Name: bottom-up in vitro methods to assay the ultrastructural organization, membrane reshaping, and curvature sensitivity behavior of septins
Uploaded: 2022-08-17T13:10:00
Description: Watch this Scientific Journal Video about Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins at JoVE.com

We thank Patricia Bassereau and Daniel L&#233;vy for their useful advice and discussions. This work benefited from the support of the ANR (Agence Nationale de la Recherche) for funding the project &#34;SEPTIME&#34;, ANR-13-JSV8-0002-01, ANR SEPTIMORF ANR-17-CE13-0014, and the project &#34;SEPTSCORT&#34;, ANR-20-CE11-0014-01. B. Chauvin is funded by the Ecole Doctorale &#34;ED564: Physique en Ile de France&#34; and Fondation pour lea Recherche M&#233;dicale. K. Nakazawa was supported by Sorbonne Universit&#233; (AAP Emergence). G.H. Koenderink was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO/OCW) through the &#8216;BaSyC-Building a Synthetic Cell&#39;. Gravitation grant (024.003.019).We thank the Labex Cell(n)Scale (ANR-11-LABX0038) and Paris Sciences et Lettres (ANR-10-IDEX-0001-02). We thank the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04).

1. Determination of membrane reshaping using giant unilamellar vesicles (GUVs)
NOTE: In this section, GUVs are generated to mimic the membrane deformations possibly induced by septins in a cellular context. Indeed, in cells, septins are frequently found at sites with micrometer curvatures. GUVs have sizes ranging from a few to tens of micrometers and can be deformed. They are thus appropriate to assay any micrometer-scale septin-induced deformations. Fluorescent lipids, as well ...

<ol>
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T., Nakos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+functions+of+actin-+and+microtubule-associated+septins.">Cellular functions of actin- and microtubule-associated septins.</a> Current Biology: CB. 31 (10), 651-666 (2021).</li><li>Salameh, J., Cantaloube, I., Benoit, B., Po&#252;s, C., Baillet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42+and+its+BORG2+and+BORG3+effectors+control+the+subcellular+localization+of+septins+between+actin+stress+fibers+and+microtubules.">Cdc42 and its BORG2 and BORG3 effectors control the subcellular localization of septins between actin stress fibers and microtubules.</a> Current Biology: CB. 31 (18), 4088-4103 (2021).</li><li>Ewers, H., Tada, T., Petersen, J. 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As stated above, a lipid mixture has been used that enhances PI(4,5)P2 incorporation within the lipid bilayer and thus facilitates septin-membrane interactions. Indeed, we have shown elsewhere25 that budding yeast septins interact with vesicles in a PI(4,5)P2-specific fashion. This lipid composition was adjusted empirically from screening multiple compositions and is now widely used by the authors. PI(4,5)P2 lipids have to be handled carefully. Stock solutions must...

Septins are cytoskeletal filament-forming proteins that interact with lipid membranes. Septins are ubiquitous in eukaryotes and essential to numerous cellular functions. They have been identified as the main regulators of cell division in budding yeast and mammals1,2. They are involved in membrane reshaping events, ciliogenesis3, and spermiogenesis4. Within mammalian cells, septins can also interact with actin and microtubules5,6,7 in a binder of Rho GTPases (BORG)-dependent manner8. In various tissues (neurons9, cilia3, spermatozoa10), septins have been identified as regulators of diffusion barriers for membrane-bound components11. Septins have also been shown to regulate membrane blebbing and protrusion formation12. Septins, being multi-tasking proteins, are implicated in the emergence of various prevalent diseases13. Their misregulation is associated with the emergence of cancers14 and neurodegenerative diseases15.
Depending on the organism, several septin subunits (two in Caenorhabditis elegans to 13 in humans) assemble to form complexes whose organization varies in a tissue-dependent fashion16. The basic septin building block gathers two to four subunits, present in two copies and self-assembled in a rod-like palindromic manner. In budding yeast, septins are octameric17,18. In situ, septins are often localized at sites with micrometer curvature; they are found at division constriction sites, at the base of cilia and dendrites, and at the annulus of spermatozoa19,20. At the membrane, the role of septins seems to be dual: they are implicated in reshaping the lipid bilayer and in maintaining membrane integrity21. Hence, investigating the biophysical properties of septin filament-forming proteins and/or subunits at the membrane is crucial for understanding their role. To dissect specific properties of septins in a well-controlled environment, bottom-up in vitro approaches are appropriate. So far, only a few groups have described the biophysical properties of septins in vitro20,22,23. Hence, as compared with other cytoskeletal filaments, the current knowledge on the behavior of septins in vitro remains limited.
This protocol describes how the organization of septin filaments, membrane reshaping, and curvature sensitivity can be analyzed19. To this end, a combination of optical and electron microscopy methods (fluorescence microscopy, cryo-electron microscopy [cryo-EM], and scanning electron microscopy [SEM]) has been used. The membrane reshaping of micrometer-sized giant unilamellar vesicles (GUVs) is visualized using fluorescence optical microscopy. The analysis of the arrangement and ultrastructure of septin filaments bound to lipid vesicles is performed using cryo-EM. Analysis of septin curvature sensitivity is carried out using SEM, by studying the behavior of septin filaments bound to solid-supported lipid bilayers deposited on wavy substrates of variable curvatures, which enables the analysis of curvature sensitivity for both positive and negative curvatures. As compared with previous analysis20,24, here, we propose to use a combination of methods to thoroughly analyze how septins can self-assemble, synergistically deform membrane, and be curvature-sensitive. This protocol is believed to be useful and adaptable to any filamentous protein that displays an affinity for membranes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850725</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine</td>
			<td>Avanti Polar Lipids</td>
			<td>840035</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath sonicator</td>
			<td>Elma</td>
			<td></td>
			<td>Elmasonic S10H</td>
		</tr>
		<tr>
			<td>Bodipy-TR-Ceramide</td>
			<td>invitrogen, Thermo Fischer scientific</td>
			<td>11504726</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals: NaCl, Tris-HCl, sucrose, KCl, MgCl2, B-casein, chloroform, sodium cacodylate, tannic acid, ethanol</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>nikon</td>
			<td></td>
			<td>spinning disk or confocal</td>
		</tr>
		<tr>
			<td>Critical point dryer</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>CPD300</td>
		</tr>
		<tr>
			<td>Deionized water generator</td>
			<td>MilliQ</td>
			<td>F1CA38083B</td>
			<td>MilliQ integral 3</td>
		</tr>
		<tr>
			<td>Egg L-&#945;-phosphatidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>840051</td>
			<td></td>
		</tr>
		<tr>
			<td>Field Emission Gun SEM (FESEM)</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Gemini SEM500</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25 %, aqueous solution</td>
			<td>Thermo Fischer scientific</td>
			<td>50-262-19</td>
			<td></td>
		</tr>
		<tr>
			<td>High vacuum grease, Dow corning</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMOD software</td>
			<td>https://bio3d.colorado.edu/imod/</td>
			<td></td>
			<td>software suite for tilted series image alignment and 3D reconstruction</td>
		</tr>
		<tr>
			<td>Lacey Formvar/carbon electron microscopy grids</td>
			<td>Eloise</td>
			<td>01883-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipids</td>
			<td>Avanti Polar Lipids</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-&#945;-phosphatidylinositol-4,5-bisphosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>840046</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal evaporator</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>EM ACE600</td>
		</tr>
		<tr>
			<td>NOA (Norland Optical Adhesives), NOA 71 and NOA 81</td>
			<td>Norland Products</td>
			<td>NOA71, NOA81</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetraoxyde 4%</td>
			<td>delta microscopies</td>
			<td>19170</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>L&#246;ser</td>
			<td></td>
			<td>15 M</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Alcatel</td>
			<td></td>
			<td>pascal 2005 SD</td>
		</tr>
		<tr>
			<td>Plasma generator</td>
			<td>Electron Microscopy Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plunge freezing equipment</td>
			<td>leica microsystems</td>
			<td></td>
			<td>EMGP</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Thermofischer</td>
			<td></td>
			<td>Tecnai G2 200 kV, LaB6</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Science</td>
			<td>22451</td>
			<td>this product is not available for purchase any longer</td>
		</tr>
		<tr>
			<td>Wax plates, Vitrex</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

GUVs deformations 
Typical confocal fluorescence images of GUVs reshaped after being incubated with septins are displayed in Figure 3, in conditions where septins polymerize. Bare GUVs (Figure 3A) were perfectly spherical. Upon incubation with more than 50 nM budding yeast septin filaments, the vesicles appeared deformed. Up to a concentration of 100 nM budding yeast septin octamers, the vesicles appeared facetted, and the deformations rema...

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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.

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 ...

Using this protocol, we visualized the organization of cytoskeletal filamentous proteins on pattern substrates displaying various curvatures. We can test the curvature sensitivity. The resolution of scanning electron microscope images is high enough so that nanometer scale organizations are visualized.The methods we use for fixation are mild enough to preserve the intra-structural organization of the protein. This remains in the scope of fundamental research to understand the behavior of cytoskeletal filaments. Begin by designing a wavy Norland Optical Adhesive, or NOA replica, in a clean room environment from wavy polydimethylsiloxane undulated patterns of 250 nanometer amplitude and two micrometer lateral periodicity.To do so, deposit five microliters of liquid NOA on a circular glass cover slip of one centimeter diameter and then place the PDMS template on the drop. Treat the assembly with UV light at 320 nanometers for five minutes to photopolymerize the liquid NOA into a thin polymer film. Once done, gently peel off the PDMS template from the cover slip with the freshly polymerized NOA.Treat the NOA film using an air plasma cleaner for five minutes to make the surface hydrophilic. Prepare a solution of small unilamellar vesicles, or SUVs, as described in the manuscript, and obtain the SUVs by resuspending a dried lipid film in the observation buffer under the sonication in a bath sonicator for five to 10 minutes until the solution is transparent. Later, insert the cover slips supporting the NOA patterns within the wells of cell culture boxes and incubate the freshly glow discharged NOA patterns with 100 microliters of one milligram per milliliter SUV solution for 30 minutes at room temperature to generate a supported lipid bilayer.To remove the unfused SUV, rinse the sides thoroughly six times with the septin low salt buffer without letting the sample completely dry between two washes. Dilute the octomeric septin stock solution in a septin low salt buffer to final concentrations, ranging from 10 to 100 nanomolar and volumes of one milliliter. Then, incubate the protein solution on the slides for one hour at room temperature.Wash the NOA slides containing fused supported lipid bilayers and incubate protein with septin low salt buffer. Replace septin low salt buffer with 2%glutaraldehyde fixative solution in 0.1 molar sodium cacodylate buffer, prewarmed at 37 degrees Celsius, and let the reaction proceed for 15 minutes, and wash the fixed sample thrice, five minutes each with 0.1 molar sodium cacodylate. After washing, incubate the sample in the fixative solution of osmium tetroxide, then the filtered tannic acid, and finally the filtered uranyl acetate for 10 minutes each.Wash the sample thrice in distilled water after every solution. Incubate the sample serially in ethanol solutions starting from 50%to 100%for two to three minutes each. Transfer the glass slides inside the critical point dryer prefilled with ethanol and follow the manufacturer's instructions for drying.As dried samples are highly hydroscopic, coat them immediately by attaching the cover slip to the stubs. Then add a strip of silver paint to the upper face of the cover slip without creating paint deglomerates. Make sure that the connection with the stub is satisfactory.When the sample evaporates completely, use an apparatus equipped with a plasma magnetron sputtering head and a rotary planetary stage, and follow standard protocols provided by the manufacturer. Perform pre-sputtering at 120 milliamperes for 60 seconds to remove the oxide layer at the surface. Then deposit 1.5 nanometers of tungsten with a film thickness monitor at 90 milliamperes and a working distance of 50 milliliters.Later, store the samples under a vacuum to protect them from the ambient air until and throughout the SEM analyses. To achieve high solution imaging by detecting the secondary electrons with the inland detector, set the accelerating voltage to three kilovolts and the beam current to 20 micrometer aperture. For observations, use resolutions ranging from 21.25 nanometers per pixel to 1.224 nanometers per pixel.Use a resolution of 5.58 nanometers per pixel for data analysis. Set the working distance between one to two millimeters for high resolution observation and around three millimeters if an increased depth of field is required. Adjust the scan speed and line integration continuously to ensure a constant signal to noise ratio with an acquisition time of around 30 to 45 seconds per image.The representative analysis illustrates the effect of the material deposited on the septin filaments on wavy PDMS patterns with 1.5 nanometers of platinum and 1.5 nanometers of tungsten. It was observed that bare giant unilamellar vesicles, or GUVs, were perfectly spherical. After the septin-induced deformation, the vesicles appeared faceted and the deformations remained static, and thus did not fluctuate.At higher concentrations of the septins, periodic deformations in the vesicles were observed. Self-assembly of the septin filaments bound to large unilamellar vesicles, or LUVs, was examined with a cryo electron microscopy image and 3D reconstruction was obtained from a tilted series. The curvature-dependent arrangement of the septin filaments was visualized by scanning electron microscopy.At low magnification, a wavy pattern with the periodicity of negative and positive curvatures was visible. The ultra-structural organization of the septin filaments was viewed at higher magnification. The NOA have to be gently peeled off to prevent degradation.After imaging, some image analysis can be performed to quantify the ultra-structure features visualized by SCM, such as filament spacing, and orientation of the filament. The present method has been applied to septins, but it could also be used for other cytoskeletal proteins or proteins that organize as long filaments and thus be sensitive to curvature.

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