eoe sub articles

EoE Sub Articles

1. Planar Lipid Bilayers
<ol>
	<li>Plasma Cleaning of the Slides 
	NOTE: Plasma cleaning removes organic contaminants and increases the hydrophilicity of the glass, ensuring efficient lipid adsorption. The following steps may change or need to be optimized depending on the plasma cleaner and glass coverslips used.
	<ol>
		<li>Purge the plasma cleaner for 5 min with oxygen to remove air from the lines and chamber. This is to ensure that, when the plasma cleaner is run, there is primarily oxygen in the lines and chamber, providing more consistent bilayer preparations.</li>
		<li>While purging, stream an inert gas, such as N2 or Ar, over the micro cover glass slides to remove dust and particulates. 
		OPTIONAL: Cover glass slides can be washed by spraying each side with 100% isopropanol followed by water. Repeat the isopropanol-water wash 3x and then dry with an N2 stream.</li>
		<li>Arrange dry cover glass slides into a ceramic cradle. Place the cradle at the back of the plasma chamber so that the coverslips are parallel with the long edge of the chamber (this keeps them in place during plasma cleaning). Run the plasma cleaner for 15 min with oxygen at maximum power.</li>
	</ol>
	</li>
	<li>Chamber Preparation 
	NOTE: The method described here uses homemade chambers from plastic PCR tubes to support the reaction volumes, but other low-adhesion materials, such as silicone wells, may also be suitable.
	<ol>
		<li>Using a razor blade or scissors, cut off the cap just below the frosted part of a 0.2 mL PCR tube (Figure 1).</li>
		<li>Paint the rim of the PCR tube with UV-activated adhesive, avoiding the inside of the tube. Gently place the PCR tube glue-down in the center of a plasma-cleaned coverslip. 
		NOTE: To avoid glue inside the chamber, use the minimum amount of glue to get a seal, and promptly treat with UV without bumping or disturbing the chamber.</li>
		<li>Place the chamber under long-wavelength UV light for 5-7 min to cure the adhesive.</li>
	</ol>
	</li>
	<li>Bilayer Formation 
	NOTE: Monodispersed SUVs should be used in this step for efficient bilayer formation. Table 1 provides recipes for all buffers used in the bilayer formation, including stock and final buffer concentrations.
	<ol>
		<li>Add 40 &#181;L of supported lipid bilayer buffer (SLBB: 300 mM KCl, 20 mM HEPES, pH 7.4, 1 mM MgCl2; Table 1), 10 &#181;L of 5 mM SUVs, and 1 &#181;L of 100 mM CaCl2 to the well. Gently shake the chambers from side to side to disrupt the SUVs, and then incubate at 37 &#176;C for 20 min. 
		NOTE: To limit evaporation, incubate in a covered Petri dish with a wet wipe.</li>
	</ol>
	</li>
	<li>Washing away excess lipids 
	NOTE: This step removes excess SUVs and acts as a buffer exchange step. It is very important not to scrape the bilayer with the pipette tip while washing; if the glass is exposed, septins and many other proteins will bind irreversibly, reducing the effective protein concentration.
	<ol>
		<li>After incubation, rinse the bilayer 6x with 150 &#181;L of SLBB, pipetting up and down to mix each time while avoiding bubbles. 
		NOTE: Add 150 &#181;L directly to the 50 &#181;L of buffer and SUVs already present for a total of 200 &#181;L in the reaction well.</li>
		<li>When ready to start incubating with the septins or a different protein of choice, wash the bilayer 6x with 150 &#181;L of reaction buffer (RXN: 33.3 mM KCl, 50 mM HEPES, pH 7.4, 1.4 mg/mL BSA, 0.14% methylcellulose, 1 mM BME). 
		NOTE: For best results, make the reaction buffer fresh and be very careful while mixing in the reaction well to avoid bubbles.</li>
		<li>On the last wash step, remove 125 &#181;L of reaction buffer, leaving 75 &#181;L in the well. Add 25 &#181;L of septins diluted in septin storage buffer (SSB: 300 mM KCl, 50 mM HEPES, pH 7.4, 1 mM BME) at the desired concentration and image by TIRF microscopy. 
		NOTE: When setting up this assay for the first time or incorporating a new lipid composition, it is good practice to ensure lipids are freely diffusing on the surface using fluorescence recovery after photobleaching (FRAP). While the rate of recovery will be different for different lipid compositions and inherently lower than for free-standing systems, the lipids should not be immobile.</li>
	</ol>
	</li>
</ol>
Table 1: Buffer components for preparation of supported lipid bilayer and reactions. Volumes of stock solutions that are incorporated into buffers and the final concentrations of each component are shown. SLB and PRB can be stored at room&#160;temperature and reused between experiments. Reaction buffer and SSB are made fresh for each experiment.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Supported Lipid Bilayer Buffer (SLBB)</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Volume</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>1.5 mL</td>
			<td>300 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>200 &#181;L</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>500 mM MgCl2</td>
			<td>20 &#181;L</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>8 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Pre-Reaction Buffer (PRB)</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Volume</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>166 &#181;L</td>
			<td>33.3 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>500 &#181;L</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>9.33 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Reaction Buffer</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Volume</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>166 &#181;L</td>
			<td>33.3 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>300 &#181;L</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>10 mg/mL BSA</td>
			<td>1.39 mL</td>
			<td>1.39 mg/mL</td>
		</tr>
		<tr>
			<td>1% Methylcellulose</td>
			<td>1.39 mL</td>
			<td>0.0014</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Up to 10 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>BME</td>
			<td>0.7 &#181;L</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td colspan="3">Septin Storage Buffer (SSB)</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Volume</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>1.5 mL</td>
			<td>300 mM</td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>500 &#181;L</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Up to 10 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>BME</td>
			<td>0.7 &#181;L</td>
			<td>1 mM</td>
		</tr>
	</tbody>
</table>

planar supported lipid bilayer assay: a microscopy-based in vitro technique to investigate septin assembly on biological membrane mimics

Source: Curtis, B. N. et al. <a href="https://www.jove.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

<img alt="Figure 1" src="/files/ftp_upload/21163/21163_Figure1.jpg" /> 
Figure 1: Schematic of reaction chamber set up. To prepare custom chambers, a 0.2 mL PCR tube is cut where the tube begins to taper and at the cap (red dashed lines). The uncut rim of the cut tube is then coated with a thin layer of UV-activated glue (blue) and placed glue-down on a coverslip.

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...

Septins are cytoskeletal proteins comprising a GTP-binding domain flanked by terminal amino and carboxyl domains, with an inherent ability to self-assemble into filaments and higher-order structures at the inner plasma membrane for diverse cellular functions.
To study septin-membrane interactions in vitro using a biological membrane mimic, obtain a cut microcentrifuge tube. Apply ultraviolet adhesive over the uncut, flat rim and position it onto a plasma-treated, hydrophilized glass coverslip. Upon ultraviolet light exposure, the adhesive cures, creating an open reaction chamber.
Transfer monodispersed zwitterionic unilamellar lipid vesicles of desired lipid composition, supplemented with mono- and divalent cation-containing buffer, into the chamber. Cations facilitate lipid vesicles to adsorb and accumulate onto the coverslip. Gently shake the chamber, initiating vesicle rupture, and incubate.
The ruptured vesicles unfold and fuse to the coverslip via their hydrophilic head groups, forming planar lipid bilayer patches. The patches&#39; edges trigger remaining vesicles to rupture, generating a continuous planar-supported lipid bilayer, SLB. Remove excess unadsorbed vesicles with suitable buffer.
Next, add fluorescently-labeled septin suspension with crowding agent-containing buffer and incubate. Crowding agents enable septins to deposit onto the SLB-coated coverslip. Under optimal conditions, septins may associate with the lipids in the SLB, assembling into organized structures.
Selectively illuminate the coverslip using total internal reflection fluorescence microscopy to excite adhered fluorescently-labeled septins on the SLB to observe septin assembly.

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148 Views. Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022) In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy. 

Video: Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics - Concept

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

JoVE Journal

Biochemistry

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 ...

Bioengineering

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Septins are cytoskeletal proteins. They interact with lipid membranes and can sense but also generate membrane curvature at the micron scale. We describe in this protocol bottom-up in vitro methodologies for analyzing membrane deformations, curvature-sensitive septin binding, and septin filament ultrastructure.

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

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