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

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

First, vortex the bottle containing silica microspheres for 15 seconds. Then, bath-sonicate for one minute, and vortex again for 15 seconds to break any clusters. Mix the beads with SLBB and 10 microliters of 5-millimolar SUVs in a low-adhesion microcentrifuge tube.
Incubate the bead-lipid mixture for one hour at room temperature, on a shaker to prevent sedimentation. In the meantime, thaw a PEGylated coverslip, and glue a cut 0.2-milliliter PCR tube to it. After incubation, centrifuge the beads for 30 seconds at the designated RCF.
After the first spin, remove 50 microliters of supernatant. Then, add 200 microliters of PRB and mix by vigorous pipetting. For the next rounds, remove 200 microliters of PRB, and add another 200 microliters after the second and third spin, and add 220 microliters of PRB after the fourth spin.
If doing a competition assay with multiple bead sizes, prepare the reaction by mixing equal volumes of each bead size, and diluting 29 microliters of this bead mixture with 721 microliters of reaction buffer. Then, add 75 microliters of diluted beads and 25 microliters of the protein diluted in SSB to the wells and mix.
If measuring septins at a steady state, incubate the mixture at room temperature for one hour, and then, image by either near-TIRF or confocal microscopy.

spherical-supported-lipid-bilayer-assay-technique-to-observe-septin

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

EoE Sub Articles

eoe sub articles

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>

Source: Curtis, B. N. et al., <a href="https://jove.unicartagenaproxy.elogim.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.

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.

58 조회수. Spherical Supported Lipid Bilayer Assay: 마이크로스피어에서 Septin 어셈블리를 관찰하는 기술

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

내레이션 대본

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