Name: reconstitution of septin assembly at membranes to study biophysical properties and functions
Uploaded: 2022-07-28T14:45:00
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 scattering<sup class...

<ol>
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L., 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=Structural+basis+for+the+geometry-driven+localization+of+a+small+protein.">Structural basis for the geometry-driven localization of a small protein.</a> Proceedings of the National Academy of Sciences. 112 (15), 1908-1915 (2015).</li><li>Pan, J., Dalzini, A., Song, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+and+phosphatidylethanolamine+lipids+exert+opposite+effects+on+membrane+modulations+caused+by+the+M2+amphipathic+helix.">Cholesterol and phosphatidylethanolamine lipids exert opposite effects on membrane modulations caused by the M2 amphipathic helix.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1861 (1), 201-209 (2019).</li><li>Beckers, D., Urbancic, D., Sezgin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+nanoscale+hindrances+on+the+relationship+between+lipid+packing+and+diffusion+in+model+membranes.">Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes.</a> The Journal of Physical Chemistry B. 124 (8), 1487-1494 (2020).</li><li>Lee, A. 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Y., 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=Membrane+curvature+underlies+actin+reorganization+in+response+to+nanoscale+surface+topography.">Membrane curvature underlies actin reorganization in response to nanoscale surface topography.</a> Proceedings of the National Academy of Sciences. 116 (46), 23143-23151 (2019).</li><li>Bridges, A. 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=Septin+assemblies+form+by+diffusion-driven+annealing+on+membranes.">Septin assemblies form by diffusion-driven annealing on membranes.</a> Proceedings of the National Academy of Sciences. 111 (6), 2146-2151 (2014).</li><li>Cannon, K. S., Woods, B. L., Crutchley, J. M., Gladfelter, A. 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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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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 m...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;&#160;</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 o...

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 ...

Research

JoVE Journal

Biochemistry

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