A human septin octamer complex sensitive to membrane curvature drives membrane deformation with a specific mesh-like organization

Abstract

Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane and decouple their role from other partners, we used a combination of bottom-up in vitro methods. We assayed their ultrastructural organization, their curvature sensitivity, as well as their role in membrane reshaping. On membranes, human septins organize into a two-layered mesh of orthogonal filaments, instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is sensitive to micrometric curvature and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarse-grained computed simulation to understand their mechanisms. Our results highlight the specific organization and behavior of animal septins at the membrane as opposed to those of fungal proteins.

Keywords: Curvature; Cytoskeleton; Membrane; Septin.

Fig. 1.

Cryo-electron tomography of septins bound to liposomes. (A–C) Slices of cryo-electron tomography (left and middle) and models (right) built by segmentation of tomograms. The left column displays slices in which the membrane is visible. The middle column displays slices in which one layer of the mesh is visualized. Septins bound to a deformed LUV (A) and a round LUV (B) are organized as mesh structures. The mesh consists of two layers of septins (C); the first (bound to membrane) and the second (bound to the first layer of septin filaments) layers of septin filaments are segmented in blue and pink, respectively, in the right image. In A,B, parts of some filaments are indicated using short green lines. Yellow and pink densities in the right columns represent membranes and septin filaments, respectively. Scale bars: 100 nm (left, middle); 200 nm (right).

Fig. 2.

SEM of septin filaments on lipid bilayers supported by undulated solid substrates. Samples were prepared with different concentrations of septins dispersed in 10 mM Tris buffer containing 75 mM NaCl on undulated solid substrates with 1.6 µm periodicity and 0.20 µm amplitude. (A,B) SEM image of septin filaments prepared at (A) 8.7 nM and (B) 26 nM concentrations in solution. Raw SEM images are shown on the left. Black arrowheads show the top of convex hill of the substrate. Segmented filaments colored according to their orientations were overlaid on the raw SEM images and are shown in the middle. Distributions of orientation at the concave and convex parts of the substrates are plotted in the graphs on the right. Representations of the obtained results are shown in the schematics above the graphs. (C) SEM images of septin filaments at 26 nM concentration in solution. An image covering a wide range is shown on the left, and images focused on mesh structures that appeared on convex regions and the circular pattern of septin filaments on concave regions are shown in the top middle and bottom middle, respectively. The corresponding segmentation results of filaments colored according to their orientations are shown on the right. (D) SEM image of septin filaments at 87 nM concentration in solution. An image covering a wide range is shown on the left, and an image focused on mesh stru­ctures that appeared on the convex part of substrates is shown on the top right. Segmentation results of filaments colored according to their orientations are shown on the bottom right. (E) Measured distances between two adjacent septin filaments at the center of convex regions. Distances d₁ and d₂ correspond to the distances between two adjacent filaments in the direction parallel to the 1D wavy line of the substrate (first layer of septins on membranes) and perpendicular to the 1D wavy line of the substrate (second layer of septins bound to the first layer of septins), respectively. Measurements were performed depending on the concentration of septins in solution (d₁=46±20 nm and d₂=55±24 nm in 26 nM, d₁=27±11 nm and d₂=19±8 nm in 87 nM). Bars show the mean±s.d. Images are representative of four experiments.

Fig. 3.

SEM of septin filaments on lipid bilayers supported by undulated solid substrates of curvatures ranging from 0 to 4.5 µm⁻¹. SEM images of septin filaments at 26 nM concentration incubated with flat and varying curvatures of undulated solid substrates. (A) Raw SEM image of septin filaments on a flat NOA71 substrate (left). Segmented filaments colored according to their orientations were overlaid on the raw SEM image and are shown in the middle panel. The distribution of filament orientations averaged from three positions is plotted in the graph on the right. (B) Raw SEM image of septin filaments on an undulated substrate with curvature ranging from −1.7 to +1.7 µm⁻¹ (2.1 µm periodicity, 0.20 µm amplitude) (top). (C) Raw SEM images of septin filaments on an undulated substrate with curvature ranging from −4.5 to +4.5 µm⁻¹ (1.9 µm periodicity, 0.45 µm amplitude). An image covering a wide range is shown on top, and images focused on the boxed regions 1 (convex) and 2 (concave) are shown in the middle panels. For B,C, segmented filaments colored according to their orientations were overlaid on the raw SEM images and are shown in the middle (B) or middle right (C) panels. The distributions of filament orientations at concave and convex parts averaged from three positions for each part are plotted in the graphs on the bottom. Black arrowheads show the tops of convex hills of the substrate. Images are representative of two sets of independent experiments.

Fig. 4.

Simulation on a wavy patterned surface. Monte Carlo simulation of our model of septin on a rigid wavy substrate, with the intrinsic curvature of septin being . The color bar represents the local mean curvature of the substrate. Panels A–C have one layer of septin, whereas panels G–I have two layers of septin. Reduction of the intrinsic curvature () (D–F) changes the alignment of septins in the valleys (blue) towards the larger radius of curvature, oblique to the direction of undulation. The septins on the hills (red) also turn and form circular structures along with the ones in the valley (F). In A–C and D–F, the amount of septins was increased gradually from left to right. In G–I, the strength of interaction (ε) between the two layers of septin was increased from left to right (ε=1, 3, 5 and 6). In I, with ε=6, the orientations in the two layers became fully perpendicular. The other parameters were κ=20, =25, κ_⊥=25 and ε_LL=2 in k_bT units, and C_⊥=0 in arbitrary units. All parameters were the same for both layers of proteins.

Fig. 5.

Characterization of the interaction between human septins and membrane. (A) Phase diagram of GUV behavior as a function of concentration of NaCl in external buffer and concentration of human septin octamers. (B) Confocal fluorescence microscopy images of slices of the GUVs. Lipids and septins are visualized in red and green, respectively, and these signals are overlaid. (1) A GUV without human septin octamers in 10 mM Tris buffer at pH 7.8 containing 70 mM NaCl. (2) An example image of a GUV found in the region ‘weak interaction’. The GUV was incubated with 0.87 nM septin in 10 mM Tris buffer containing 70 mM NaCl. (3,4) Examples of GUVs partially covered by septins. GUVs were incubated with 4.4 nM septins in 10 mM Tris buffer containing 70 mM NaCl. Image 4 is a 2D image and recorded at the bottom of the surface of a GUV. (5,6) Deformed GUVs found in the region ‘strong interaction’ in the phase diagram. 2D- and 3D-reconstituted images of GUVs with 176 nM septins in 10 mM Tris buffer containing 70 mM NaCl. White arrows indicate excess of septins polymerized in solution. (7) A deformed GUV with 36 nM of dark septins in 10 mM Tris buffer containing 70 mM NaCl. (8) Ring-like organization of septins bound to membranes at high-salt condition. An image of 3D-reconstituted GUV interacting with 520 nM septins in 10 mM Tris containing 250 mM NaCl. Scale bars: 5 µm. Images are representative of four experiments. (C) Distance between peaks of bumps in deformed GUVs (n=18 vesicles). Different concentrations of septins are plotted on the graph. Each gives a similar distribution (see Fig. S5A). The image shows detail of a deformed GUV. Scale bar: 5 µm.

Fig. 6.

Simulation describing membrane deformations by nematically ordered filaments. (A) Simulation of septin on a deformable vesicle. Interaction between the second layer and the membrane is reduced gradually from model A to model E. The observed wavy surface emerges when only the first layer interacts with the membrane (models D and E). The ratio of the corresponding induced bending rigidities for both layers and are 1 (in model A), 0.5 (in model B), 0.1 (in model C), and 0 (in models D and E). κ_|| and κ_⊥ values are 5, 10, 15, 25 and 40 for models 1–5, respectively. The other parameters are κ=20, ε_LL=1 and ε=1 (in k_bT units), =−1 and C_⊥=0. All parameters are the same for both layers of proteins. The color bar indicates the local mean curvature of the surface. Note that number of dips saturate at higher κ_||. (B) Surface morphologies are shown from simulations performed at different vesicle volumes, keeping all other parameters fixed, corresponding to the configuration 4 shown in A. This shows that the distances between the surface dips remain approximately the same for different-sized vesicles. (C) Phase diagram of vesicle shapes as a function of anisotropic bending modulus κ_|| and intrinsic curvature . The experimentally observed undulated GUV shapes emerge when both these parameters are high, whereas handful of buds and tubes occur when both these parameters are small (see detailed explanation in the main text). The dotted line indicates the threshold between the observed experimental deformations and simulated deformations.

Fig. 7.

Model of orthogonal network assembly. Schematic of octameric septins (top) and a possible model of the orthogonal network array of human septin filaments. Note that the orientation of parallel coiled coils in the second (upper) layer of septins could diverge from this proposal in terms of orientation.