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 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.
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.
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 as fluorescently labeled septins (using Green Fluorescent Protein [GFP]), are used to follow the behavior of both lipids and proteins via fluorescence microscopy.
Figure 1: Electro-formation of GUVs. (A) Schematic representation of the electro-formation process using platinum wires. (B) Picture of the Teflon home-made device assembled with platinum wires used for generating GUVs by electro-formation. Wires are 0.5 mm in diameter and 3 mm apart. (C) GUVs (spherical objects) observed by transmission optical microscopy during the growth process. The opaque zone at the bottom of the image is the platinum wire. (D) GUVs (round fluorescent objects) observed with fluorescent microscopy during the growth on the platinum wire. Scale bars = 100 μm. Please click here to view a larger version of this figure.
2. Analysis of ultrastructural organization of septin filaments by cryo-electron microscopy
NOTE: Vesicles are not suitable for imaging with standard electron microscopy methods. Indeed, samples are dried using standard negative stain methods. Upon dehydration, vesicles are likely to undergo unspecific deformations, frequently resulting in lipid protrusions. Cryo-electron microscopy is thus a much better strategy to observe specific deformations of vesicles. Using cryo-EM, the samples are embedded within a thin (~100-200 nm) layer of vitrified ice, which preserves the samples close to the native state. GUVs are, however, too large (several tens of micrometers) to be embedded within thin ice and thus imaged by transmission electron microscopy. Hence, large unilamellar vesicles (LUVs), whose diameters range from ~50-500 nm, are generated to determine how septins can deform vesicles and how they arrange on vesicles.
3. Analysis of curvature sensitivity of septin using SEM
NOTE: To understand how septins can be sensitive to micrometer curvatures, an in vitro approach has been used to incubate septin filament complexes with solid-supported lipid bilayers deposited on micrometer-scale wavy undulated patterns.
Figure 2: Effect of the material deposited on septin filaments on wavy PDMS patterns. SEM of septin filaments coated by sputtering with either (A) 1.5 nm of platinum, exhibiting the "arid cracked soil" pattern typical of a lack of cohesivity between the clusters of platinum nuclei, or (B) 1.5 nm of tungsten covered with a smooth and cohesive layer. Scale bar = 200 nm. White square boxes represent the magnified views on the lower right. The spherical globules are small lipid vesicles interacting with the septins. Please click here to view a larger version of this figure.
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 remained static and thus did not fluctuate (Figure 3B). Above 200 nM budding yeast septins, with the membrane being saturated with septins, periodic deformations were observed (Figure 3C). Using budding yeast septins, spikes were visualized with a periodicity varying from 2 µm to 6 µm and with an amplitude of about 1 µm. Hence, septins strongly reshape membranes in a specific fashion. These amplitudes and associated curvatures of the observed deformations strikingly reflect the affinity of septins for micrometer curvatures observed in cells. GUVs, being deformable, are thus appropriate to assay how proteins reshape membranes.
Figure 3: Septin-induced deformation of GUVs. (A) Equatorial plane of control GUVs labeled with 0.5% rhodamine PE and imaged by confocal microscopy. Scale bar = 3 µm. (B) GFP-septins (100 nM) bound to a deformed GUV and imaged by fluorescence confocal microscopy. Scale bar = 1 µm. (C) GFP-septins (600 nM) bound to a deformed GUV and imaged by fluorescence spinning disk microscopy. Scale bar = 10 µm. The GUVs are made of a molar ratio of 56.8% EggPC, 15% cholesterol, 10% DOPE, 10% DOPS, 8% brain PI(4,5)P2, and 0.2% Bodipy-TR-Ceramide. The observation buffer includes 75 mM NaCl and 10 mM Tris (pH = 7.8). Please click here to view a larger version of this figure.
Self-assembly of septin filaments bound to LUVs imaged by cryo-EM
As compared with fluorescence microscopy, cryo-EM provides better spatial resolution. Individual septin filaments and lipid bilayers can thus be discerned unambiguously in images. Figure 4A displays an image of LUVs decorated with budding yeast septin filaments (some of them being highlighted in blue) at 50 nM. Parallel sets of filaments, about 10 nm apart, were observed on the LUVs. This typical image is a 2D projection. Hence, to decipher any 3D deformation and to know whether septin filaments directly interact with the membrane, cryo-electron tomography was carried out (Figure 4B and Figure 4C). Figure 4B displays a slice in a 3D reconstruction, while Figure 4C presents a segmentation of the same area highlighting the membrane in yellow and the septin filaments in blue. As seen on the side view, septin filaments remained essentially straight bound to the liposome and the vesicle was considerably flattened as compared with the naked spherical vesicles.
Figure 4: Self-assembly of septin filaments bound to LUVs. (A) Cryo-EM image of septin filaments bound to a vesicle. Some of the filaments are highlighted in blue for visibility. The dark dots are gold fiducials usually used to align data for tomography. Scale bar = 100 nm. (B) and (C) 3D reconstruction obtained from a tilted series. The lipids are highlighted in yellow, while the septins are segmented in blue. Scale bars = 200 nm. Please click here to view a larger version of this figure.
Curvature-dependent arrangement of septin filaments visualized by SEM
Septins localize in cells at sites displaying micrometer curvatures (e.g., at the cell division furrow, the base of cilia, etc.). In addition, in vitro studies show that septins, as seen above, can reshape membranes so they eventually display micrometer curvatures. To assay the curvature sensitivity of septins for both positive (convex) and negative (concave) curvatures, septins were incubated with a supported lipid bilayer fused with the wavy patterns described above. The outcome is displayed in Figure 5. Figure 5A recapitulates the different steps required to obtain the samples. Figure 5B presents a wavy pattern at low magnification, where the periodicity of negative and positive curvatures is clearly visible. Two successive waves are indicated by orange solid lines. Defects approximately orthogonal to the waves are indicated by white arrows. Figure 5C and Figure 5D display the ultrastructural organization of septin filaments at higher magnification. The septins assembled into sets of parallel filaments, whose orientation depended on the curvature (see filaments highlighted in blue). Indeed, the septin filaments were not oriented randomly. Instead, septins can bend when interacting with negative (concave) curvatures to follow the imposed curvature. Conversely, on positive curvatures, the septin filaments remained straight, unbent, and aligned along the convex waves. Hence, septins can interact with both positive and negative curvatures but adopt specific organizations on given curvatures. This methodology thus appears particularly relevant to highlight the curvature-sensitivity of binding and self-assembly of filamentous proteins.
Figure 5: Curvature-dependent arrangement of septin filaments. (A) Schematic representation of the steps required to obtain the "wavy" samples meant to analyze septin curvature sensitivity through SEM. (B) Low magnification SEM image of a wavy pattern with bound septin filaments that are not resolved at this resolution. Two consecutive waves are highlighted in orange. Orthogonal defects are indicated by white arrows. Scale bar = 10 µm. (C) SEM image at 10,000x magnification where septin filaments are visible. Scale bar = 1 µm. (D) SEM image at 20,000x magnification. Some of the filaments are highlighted in blue for visibility. Scale bar = 200 nm. The spherical objects present in the images are small vesicles interacting with the septins and substrate. The septin concentration was set at 200 nM in B–D. Please click here to view a larger version of this figure.
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 be aliquoted in small volumes so that a specific vial is not opened more than twice for pipetting. Besides, lipid aliquots should be stored under argon to prevent lipid oxidation. Finally, once the lipid mixes are solubilized in an aqueous solution, the mixture must be used at once and cannot be stored for further experiments. Notably, after GUV reconstitution or supported lipid bilayer fusion, the interaction between septins and lipids greatly reduced after 2 h.
Some of the results obtained with octameric budding yeast septin complexes (enclosing Cdc10, Cdc11, Cdc12, and Cdc3) have been presented here; however, the experimental procedures followed to analyze the behavior of septin complexes from other species should be identical.
To generate GUVs, we prefer the electro-formation method using platinum wires over electro-formation on ITO plates or PVA swelling. As demonstrated earlier25, this method reproducibly produces unilamellar GUVs of good quality without any defects.
The wavy substrates can be designed with a wide range of curvatures. After screening various designs, patterns with a periodicity of 2 µm and an amplitude of 250 nm, which generate curvatures ranging from -3.5 µm-1 to 3.5 µm-1 (± 0.5 um-1), have been consistently used. Using patterns with a short periodicity and higher amplitude, the lipid bilayers did not nicely conform to the surface. Often, in the case of higher curvature, the bilayer would be suspended in between two convex waves instead of being fully supported on the NOA substrate. With lower amplitudes and larger periodicity, septins organized with random nematic-like orientations, indicating that the patterns were too flat. Besides, using wavy patterns instead of cylindrical tubes20 or spheres20 has the benefit that both positive and negative curvatures can be tested simultaneously. In the future, examining the behavior of filaments on gaussian "saddle-like" curvatures would be relevant to mimic the curvature of more realistic cellular contexts.
In solution, mixing LUVs with septins induced the generation of septin-lipid aggregates even at low concentrations of proteins and lipids. The samples thus were heterogeneous, and it is wise to look for thinner ice domains where smaller and more defined septin-vesicle objects are found. This is particularly crucial when choosing areas of interest where cryo-tomography will be performed. Often, even at low magnifications, vesicles displaying protrusions of strong deformations, as compared with spherical vesicles, are more likely to have a high density of septin filaments bound. This protocol focuses on obtaining a description of the global ultrastructure of filaments on biomimetic membranes. Currently, higher resolutions are reachable. The resolution limitation results from the limited number of views available by imaging. However, this remains beyond the scope of this protocol.
We have shown through this protocol that a combination of complementary methods is essential to understand and dissect how the specific arrangement of cytoskeletal septin filaments can sense and reshape membranes in a curvature-dependent manner.
The authors have nothing to disclose.
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).
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 |