Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
We thank Patricia Bassereau and Daniel Lévy for their useful advice and discussions. This work benefited from the support of the ANR (Agence Nationale de la Recherche) for funding the project "SEPTIME", ANR-13-JSV8-0002-01, ANR SEPTIMORF ANR-17-CE13-0014, and the project "SEPTSCORT", ANR-20-CE11-0014-01. B. Chauvin is funded by the Ecole Doctorale "ED564: Physique en Ile de France" and Fondation pour lea Recherche Médicale. K. Nakazawa was supported by Sorbonne Université (AAP Emergence). G.H. Koenderink was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO/OCW) through the ‘BaSyC-Building a Synthetic Cell'. 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).
Materials
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine | Avanti Polar Lipids | 850725 | |
| 1,2-dioleoyl-sn-glycero-3-phospho-L-serine | Avanti Polar Lipids | 840035 | |
| Bath sonicator | Elma | Elmasonic S10H | |
| Bodipy-TR-Ceramide | invitrogen, Thermo Fischer scientific | 11504726 | |
| Chemicals: NaCl, Tris-HCl, sucrose, KCl, MgCl2, B-casein, chloroform, sodium cacodylate, tannic acid, ethanol | Sigma Aldrich | ||
| Confocal microscope | nikon | spinning disk or confocal | |
| Critical point dryer | Leica microsystems | CPD300 | |
| Deionized water generator | MilliQ | F1CA38083B | MilliQ integral 3 |
| Egg L-α-phosphatidylcholine | Avanti Polar Lipids | 840051 | |
| Field Emission Gun SEM (FESEM) | Carl Zeiss | Gemini SEM500 | |
| Glutaraldehyde 25 %, aqueous solution | Thermo Fischer scientific | 50-262-19 | |
| High vacuum grease, Dow corning | VWR | ||
| IMOD software | https://bio3d.colorado.edu/imod/ | software suite for tilted series image alignment and 3D reconstruction | |
| Lacey Formvar/carbon electron microscopy grids | Eloise | 01883-F | |
| Lipids | Avanti Polar Lipids | ||
| L-α-phosphatidylinositol-4,5-bisphosphate | Avanti Polar Lipids | 840046 | |
| Metal evaporator | Leica microsystems | EM ACE600 | |
| NOA (Norland Optical Adhesives), NOA 71 and NOA 81 | Norland Products | NOA71, NOA81 | |
| Osmium tetraoxyde 4% | delta microscopies | 19170 | |
| Osmometer | Löser | 15 M | |
| Plasma cleaner | Alcatel | pascal 2005 SD | |
| Plasma generator | Electron Microscopy Science | ||
| Plunge freezing equipment | leica microsystems | EMGP | |
| Transmission electron microscope | Thermofischer | Tecnai G2 200 kV, LaB6 | |
| Uranyl acetate | Electron Microscopy Science | 22451 | this product is not available for purchase any longer |
| Wax plates, Vitrex | VWR |
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