5. Cell Deposition (see reference 7 for details)
<ol>
	<li>Carefully deposit 400 &#181;L of the cell suspension into the chamber. Incubate the cells for 30 min at 37 &#176;C.</li>
	<li>Fix the cell with 2% of paraformaldehyde (PFA) for 15 min at 37 &#176;C. Replace the PFA with PBS by repeated removal and addition of 500 &#181;L of PBS.</li>
</ol>
6. Observation
<ol>
	<li>Proteo-lipidic Nano-pattern and SLB fluidity
	<ol>
		<li>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.</li>
		<li>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=<img alt="Equation" src="/files/ftp_upload/55060/55060eq1.jpg" />).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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-propanol treatment do not render the glass sufficiently hydrophilic and therefore do not support the formation of a large-coverage bead monolayer. At the same time, the surface of the slide needs to be compatible with the subsequent step of formation of the SLB by Langmuir-Blodgett technique 7,15,16. The next critical step is the deposition and removal of aluminum. If the deposition is via sputtering technique, as done here, the target should to be doped with silicon (at 1%). Otherwise, if the target is made of pure aluminum, the deposited layer is hard to remove with the alkali hydroxide solution as described above, probably due to the formation of aluminum oxide and interpenetration between deposited aluminum layer and glass slide substrate. The duration of deposition determines the size of the protein dots 9, however, here we have worked with a single deposition time and therefore single dot size. The sample can be stored under ambient conditions for several months after aluminum deposition and for about a week after the deposition of BSA-biotin.
The second crucial step concerns the deposition of the SLB. Cleaning of the glass cover-slide is again a crucially important point. As is the case for any Langmuir-Blodgett deposition work, all the material used should be made of glass or Polytetrafluoroethylene (PTFE) and should be scrupulously clean. After deposition of the first lipid monolayer, the cover-slides can be stored for a couple of days but after the deposition of the second monolayer, they need to be used immediately.
While we have demonstrated the protocol for creating anti-CD3 nano-dots for use in T lymphocyte adhesion studies 9,13, the procedure is highly flexible and can be adapted for any biotinylated protein. The composition of the lipid bilayer can be easily changed and it can be further functionalized if desired. One important point to consider is the possible unspecific absorption of proteins, especially on the lipid bilayer surrounding the dots.
The main limitation of the technique arises from the use of colloidal-bead self-assembly for the primary mask. Being a bottom-up technique, it shares some of the problems of all such approaches for example, lack of flexibility and full control over the pattern shape. The pattern lattice necessarily reflects the symmetry of the bead mask and is therefore always hexagonal. The shape of the pattern motif is a circle and is determined by a combination of bead-size and the duration of aluminum deposition 9,13. Alternative techniques for controlling the dot size have also been suggested 14,17,18.
Substrates nano-patterns with proteins, protein-fragments or peptides have been extensively used in the past to probe cell-surface interactions, especially adhesion 19 and migration 20. Pioneering work has shown that tissue-forming cells fail to spread on patterns with a pitch greater than a given threshold 21, and further investigation showed that the length-scale of this phenomenon is set by the size of talins, which are instrumental in linking integrin receptors to the actin cytoskeleton 22. However, in all these studies, the proteins were linked to gold nano-particles, which were themselves immobilized on glass.
In the context of T cells, proteo-lipidic membranes, typically mimicking antigen presenting cells, have been extensively used to understand fundamental aspects of T cell function 6. Ingenious nano-patterning techniques have been used to create corrals with metal barriers separating protein functionalized SLB patches, which have provided insight into the structure and connectivity of the T cell/APC interface 23. This kind of nano-patterning is however very different from the protein nano-dots proposed here. Recently, nano- clusters were created using chemical linkage of the protein functionalized lipids 3, which shed light on the consequence of receptor clustering. The advantage was that, unlike the present case, the clusters could in principle be themselves mobile. However, such spontaneously formed clusters are necessarily less well controlled in terms of size and density than pre-formed protein nano-dots described here.
We envisage that the protein nano-dot decorated SLBs presented here can be used to investigate different aspects of cell-cell adhesion. One obvious question that arises is whether, as described above for tissue forming cells 19, lymphocytes too have an intrinsic length-scale associated with adhesion. Preliminary results seem to indicate that at least when the adhesion is mediated by the TCR complex, the density of ligands rather than spacing is the defining parameter for spreading and activation 10,11,12,13. Whether inclusion of mobile ligands in the surrounding SLB impacts this observation and how the mobile and immobile fractions work together is a possible question, which was partly addressed using self-assembled SLB bound clusters 24. Another interesting application will be in the context of mechano-sensing where cell adhesion/activation on mobile and immobilized ligands were shown to be different not only for T cells 7,25 but also for cells that habitually adhere to the extra cellular matrix 26.
The two main advantages of these proteo-lipidic patterns are the compatibility of the substrates with advanced optical microscopy and the ease of preparation which makes them compatible with use-and-throw applications. Subsequent to aluminum deposition, all the preparation steps can be carried out on a standard wet-lab bench. In the future, it can be envisaged that the aluminum-coated and glass-supported bead-masks produced in a specialized facility are transferred and stored in biology laboratories for use as and when required. With this in view, we believe that these substrates have the potential to become the platform of choice for studying the interaction of cells with controlled nano-patterned proteo-lipidic membranes.

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><tbody><tr><td>Glass coverslips</td><td>Assistent, Karl Hecht KG&amp;nbsp;</td><td></td><td></td></tr><tr><td>Observation chamber</td><td>Home made</td><td></td><td></td></tr><tr><td>Alkaline surfactant concentrate (Hellmanex)</td><td>Hella Analytics</td><td>9-307-011-4-507</td><td></td></tr><tr><td>Ultra-sonicator</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Desiccator</td><td>Labbox</td><td></td><td></td></tr><tr><td>Crystallizer&amp;nbsp;</td><td>Shott</td><td></td><td></td></tr><tr><td>Neutravidine</td><td>Thermo Fischer Scientifique</td><td>84607</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Sigma-aldrich</td><td>P3813</td><td></td></tr><tr><td>Water MQ&amp;nbsp;</td><td>ELGA, Veolia France</td><td></td><td></td></tr><tr><td>Silica beads</td><td>Corpuscular Inc</td><td>147114-10</td><td></td></tr><tr><td>APTES</td><td>Sigma-aldrich</td><td>A3648</td><td></td></tr><tr><td>BSA-Biotin</td><td>Sigma-aldrich</td><td>A8549</td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850375C</td><td></td></tr><tr><td>Dansyl-PE</td><td>Avanti Polar Lipids</td><td>810330C</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-aldrich</td><td>650471</td><td></td></tr><tr><td>Gastight syringe&amp;nbsp;</td><td>Dominique Dustcher , France</td><td>74453</td><td></td></tr><tr><td>Film balance</td><td>NIMA</td><td>Medium</td><td></td></tr><tr><td>Microscope</td><td>Zeiss, Germany</td><td>TIRF-III system</td><td></td></tr><tr><td>Aluminium Target&amp;nbsp;</td><td>Kurt J. Lesker Compagny, USA</td><td></td><td></td></tr><tr><td>Radio Frequency Magnetron sputtering Syst&amp;egrave;me&amp;nbsp;</td><td>modified SMC 600 tool by ALCATEL , France</td><td></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 in SLB imaging channel and the dots seen in the NAV imaging channel. Analysis of continuous photo bleaching data shows that the lipids in the patterned bilayer remain fluid and have a typical diffusion constant of 5 &#181;m&#178;/s (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/55060/55060fig1.jpg" /> 
Figure 1: Schematic representation of fabrication steps. (a) Primary bead mask. Inset shows a scanning electron microscopy (SEM) image of the mask, made with beads of 2 &#181;m diameter and covered with a thin layer of aluminum. Parameters used in this image are: nominal aluminum thickness 200 nm deposed at 400 W RF, initial pressure of 9 x 10-7 Torr, Argon flux 10 sccm, process pressure 6.2 mTorr, imaged at acceleration voltage of 5 kV. The images confirm observation made with optical microscopy (image not included) before aluminum deposition that the beads are arranged in a monolayer centered hexagonal lattice on the glass-slide. (b) Secondary mask of aluminum created by the sputter deposition after removal of the primary bead-mask. (c) Deposition of organosilane and BSA-biotin though the secondary mask. (d) Removal of aluminum revealing nano-dots of BSA-biotin. (e) Deposition of SLB. (f) Binding NaV to BSA-biotin. (g) Binding anti-CD3 to NaV. Inset shows epi-fluorescence image of the nano-dots arrays. Here the NaV is fluorescently labelled. Typical dot-size is 600&#177;100 nm and typical spacing is 1,900 &#177; 80 nm. <a href="http://cloudflare.jove.com/files/ftp_upload/55060/55060fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55060/55060fig2.jpg" /> 
Figure 2: Complementarity of the nano-dot and SLB pattern. (a,b) Epi-fluorescence images of fluorescent NaV nano-dots and of SLB with fluorescent tracer lipids. (c) Composite image of the NaV dots (red) in the sea of SLB (green) shows perfect complementarity of the NaV and SLB. Fast Fourier Transform (FFT) image in the inset indicates long-range order. Scale bar: 4 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55060/55060fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55060/55060fig3.jpg" /> 
Figure 3: Quantification of lipid diffusion in the SLB. (a) Epi-fluorescence image of a SLB before bleaching. The protein dots show up as dark holes in a bright sea of lipids. The field-diaphragm limits the illuminated area. (b) Epi-fluorescence of SLB after bleaching continuously for 50 s. The halo visible inside the region delimited by the field-diaphragm indicates that the lipids are mobile. Scale bar: 10 &#181;m. (c) Average intensity profile along the edge of the field-diaphragm (top) and the decay of intensity over time during the beaching process (bottom). This data is analyzed to extract the diffusion constant, which is typically 5 &#181;m&#178;/s.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.9K Views.  CNRS, UMR 7325, CINaM. 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. 

Video: Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biochemistry

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Chemistry

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

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 ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies.

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

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 ...

Light-Controlled Reversible Protein Patterning on Lipid Membranes

Dynamic Light-Induced Protein Patterns at Model Membranes

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

Author Spotlight: Photo Switchable Protein Recruitment for Reversible Patterning in Artificial Cellular Systems

Here, a protocol is described for generating light-regulated and reversible protein patterns with high spatiotemporal precision at artificial lipid membranes. The method consists of the localized photoactivation of the protein iLID (improved light-inducible dimer) immobilized on model membranes that, under blue light, binds to its partner protein Nano (wild-type SspB).

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Summary

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Chapters in this video

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

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

Dynamic Light-Induced Protein Patterns at Model Membranes