Name: reconstitution of septin assembly at membranes to study biophysical properties and functions
Uploaded: 2022-07-28T14:45:00.000+00:00
Duration: 6 min 32 s
Description: Watch this Scientific Journal Video about Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions at JoVE.com

This work was supported by the National Institutes of Health (NIH) Grant no. R01 GM-130934 and National Science Foundation (NSF) Grant MCB- 2016022. B.N.C, E.J.D.V., and K.S.C. were supported in part by a grant from the National Institute of General Medical Sciences under award T32 GM119999.

NOTE: Forming supported lipid bilayers requires the preparation of monodispersed small unilamellar vesicles (SUVs). Please refer to a previously published protocol24 on SUV formation. Briefly, all SUVs are formed by probe sonication for 12 min in total at 70% amplitude via 4 min sonication periods followed by 2 min rest periods in ice-water. SUV solutions must be well clarified and monodispersed in size. Size distributions of SUVs can be measured, for example, by dynamic light scattering25.
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 2).</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 microscopy26. 
		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 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>
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 temperature and reused between experiments. Reaction buffer and SSB are made fresh for each experiment.
2. 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. Table 2 shows the corrected volumes of beads and buffer to maintain 5 mm2 of total membrane surface area. This protocol expands on a previously published method8,18.
<ol>
	<li>Bilayer formation 
	NOTE: As for planar bilayers, it is important to have high-quality SUVs for robust bilayer formation. Table 2 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 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 2) 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 in step 1.2. 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. For a similar passivation protocol, please see the work of Gidi et al.27. 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). Table 3 lists the spin speeds in RCF for a range of bead diameters.
	<ol>
		<li>Spin beads at the designated RCF for 30 s (Table 3). 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 
	NOTE: This reaction is designed to preserve the total membrane surface area of the reaction at 5 mm2 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.
	<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.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bead Diameter&#160;(&#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: Normalized volumes of microspheres. 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>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 3: Sedimentation velocities for microspheres of varying diameters. For each bead diameter, the shown minimum sedimentation velocities were used to pellet the beads for washing away unbound liposomes.
3. Rod supported lipid bilayers
NOTE: In contrast to the other assays presented here, the rod assay does not allow for careful control of the total membrane surface area. One can be consistent in amounts and volumes between experiments, but because this results in rods of different lengths and diameters, it is difficult to extrapolate the total membrane surface area in the reaction. Thus, while this is an excellent assay for exploring curvature sensing with multiple curvatures on a single surface and has been useful for exploring septin ultrastructure, it is not recommended for kinetic measurements. This method was previously reported18 and is being expanded upon here.
<ol>
	<li>Obtaining borosilicate rods from glass microfiber filters
	<ol>
		<li>Tear a single 42.5 mm grade GF/C glass microfiber filterinto small pieces and place them into a 100 mL beaker with 60 mL of 100% ethanol. Bath-sonicate until the solution becomes opaque; this can take as little as 20-30 min with a finely shredded filter. 
		NOTE: Sonicate thoroughly to break up big chunks; the more dissociated/opaque, the better.</li>
		<li>Cover the solution with film and store the solution at room temperature overnight.</li>
	</ol>
	</li>
	<li>Forming bilayers on the rods 
	NOTE: This step is performed with excess lipids to achieve complete rod coverage as it is impossible to quantify the total surface area in this method.
	<ol>
		<li>The next day the ethanol solution will be settled (step 3.1.2.), so mix it well before using. Combine 10 &#181;L of rod solution and 70 &#181;L of SLBB.</li>
		<li>Spin at top speed in a mini centrifuge for 30 s and remove 50 &#181;L of the supernatant. Resuspend in another 50 &#181;L of SLBB and repeat the spin for a total of four washes to dilute out the ethanol. 
		NOTE: Rod supports will not form a solid pellet at the bottom of the tube. They are instead smeared along the side of the tube; this makes them difficult to completely preserve when washing. Since the total surface area in this assay is not controlled, it is fine if they are disturbed.</li>
		<li>Combine 10 &#181;L of well-mixed rod solution with 50 &#181;L of 5 mM SUVs and 20 &#181;L of SLBB. Incubate for 1 h at room temperature, rotating end-over-end as when forming bilayers on the microspheres.</li>
	</ol>
	</li>
	<li>Washing away excess lipids
	<ol>
		<li>Similar to step 3.2.2., spin the lipid-rod solution at top speed in a mini centrifuge for 30 s to pellet the membrane-coated rods out of the solution.</li>
		<li>After the first spin, remove 50 &#181;L of the 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, add 220 &#181;L of PRB, and pipette vigorously to mix.</li>
	</ol>
	</li>
	<li>Preparing the reaction
	<ol>
		<li>In a 1.5 mL microcentrifuge tube, mix 721 &#181;L of reaction buffer with 29 &#181;L of rod solution. Mix well.</li>
		<li>In a 0.2 mL PCR tube, combine 75 &#181;L of rods in reaction buffer and 25 &#181;L of septins at the desired concentration, diluted in SSB. Allow it to incubate at room temperature for at least 1 h.</li>
	</ol>
	</li>
	<li>Preparing for scanning electron microscopy
	<ol>
		<li>After incubation, add the 100 &#181;L septin-rod mixture onto a round, PEG-coated 12 mm coverslip and incubate at room temperature for 1 h in a closed Petri dish.</li>
		<li>Fix the sample by filling the bottom of the Petri dish (10 mL should be enough) with 2.5% glutaraldehyde in 0.05 M sodium cacodylate (NaCo), pH 7.4, for 30 min. Aspirate the liquid with a glass pipette. Wash the sample by incubating it with 10 mL of 0.05 M NaCo for 5 min and repeat for a total of two washes.</li>
		<li>Postfix in 0.5% OsO4 cacodylate buffer for 30 min, and then wash 3x in 0.05 M NaCo (5 min/wash).</li>
		<li>Incubate the sample with 1% tannic acid for 15 min, and then wash in NaCo 3x. Incubate for 15 min in 0.5% OsO4 and wash 3x with NaCo.</li>
		<li>Dehydrate the samples using increasing ethanol concentrations: 30% EtOH for 5 min, 2x; 50% EtOH for 5 min; 75% EtOH for 5 min; and 100% EtOH for 5 min, 2x. Follow with another 10 min incubation.</li>
		<li>Incubate in transition fluid (hexamethyldisilazane) 3x (5 min, 10 min, then 5 min).</li>
		<li>Allow to air dry, then place the samples in a desiccator until sputter-coating. Sputter-coat the 4 nm layer with a gold/palladium alloy and then image on a scanning electron microscope.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+septin+cortex+at+the+yeast+mother-bud+neck.">The septin cortex at the yeast mother-bud neck.</a> Current Opinion in Microbiology. 4 (6), 681-689 (2001).</li><li>Ong, K., Wloka, C., Okada, S., Svitkina, T., Bi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+dynamic+remodelling+of+the+septin+cytoskeleton+during+the+cell+cycle.">Architecture and dynamic remodelling of the septin cytoskeleton during the cell cycle.</a> Nature Communications. 5, 1-10 (2014).</li><li>Bertin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4,5-bisphosphate+promotes+budding+yeast+septin+filament+assembly+and+organization.">Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin filament assembly and organization.</a> Journal of Molecular Biology. 404 (4), 711-731 (2010).</li><li>Bridges, A. 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The authors have no conflicts of interest.

Cell membranes take on many different shapes, curvatures, and physicochemical properties. In order to study the nanometer-scale machinery through which cells build micrometer-scale assemblies, it is necessary to design minimal reconstitution systems of membrane mimetics. This protocol presents techniques that precisely control both membrane curvature and composition while allowing the user to easily take quantitative fluorescence measurements using widely available microscopy techniques.
The most critical components of this protocol are assembling wells on the appropriate surface and handling the lipids. The wells described here are handmade using PCR tubes and UV-activated adhesives; it is important not to get glue inside of the reaction area during assembly. This can be checked for during the experiment by imaging along the edges of the well and looking for high protein adsorption onto the cover glass. The user may choose to use alternative materials for wells, such as silicone, which can be ordered commercially, but the presented method provides sturdy wells that will not shift or leak and can support larger reaction volumes. The surface of the cover glass, in our hands, has been very important for forming planar bilayers. Optimization of plasma cleaning times or even the brand of cover glass may be needed to see even formation of the bilayers, as appropriate surface treatment and charge are critical to vesicle adsorption and fusion31,32,33. When setting up these experiments for the first time or working with new lipid compositions, fluorescence recovery after photobleaching (FRAP) experiments should be used to assess the fluidity of the membrane compared to free-standing systems30.&#160;It is expected that support systems will be less fluid than their free-standing counterparts but not immobile12.
When assessing the bilayer quality by fluorescence, it is important to look for unburst liposomes or defects in the bilayer (Figure 3C), as these can alter local protein adsorption. The following troubleshooting options may be considered. a)&#160;One can adjust the SUV preparation to improve bilayer quality. Sonication, freeze-thaw, and extrusion are all commonly used methods that may vary in ease and success based on lipid compositions. In our hands, a fully clarified SUV solution with a monodispersed size distribution results in the most homogeneous bilayers. Dynamic light scattering (DLS) can be used to assess size distribution25.&#160;b) One can increase the difference in salt concentration between the inside of the SUVs and the external buffer. By reducing the internal ion concentration (or increasing the external one), osmotic stress on the SUVs increases the likelihood of rupture31. Alternatively, altering the monovalent cation present may change the kinetics of SLB formation and aid in optimizing SLBs of new lipid compositions34. c) One can increase the number or force of washes. In the authors&#39; experience, vigorous mixing during the washes leads to cleaner bilayers and does not appear to be disruptive; instead, the biggest issue comes from accidentally scraping the bilayer with pipette tips, which will appear as a gash in the membrane. For curved bilayers, it is best to minimize handling with narrow pipette tips and mix gently when performing the wash steps, especially for larger (&#62;1 &#181;m) beads, as the membrane may be sheared from the glass beads. If persistent gaps in the spherical membrane bilayers are observed (Figure 4C), increasing the width of the pipette tips (by cutting off the end of the tip or buying wide tips) and reducing handling as much as possible, while still completely mixing the bead solutions, can help. One common technical issue with this technique is the tendency of the beads to clump together (Figure 4D). This can be partially resolved by increasing the sonication time before bilayer formation; however, some clustering is unavoidable as membrane-coated beads may also tend to clump together over time in the well. Clumped beads should be avoided for quantitative analyses.
Limitations to quantitative measurements of single-molecule dynamics on planar bilayers include the need to stay at low concentrations in order to accurately track and count the particles. However, this can be rectified by under-labeling the protein of interest during acquisition to reduce the number of particles visible during tracking35. Additionally, spherical and rod SLBs were developed specifically to quantify the curvature sensitivity of proteins on micrometer-scale membranes, and the silica microspheres used here are only available down to 100 nm in diameter, at which point they are diffraction-limited puncta when viewed by light microscopy. Thus, those looking to assess precise curvature specificity on smaller diameters will be unable to do so using this method. However, this technique will still provide valuable information about the trend of curvature preference and allow for the dissection of curvature-dependent assemblies.
Compared to free-standing lipid bilayer systems, this technique is amenable to a wide range of buffer conditions and does not require careful osmotic balancing during preparation. Additionally, because it uses glass supports, the bilayers readily sink to the bottom of the well, removing the need for tethering or the use of sucrose or other thickening agents36. While free-standing lipid bilayer systems provide a useful means of investigating membrane deformation by membrane-binding proteins, it is difficult to study the relationship between membrane curvature and complex assembly. Unlike easily deformable membrane systems, this approach allows the user to use a variety of lipid compositions without the intrinsic shape of individual lipid species dictating the global geometry of the membrane. Lastly, rod-shaped SLBs allow sampling of multiple curvatures on the same continuous membrane for proteins of interest (Figure 5B), which is uniquely informative for evaluating curvature-dependent assemblies or the colocalization of multiple components.

The shapes of cells and many of their internal compartments are dependent on the lipid membranes that surround them. Membranes are viscoelastic structures that can be deformed through interactions with proteins, lipid sorting, and acting internal and external forces to generate a variety of shapes1,2,3,4. These shapes are often described in terms of membrane curvature. Cells use a diverse suite of proteins capable of preferentially assembling onto, or &#34;sensing&#34;, particular membrane curvatures to ensure defined spatio-temporal control over processes including cell trafficking, cytokinesis, and migration5,6. The dynamics of cell machinery at the membrane are notably difficult to observe due to the difficulty of balancing time and spatial resolution with cell health. While super-resolution techniques can offer a detailed view of such structures, they require lengthy acquisitions that are not amenable to the timescales of assembly/disassembly for most machinery. Additionally, the molecular complexity of these assemblies in their native environment and the multitude of roles a single component can play make minimal reconstitution systems a valuable tool for studying the functional capacity of molecules.
Minimal membrane mimetics have been developed to study membrane properties and protein-membrane interactions outside of the cell. Membrane mimetics vary from free-standing lipid bilayers, such as liposomes or giant unilamellar vesicles, to supported lipid bilayers (SLBs)7,8,9,10. SLBs are biomimetic membranes anchored to underlying support, typically composed of glass, mica, or silica11,12.&#160;A variety of geometries can be used, including planar surfaces, spheres, rods, and even undulating or micropatterned substrates to probe protein-membrane interactions on both concave and convex curvatures simultaneously13,14,15,16,17,18. Bilayer formation begins with vesicle adsorption onto a hydrophilic surface, followed by fusion and rupture to form a continuous bilayer (Figure 1)19. Supported bilayers are particularly amenable to light and electron microscopy, providing both better time and spatial resolution than is often achievable in cells. Curved SLBs especially provide an attractive means to probe protein curvature sensitivity in the absence of significant membrane deformation, allowing one to distinguish between curvature sensing and curvature induction, which are often impossible to separate in free-standing systems.
Septins are a class of filament-forming cytoskeletal proteins well known for their ability to assemble on positively curved membranes6,18,20. Over the course of the cell cycle in yeast, septins assemble into a ring and must rearrange to form the hourglass and double ring structures associated with bud emergence and cytokinesis, respectively21. While beautiful work has been done using platinum replica electron microscopy to observe septin architecture at varying cell cycle stages22, watching septin assembly over time using light microscopy in yeast has met with limited spatial resolution. Previous work on septins using lipid monolayers visualized by transmission electron microscopy (TEM) was able to reconstitute several interesting septin structures such as rings, bundles, and gauzes23. However, EM techniques are likewise limited in their temporal resolution, unlike fluorescence microscopy. In order to better resolve the kinetic parameters of the multi-scale process of septin assembly, we turned to supported membrane mimetics, where one can carefully control membrane geometry, sample conditions, and imaging modality.
The protocols described here use planar or curved SLBs, purified protein, and a combination of microscopy techniques. Quantitative fluorescence confocal microscopy and total internal reflection fluorescence microscopy (TIRFM) were used to measure both bulk protein binding onto various membrane curvatures, as well as to measure the binding kinetics of single molecules. Furthermore, this protocol has been adapted to be used with scanning electron microscopy (SEM) to examine protein ultrastructure on different membrane curvatures. While the focus of these protocols is on the septin cytoskeleton, the protocols can be easily modified to investigate the curvature sensitivity of any protein the reader finds interesting. Additionally, those working in fields such as endocytosis or vesicular trafficking may find these techniques useful for probing the curvature-dependent assemblies of multi-protein complexes.

<table><tbody><tr><td>0.2 mL PCR Tubes with flat cap, Natural</td><td>Watson</td><td>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>EMS Glutaraldehyde Aqueous 25%, EM Grade</td><td>VWR</td><td>16220</td><td></td></tr><tr><td>EMS Sodium Cacodylate Buffer</td><td>VWR</td><td>11652</td><td></td></tr><tr><td>Ethanol, 200 proof</td><td>Fisher Scientific</td><td>04-355-223EA</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375-1KG</td><td></td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma-Aldrich</td><td>440191</td><td></td></tr><tr><td>Magnesium chloride</td><td>VWR</td><td>7791-18-6</td><td></td></tr><tr><td>Methyl cellulose 4000cp</td><td>Sigma-Aldrich</td><td>M052-100G</td><td></td></tr><tr><td>Microglass coverslips for planar bilayers</td><td>Matsunami</td><td>Discontinued</td><td>22x22</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>Osmium tetroxide</td><td>Millipore Sigma</td><td>20816-12-0</td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>Plasma Cleaner</td><td>Plasma Etch</td><td>PE-25</td><td>Voltage: 120V, 60Hz. Current: 15 AMPS</td></tr><tr><td>Potassium chloride</td><td>VWR</td><td>0395-1kg</td><td></td></tr><tr><td>Round coverglass, #1.5 12mm&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>64-0712</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><tr><td>WhatmanGlass Microfiber Filter Paper</td><td>VWR</td><td>28455-030</td><td>42.5 mm diameter, Grade GF/C</td></tr></tbody></table>

reconstitution of septin assembly at membranes to study biophysical properties and functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Following the preparation of each SLB, septins or the protein of interest may be incubated with the desired support and imaged via TIRFM, confocal microscopy, or SEM. The results shown here use septins recombinantly expressed and purified from E. coli17. Using TIRFM on planar SLBs, it is possible to determine the length of filaments and their flexibility, measure the diffusion coefficients and observe assembly over time28,29. In order to collect the highest quality measurements, it is first necessary to ascertain the quality of bilayers, especially when preparing them for the first time or when changing lipid compositions. A visual inspection of the protein distribution on the bilayers by TIRFM can help identify regions of the bilayer that have been scratched or are malformed. Protein distribution should be homogeneous (Figure 3A), and there should not be holes or gaps in the bilayer (Figure 3B).&#160;It is best to avoid membranes with holes, which can form from dust contamination or smudges from slide handling, as this can change the protein distribution in other areas of the bilayer. To visualize the membrane itself, trace rhodamine-PE can be incorporated into SUVs for bilayer formation. In high-quality bilayers, the field will appear even (images not shown), but if liposomes are old or if washes are not stringent enough, unburst and tubulated liposomes may accumulate on the surface (Figure 3C). Additionally, while solid supports will hamper the free diffusion of lipids8, the lipids should not be immobile. FRAP experiments can be used to assess the mobility of lipids on planar bilayers, which may vary by composition and should show recovery rates in the order of seconds30.
Spherical supports can be used to examine protein binding on membranes of defined curvatures either in isolation (one curvature) or with several membrane curvatures in the same well to observe competition between curvatures6,18. Near-TIRFM on beads &#62;1 &#181;m can also be used to measure the number of association events for a given area27. As with planar bilayers, we use rhodamine-PE to look for smooth bilayer deposition (Figure 4A), i.e., no lipid clumps, which indicate multi-lamellarity or unburst liposomes (Figure 4B), and no gaps in the bilayer (Figure 4C). For measuring the total protein adsorption or the protein adsorption over time on curved surfaces, it is necessary to isolate individual beads from each other in order to create a discrete volume for which sum lipid intensity and sum septin intensity can be measured. Thus, the beads should be well-separated rather than clumped together as in Figure 4D. We address troubleshooting options for all of these potential issues in the discussion section.
This SLB assay can also be applied to rod supports, which offer an environment for proteins to sample multiple curvatures on a single surface. Pairing this assay with scanning electron microscopy allows the user to examine curvature preference, alignment, and length distribution of septins18 or other proteins of interest. While similar to the other assays presented here, the rod assay does not allow for careful control of total membrane surface area because of the heterogeneous nature of the substrate that is derived from filter paper. The material properties of the filter paper used here result in rods of different lengths and diameters (Figure 5A); this is useful for exploring protein curvature sensing and organization on curved surfaces (Figure 5B), but because the ratio of protein to the membrane cannot be controlled, the rods are of limited utility for generating saturation-binding curves or measuring parameters such as binding constants.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64090/64090fig01.jpg" /> 
Figure 1: Overview of supported lipid bilayer formation on supports with various curvatures. SUVs are incubated with solid supports of different geometries in order to change the curvatures available for sampling on a given membrane. SUVs adsorb onto the solid support surface and rupture to create lipid bilayers. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64090/64090fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64090/64090fig02.jpg" /> 
Figure 2: 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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64090/64090fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64090/64090fig03.jpg" /> 
Figure 3: Representative TIRF micrographs of planar bilayers. (A) A representative image of a high-quality lipid bilayer (75% DOPC, 25% Soy PI)with non-polymerizable septins bound (green). Protein is not clustering in any specific regions, and there are no liposomes attached to the membrane and no holes. (B) This bilayer was made with poorly cleaned glass coverslips and exhibits what appear to be &#34;holes&#34; (white arrows) in the membrane where there are fewer septins. Septins can be seen crowded at the edges of these defects in the bilayer (denoted by the white arrows in the zoomed region on the right). (C) A representative image of a low-quality bilayer using trace rhodamine-PE. Unburst liposomes and tubulated lipids are visible on the surface. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64090/64090fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64090/64090fig04.jpg" /> 
Figure 4: Representative micrographs of lipid-coated microspheres. Representative images from competition assays where lipid-coated beads (75% DOPC, 25% Soy PI, 0.1% Rhodamine PE)with diameters of 0.3 &#181;m, 0.5 &#181;m, 1 &#181;m, 3 &#181;m, and 5 &#181;m were mixed. All images are maximum Z projections. (A) Representative image of a high-quality mixture of lipid-coated microspheres. The spherical supports are evenly coated by the membrane, and there are few bead clusters. (B)&#160;Representative image of uneven membrane coating, likely caused by insufficient washing of excess lipids. (C) Representative image of beads with gaps in membrane coverage (white arrows) due to improper handling. (D) Representative image of densely clustered beads, likely caused by insufficient mixing throughout the procedure, especially when combining the different beads together. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64090/64090fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64090/64090fig05.jpg" /> 
Figure 5: Representative electron micrographs of lipid and septin-coated rods. (A)&#160;SEM image showing the distribution of lipid-coated (75% DOPC, 25% Soy PI, 0.1% Rhodamine PE) rod lengths and diameters. (B)&#160;The edge of an isolated membrane-coated rod with septin filaments aligned along the axis of positive curvature. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64090/64090fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions at JoVE.com

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

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Biochemistry

2.2K Views.  University of North Carolina at Chapel Hill. Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Video: Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

In vitro reconstitution of cytoskeletal proteins is a vital tool to understand the basic functional properties of these proteins. The present paper describes a protocol to purify and assess the quality of recombinant septin complexes, which play a central role in cell division and migration.

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Biology

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.

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic ...

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.bdigitaluss.remotexs.co/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.

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

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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.bdigitaluss.remotexs.co/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

Septins are protein filaments forming the cytoskeleton along with the microtubules, microfilaments, intermediate filaments, and other accessory proteins. In 1971 while studying the cell division cycle in mutant Saccharomyces cerevisiae Harwell et al. first identified the septin-related genes playing a crucial role in yeast cytokinesis. Fluorescence microscopy revealed that these proteins localize at the budding neck as rings. These ring-like proteins were then named Septins by John Pringle, and the characterization of these highly conserved cytoskeletal proteins began in the 1980s.
Structure and formation of Septins
Septin monomers can be broadly divided into a variable N-terminal domain, a GTP hydrolyzing domain, and a coiled-coil C-terminal domain. The GTP-binding domain consists of three GTP binding motifs, first G1 or P-loop or Walker A box; second the G3 Switch II and the third G4 for selective binding of GTP. The GTP binding domain ends in a septin unique region (SUR), which comprises 50 amino acid residues and is responsible for helping in septin filament formation.
Classification of Septins
The type and number of septins vary among different organisms. For example, S. cerevisiae has seven, while humans have thirteen. Based on protein sequence similarity, septins are classified into four groups: SEPT2, SEPT3, SEPT6, and SEPT7. In humans, group SEPT2 comprises SEPT1, SEPT2, SEPT4, and SEPT5; group SEPT3 has SEPT3, SEPT9, and SEPT 12; group SEPT6 has SEPT6, SEPT8, SEPT10, and SEPT11; and lastly, group SEPT7 only has SEPT7.

Septins are protein filaments forming the cytoskeleton along with the microtubules, microfilaments, intermediate filaments, and other accessory proteins. ...

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Unit 1

The Cytoskeleton II: Microtubules and Intermediate Filaments

KEY TERMS AND CONCEPTS

Role of Septins

Septins are the recently discovered fourth major protein component of the cytoskeleton, along with microfilaments, microtubules, and intermediate filaments. These proteins can associate with other cytoskeletal filaments and carry out varied roles or can be free-floating in the cytoplasm.
Cellular Functions of Septins 
Recent studies have revealed the multifaceted roles of septins in various cellular processes such as cytokinesis, ciliogenesis, and neurogenesis. Septins act as scaffolds and promote protein-protein interactions; for example, in S. cerevisiae, septin scaffolding protein helps recruit different filament proteins to form contractile rings during cytokinesis.
Septin was first found in the sperm flagella&#39;s annulus, which compartmentalizes the anterior and posterior tail regions. Mutation in the septin-forming genes like SEPT12 affects sperm motility and its structural integrity. Other septins like SEPT2 are involved in forming a diffusion barrier between the cilia and the cytoplasm.
Septins also play a role in stabilizing the membrane by binding and modifying the membrane and action-myosin interactions.
Diseases Related to Septins
Studies have shown septins are associated with proteins involved in neurodegenerative diseases like Parkinson&#39;s and Alzheimer&#39;s. In Parkinson&#39;s disease, septin interacts with parkin, an E3 ubiquitin ligase. Septins&#39;&#160;mutation has also been linked with tumorigenesis or cancer. Studies on cancerous cells suggest septin mutations are also related to resistance to cells, metastasis, proliferation, and angiogenesis.

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

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Chapters in this video

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

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

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

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

Septins

Role of Septins