Name: Video: Test dwuwarstwowy lipidów ze sferycznym wsparciem: technika obserwacji składania się septyny na mikrosferach - Concept
Uploaded: 2023-04-30T15:01:39.000+00:00
Duration: 2 min 1 s
Description: 136 Views. Źródło: Curtis, B. N. et al., Rekonstrukcja zespołu septyny w błonach w celu zbadania właściwości i funkcji biofizycznych. J. Vis. Exp. (2022). Ten film opisuje technikę badania składania białek septyny na mikrosferach pokrytych dwuwarstwami lipidowymi. Technika ta pomaga w zrozumieniu zależnych od krzywizny zespołów białek na zakrzywionych powierzchniach.

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1. Spherical supported lipid bilayers
NOTE: This protocol uses silica microspheres suspended in ultrapure water at 10% density. For any work on the kinetic parameters of protein assembly, it is important to strictly control the total membrane surface area between experiments and curvatures.&#160;Table 1 shows the corrected volumes of beads and buffer to maintain 5 mm2&#160;of total membrane surface area.
<ol>
	<li>Bilayer formation 
	NOTE: As for planar bilayers, it is important to have high-quality SUVs for robust bilayer formation.&#160;Table 1&#160;lists the volume of beads from a 10% density solution and their respective SLBB volumes that are used to obtain a final surface area of 5 mm2&#160;for a range of bead diameters.
	<ol>
		<li>Silica microspheres (also referred to as beads) tend to settle and clump together; to mix the beads and break up any clusters, vortex the bottle for 15 s, then bath-sonicate for 1 min, and vortex again for 15 s.</li>
		<li>Mix the appropriate volume of beads (Table 1) with the corresponding volume of SLBB and 10 &#181;L of 5 mM SUVs in a 0.5 mL low-adhesion microcentrifuge tube.</li>
		<li>Rock the bead-lipid mixture end-over-end for 1 h at room temperature. Ensure that this incubation is done on a rotator to prevent sedimentation.</li>
	</ol>
	</li>
	<li>Prepare chambers on PEGylated coverslips 
	NOTE: As for planar bilayers, homemade chambers from plastic PCR tubes are used to support the reaction volumes, but other low-adhesion materials, such as silicone wells, may work just as well.
	<ol>
		<li>While the beads are rocking, thaw a PEGylated coverslip at room temperature. Cut a 0.2 mL PCR tube and glue it to the coverslip as described. For 24 mm x 50 mm slides, up to 10 chambers can feasibly fit on one slide. 
		NOTE: This protocol uses 24 mm x 50 mm PEGylated glass coverslips that are prepared in advance. Briefly, glass coverslips are thoroughly cleaned through sonication in acetone, methanol, and 3 M KOH. Coverslips are then functionalized with n-2-aminoethyl-3-aminopropyltrimethoxysilane and covalently linked to mPEG succinimidyl valerate. Finished PEGylated coverslips are stored vacuum-sealed at &#8722;80 &#176;C. While PEGylated glass coverslips are suggested here, other means of passivation, such as BSA or poly-L-lysine methods, may be effective.</li>
	</ol>
	</li>
	<li>Washing away excess lipids 
	NOTE: This step functions to remove excess SUVs and perform a buffer exchange. Beads are washed 4x in total with pre-reaction buffer (PRB: 33.3 mM KCl, 50 mM HEPES, pH 7.4).&#160;Table 2&#160;lists the spin speeds in RCF for a range of bead diameters.
	<ol>
		<li>Spin beads at the designated RCF for 30 s (Table 2). If using multiple bead sizes, keep beads rocking until it is their time to be washed. 
		NOTE: It is not worth sedimenting larger beads in the same spin as smaller beads with the smaller bead sedimentation velocity to save time. This can cause beads to stick to each other and compromise the integrity of the bilayer.</li>
		<li>After the first spin, remove 50 &#181;L of supernatant and add 200 &#181;L of PRB. After the second and third spins, remove 200 &#181;L and add 200 &#181;L of PRB. After the fourth spin, remove 200 &#181;L and add 220 &#181;L of PRB. Pipette vigorously for each wash. 
		NOTE: The final resuspension volume is different than the washes. Do not vortex the beads after incubation with the lipids as this can leave gaps in the bilayers. To avoid sedimentation while working with the other bead sizes or setting up other parts of the assay, place the bead mixtures back on the end-over-end rotator.</li>
	</ol>
	</li>
	<li>Preparing the reaction
	<ol>
		<li>If doing a competition assay with multiple bead sizes, make a 1:1 mixture by mixing equal volumes of each bead size together. Then, mix 29 &#181;L of the desired bead mixture (or of a single bead) with 721 &#181;L of RXN buffer. 
		NOTE: It is important to thoroughly mix the beads at each step with a pipette to avoid dense clusters of beads; this will result in a more even bead distribution and accurate total surface area.</li>
		<li>Mix 75 &#181;L of diluted beads and 25 &#181;L of protein diluted in SSB to the wells. 
		NOTE: The authors typically image yeast septin complexes at 1-50 nM.</li>
		<li>If measuring septins at a steady state, incubate at room temperature for 1 h, and then image by either near-TIRF or confocal microscopy. 
		NOTE: This reaction is designed to preserve the total membrane surface area of the reaction at 5 mm2&#160;and to produce a final reaction condition of 100 mM KCl, 50 mM HEPES, 1 mg/mL BSA, 0.1% methylcellulose, and 1 mM BME.</li>
	</ol>
	</li>
</ol>
Table 1: Normalized volumes of microspheres.&#160;In order to maintain an equal surface area of each bead size and to keep the total membrane surface area consistent between experiments, volumes for each bead size and buffer that normalized the total surface area were calculated.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bead Diameter (&#181;m)</td>
			<td>Volume of well-mixed beads (&#181;L)</td>
			<td>Volume of SLB buffer (&#181;L)</td>
			<td>Volume of SUVs (&#181;L)</td>
		</tr>
		<tr>
			<td>6.46</td>
			<td>8.94</td>
			<td>61.1</td>
			<td>10</td>
		</tr>
		<tr>
			<td>5.06</td>
			<td>7</td>
			<td>63</td>
			<td>10</td>
		</tr>
		<tr>
			<td>3.17</td>
			<td>4.39</td>
			<td>65.6</td>
			<td>10</td>
		</tr>
		<tr>
			<td>0.96</td>
			<td>1.33</td>
			<td>68.7</td>
			<td>10</td>
		</tr>
		<tr>
			<td>0.54</td>
			<td>0.75</td>
			<td>69.3</td>
			<td>10</td>
		</tr>
		<tr>
			<td>0.31</td>
			<td>0.43</td>
			<td>69.6</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 2: Sedimentation velocities for microspheres of varying diameters.&#160;For each bead diameter, the shown minimum sedimentation velocities were used to pellet the beads for washing away unbound liposomes
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bead diameter (&#181;m)</td>
			<td>Sedimentation velocity (RCF)</td>
		</tr>
		<tr>
			<td>0.31</td>
			<td>4.5</td>
		</tr>
		<tr>
			<td>0.54</td>
			<td>4.5</td>
		</tr>
		<tr>
			<td>0.96</td>
			<td>2.3</td>
		</tr>
		<tr>
			<td>3.17</td>
			<td>0.8</td>
		</tr>
		<tr>
			<td>5.06</td>
			<td>0.3</td>
		</tr>
	</tbody>
</table>

<table><tbody><tr><td>0.2 mL PCR Tubes with flat cap, Natural</td><td>Watson</td><td>&amp;nbsp;137-211C(EX)</td><td></td></tr><tr><td>0.5 mL low adhesion tubes</td><td>USA Scientific</td><td>1405-2600</td><td></td></tr><tr><td>Beta mercaptoethanol (BME)</td><td>Sigma-Aldrich</td><td>M6250-100ML</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4612-25G</td><td></td></tr><tr><td>Coverglass for making PEGylated coverslips</td><td>Thermo Scientific</td><td>152450</td><td>Richard-Allan Scientific SLIP-RITE Cover Glass 24x50 #1.5</td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850375</td><td></td></tr><tr><td>Egg Liss Rhodamine PE</td><td>Avanti Polar Lipids</td><td>810146</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375-1KG</td><td></td></tr><tr><td>Methyl cellulose 4000cp</td><td>Sigma-Aldrich</td><td>M052-100G</td><td></td></tr><tr><td>Mini centrifuge</td><td></td><td></td><td></td></tr><tr><td>Non-Functionalized Silica Microspheres</td><td>Bangs Laboratories, Inc.</td><td>Depends on size: SS0200*-SS0500*</td><td>Silica in aqueous suspension</td></tr><tr><td>Optical Adhesive</td><td>Norland Thorlabs</td><td>NOA 68</td><td>Flexible adhesive for glass or plastics</td></tr><tr><td>Potassium chloride</td><td>VWR</td><td>&amp;nbsp;0395-1kg</td><td></td></tr><tr><td>Sonicator bath</td><td>Branson</td><td>1510R-MT</td><td>Bransonic Ultrasonic cleaner. 50-60 Hz. Output: 70W</td></tr><tr><td>Soy PI</td><td>Avanti Polar Lipids</td><td>840044</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Eppendorf</td><td>22331</td><td></td></tr><tr><td>UV Lamp</td><td>Spectroline</td><td>ENF-260C</td><td>115 Volts, 60 Hz, 0.20 AMPS</td></tr></tbody></table>

spherical supported lipid bilayer assay: a technique to observe the septin assembly on microspheres

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).
This video describes the technique of studying the septin protein assembly on microspheres coated with lipid bilayers. This technique helps in understanding the curvature-dependent assemblies of protein on curved surfaces.

Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres

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

Septins are key cytoskeletal proteins that bind to curved plasma membrane surfaces.
To observe in vitro septin assembly on microspheres, first, take a suspension of uniformly sized, microscopic silica beads, acting as solid support for bilayer formation. Vortex this suspension to break any bead clusters.
Supplement it with small unilamellar vesicles, SUVs - spherical vesicles surrounded by a single lipid bilayer. Incubate with constant agitation, preventing microspheres from settling.
During incubation, the outer polar head groups facilitate the adsorption of SUVs onto the hydrophilic microsphere surface. This interaction causes SUVs to rupture, followed by the fusion of SUV lipids onto the curved microsphere surface as a continuous layer, resulting in a spherical, supported lipid bilayer, SLB, formation.
Wash to remove excess unbound lipids and resuspend in reaction buffer. Transfer the spherical SLB suspension onto a small region of a polyethylene glycol-coated surface, to prevent non-specific adsorptions. Add a fluorescently-labeled septin suspension to the chamber and incubate.
Septins form hetero-oligomers that recognize the positive curvature of spherical SLBs, polymerizing to form filaments. Eventually, septin filaments interact with each other, leading to the formation of higher-order structures.
Observe septin-coated SLBs under a high-resolution fluorescence microscope. The presence of fluorescence indicates septin&#8217;s curvature-sensitive deposition.

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Biochemical assays

136 Views. Źródło: Curtis, B. N. et al., Rekonstrukcja zespołu septyny w błonach w celu zbadania właściwości i funkcji biofizycznych. J. Vis. Exp. (2022). Ten film opisuje technikę badania składania białek septyny na mikrosferach pokrytych dwuwarstwami lipidowymi. Technika ta pomaga w zrozumieniu zależnych od krzywizny zespołów białek na zakrzywionych powierzchniach. 

Video: Test dwuwarstwowy lipidów ze sferycznym wsparciem: technika obserwacji składania się septyny na mikrosferach - 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

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

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Biochemistry

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 ...

Bioengineering

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 ...

Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres

Transkrypcja

Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres