Membrane reshaping by micrometric curvature sensitive septin filaments

Abstract

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.

Fig. 1

Micropipette experiment and GUV mechanics. a Illustration of a typical micropipette experiment where R_v, R_p, L_p, and ΔP, are, respectively, the vesicle radius, the pipette radius, the tongue length, and the applied pressure. b Confocal images of a GUV held by a micropipette at constant tension, where lipids are labeled in red and septins in green. The first image corresponds to t = 0, before addition of septins and the second image to t = 10 min; the dashed line represents the position of the tongue length at t = 0. Scale bar = 10 μm. c Typical curve representing the relative change over time in area (red squares) and volume (black triangles), as a response to the binding of septins on a GUV held by a micropipette. The red squares correspond to the density of bound septins. Error bars represent s.d. (standard deviations). d Mean curve of the change of area as a function of bound septin density (N = 56). e Mean curves of the change of area as a function of applied tension without septins (red dots, N = 37) and at fixed septin density d = 3000–6000 µm⁻¹ (green squares, N = 27). Dotted lines represent linear fits. Error bars represent s.d. f Box plot of measured bending moduli. Boxes represent the 25–75% range, crosses are min and max values, whiskers are standard deviations and squares are mean values

Fig. 2

Septins induce deformations on GUVs. a 3D reconstructions of confocal spinning disk images of GUVs in a solution of 200 nM septins (red = lipids, green = septins). GUVs appear deformed and undulated. b 3D reconstructions of confocal spinning disk images of GUVs in a solution of 600 nM septins. Spikes appear on the surface of the GUVs. c Confocal image of a spiky GUV in a solution of 600 nM septins. The different parameters R (radius of curvature), A (height) and λ (distance) of the spike are highlighted. d Distribution of parameters height, A, (blue); radius of curvature, R (green); and distance, λ (red) of the spike on GUVs (N_vesicles = 35). e 3D reconstruction of spinning disk image of a GUV in a solution of 600 nM septins at high salt concentration (300 mM NaCl). f Sketch of a septin filament bound to a curved membrane. At a constant volume, a bent filament shows a greater surface towards curved membrane. All scale bars are 10 µm

Fig. 3

Cryo-electron microscopy of septins bound to LUVs. a Cryo-EM images of septins bound to vesicles. Stars indicate vesicles with septins bound. Left: flattened vesicles with protrusions. The arrow points to septin networks of filaments. Insert 1: Control without septins, insert 2: After septin addition. Middle: example of a ruptured vesicle. The arrows point at the broken membrane. Right: vesicle covered with an array of parallel filaments. Scale bars = 50 nm, inserts: circular holes of 1 µm in diameter. b Electron cryo-tomography: segmentation within cryo-tomograms of septins bound to vesicles. The membrane is highlighted in yellow. Filaments are modeled in blue. Middle and left image correspond to a vesicle covered with septin filaments. Right image: deformed membrane covered with filaments. Scale bars = 200 nm. c Cryo-tomography of septin filaments around protrusions. Left: slice within a tomogram with the segmentation highlighted in the middle panel. Right: filaments wrapping around a protrusion. Scale bars = 200 nm. d Cryo-tomography of septins rupturing vesicles. Left (slice in a tomogram) and middle (segmentation only): filaments breaking the membrane. Scale bars = 200 nm. Right: filaments inside a vesicle aligning within a protrusion. Scale bars = 100 nm

Fig. 4

Patterned substrates. a Schematic representation of the wrinkled substrates (blue) and lipid bilayer (red). b Relative septin density (green) on SLB deposited on the patterned substrates as a function of the curvature (N = 202). The red and orange curves correspond to the lipid signals as controls (Bodipy-TR-ceramide and fluorescent PI(4,5)P2, respectively). Error bars represent s.d. c Low magnification SEM image of a representative patterned substrate. Scale bar is 10 µm. d, e SEM image of a patterned substrate incubated with a septin bulk solution at 200 nM. Scale bar is 1 µm in d and 300 nm in e. f, g Color-labeled image of the septin orientation with respect to the wrinkle substrate of image d and e, respectively. The inset shows the angular distribution of pixels at the top (purple) (positive curvature) and at the bottom (green) (negative curvature of the substrate). The white arrow points to a convex surface where septins align and bundle parallel to the undulations. h SEM image of a patterned substrate incubated with mutant septin solution at 200 nM. Scale bar is 200 nm. The inset shows the angular distribution of septins. No preferential orientation is found on positive or negative curvature. i Schematic representation of the wavy substrates with septin filaments following the null curvature at the top, positive, (green) and the bottom, negative, curvature (purple) of the wrinkled substrate

Fig. 5

Schematic representation of model predictions for septin filaments at the bud neck of S. cerevisiae. a Filaments on a spherical geometry arranged radially. b Filaments on a barrel-like geometry arranged along the height of the barrel. c At the onset of the constriction, single filaments are arranged circumferentially while double filaments are arranged perpendicular to the single filaments. d Upon further constriction, all filaments at the center of the bottleneck are arranged circumferentially while the organization remains the same at the edges. e Under extreme constriction, filament bundles appear