# Septin filament assembly assist the lateral organization of membranes

Fatima El Alaoui, Isabelle Al-Akiki, Sandy Ibanes, Sébastien Lyonnais, David Sanchez-Fuentes, Rudy Desgarceaux, Chantal Cazevieille, Marie-Pierre Blanchard, Andrea Parmeggiani, Adrian Carretero-Genevrier, et al.

► To cite this version:

Fatima El Alaoui, Isabelle Al-Akiki, Sandy Ibanes, Sébastien Lyonnais, David Sanchez-Fuentes, et al.. Septin filament assembly assist the lateral organization of membranes. Structure, 2025, 33 (3), pp.451-464. 10.1016/j.str.2025.01.005 . hal-04782300

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Septin filament assembly assist the lateral organization of membranes

Fatima El Alaoui¹, Isabelle Al-Akiki¹, Sandy Ibanes², Sébastien Lyonnais³, David Sanchez-Fuentes⁴, Rudy Desgarceaux⁴, Chantal Cazevieille⁵, Marie-Pierre Blanchard⁶, Andrea Parmeggiani⁷, Adrian Carretero-Genevrier⁴, Simonetta Piatti², Laura Picas¹,*.

¹Institut de Recherche en Infectiologie de Montpellier (IRIM). Université de Montpellier – CNRS UMR 9004, Montpellier, France.

²Centre de Biologie Cellulaire de Montpellier (CRBM). Université de Montpellier – CNRS UMR UMR 5237, Montpellier, France.

³Centre d'Etudes des Maladies Infectieuses et Pharmacologie Anti-Infectieuse (CEMIPAI), Université de Montpellier, UAR 3725 CNRS, Montpellier, France.

⁴Institut d'Électronique et des Systèmes (IES), Université de Montpellier – CNRS UMR 5214, Montpellier, France.

⁵COMET Electron Microscopy Platform, Institute for Neurosciences of Montpellier (INM), Université de Montpellier, INSERM U 1298, Montpellier, France.

⁶Montpellier Ressources Imagerie, BioCampus Montpellier, Université de Montpellier, CNRS, INSERM, Montpellier, France.

⁷Laboratoire Charles Coulomb (L2C), Université de Montpellier, CNRS, Montpellier, France.

<footnote>*Correspondance sent to: laura.picas@irim.cnrs.fr</footnote>
31 **ABSTRACT**

32 Compartmentalized interactions of plasma membrane components are essential to support
33 many cell functions, from signaling to cell division, adhesion, migration, or phagocytosis.

34 Cytoskeletal-membrane interactions play an important role in forming membrane
35 compartments, and this feature has been primarily studied through the actin cytoskeleton.

36 Unlike actin, septins directly interact with membranes, acting as scaffolds to recruit proteins
37 to specific cellular locations and as structural diffusion barriers for membrane components.

38 However, how septins interact with and remodel the local membrane environment is unclear.

39 Here we combined minimal reconstituted systems based on fluorescence microscopy and
40 quantitative atomic force microscopy together with live yeast cell imaging and STED
41 microscopy to study septin-mediated membrane organization. Our results show that septins
42 self-assembly into filament-based sub-micrometric patches and high-order structures prompt
43 their membrane-organizing role *in vitro* and in yeast cells, respectively. Furthermore, we
44 show that the polybasic domain of Cdc11, in addition to the amphipathic helix of Cdc12,
45 plays an essential role in supporting the membrane remodeling and curvature-sensing
46 properties of yeast septins. Collectively, our work provides a framework for understanding
47 the molecular mechanisms by which septins can support cellular functions intimately linked
48 to membranes.

49

50
## 51 INTRODUCTION

The architecture of the cell cortex is essential to support many cellular processes, such as cell division, trafficking and motility (1, 2). In eukaryotic cells, the cortex is made of a dynamic composite encompassing the plasma membrane and its interacting proteins that is linked to the underlying cytoskeletal network. This cytoskeleton-membrane adhesion has two major roles: mechanical, to support changes in cell shape, for instance during cell blebbing or filopodia protrusion, and biochemical, as it provides a spatial and temporal organizing frame for membrane-associated components (3–5). In the latter case, the cytoskeleton can act as a microscopic structural diffusion barrier for membrane components, as reported for neurons, the base of cilium or the cleavage furrow of dividing cells (5–7). Furthermore, the association of trans-membrane proteins with the underlying actin meshwork is involved in the mesoscale-domain compartmentalization of the cell membrane (5, 8). *In vitro*, the actin cytoskeleton was shown to influence the phase behavior and microdomain formation at membranes depending on the type of anchoring of lipids and proteins (9), the effect of actin polymerization (10), and the stress induced by molecular motors such as myosin (11). The cytoskeleton-mediated compartmentalization of the membrane appears essential to facilitate biomolecular reactions (12, 13) and thus, may be very important for cellular signaling (8, 14). For instance, phosphoinositide signaling, including the receptor G protein/PLC pathway and the PI4K pathway, appears concentrated at liquid-ordered (Lo) domains enriched with cholesterol (14). Also, the activation of type I tyrosine kinase epidermal growth factor (EGF) receptor appears to depend on the cortical actin organization and to polarize at specific cellular locations (13). The type of cytoskeleton cross-linking to the plasma membrane plays an important regulatory role in the formation of specialized membrane compartments (7). For instance, in the case of actin, the interaction with the membrane is not direct but mediated by cytoplasmic proteins, such as the ezrin, radixin and moesin (ERM) family, myosin 1b, anillin and septins (15–20) that bind both actin and lipids, typically phosphatidylinositol-4,5-bisphosphate(PI(4,5)P₂) (21). In the case of septins, their organization into octamer-based filaments is essential for their role as actin-membrane linkers in human cells (20). Furthermore, the density and adhesion
79 strength of the cytoskeletal linkers are crucial parameters to prevent the cell cortex slippage
80 during mechanically active processes, such as actomyosin contraction, and allow sustained
81 membrane deformations (22–24).

82 Septins, the fourth cytoskeleton component, might execute a central role in stabilizing the cell
83 cortex architecture during actomyosin contraction. Furthermore, septin organization at the
84 collar of dividing yeast cells was shown to scaffold actomyosin ring assembly for cytokinesis
85 (25–28).

86 Septins are a family of cytoskeletal proteins highly conserved in animal cells and fungi. They
87 share a common structural organization consisting of a core GTP-binding domain, a septin-
88 unique element and variable N- and C-terminal extensions (29, 30). Septins form
89 heteromeric complexes composed by pairs of different septins and higher-order structures,
90 such as non-polar filaments and rings. These septin structures can be localized at cellular
91 protrusions, such as at the base of cilia and dendritic spines or the bud neck of budding
92 yeast cells, where they act as scaffolds to promote protein-protein interactions and to
93 maintain subcellular compartmentalization by forming diffusion barriers for membrane-
94 associated proteins (6) and, recently, for PI(4,5)P₂ lipids (31). Septins have been shown to
95 interact with actin (18, 32), microtubules (33, 34) and to associate with actin-binding proteins,
96 such as anillin and non-muscle myosin II (32, 35). Furthermore, septins were shown to bind
97 membranes and this interaction influences, in turn, septin organization into higher-order
98 structures (17, 36). Septins interact with membranes containing anionic lipids, such as
99 PI(4,5)P₂, phosphatidylserine and cardiolipin (17, 37–39), and display a strong affinity for
100 micron-scale membrane curvatures (40–43). Truncations within the N-terminal domain of
101 septins, which contains a polybasic amino-acid region, showed its role for phosphoinositide
102 selection and membrane association (37, 44–46). In addition, the C-terminal amphipathic
103 helix of Cdc12, which is responsible for septin-curvature sensing (38, 40, 41), was recently
104 reported to facilitate membrane interaction *in vitro* (47).

105 Because the lipid composition and topology of cellular membranes is heterogeneous, these
106 observations suggest that the appearance of specific septin organizations might be sensitive
107 to the local membrane environment. However, it is unclear if all septin architectures or
108 specific ones would, in turn, affect the lateral organization and compartmentalization of
109 membranes.

110 We use a combination of time-lapse and super-resolution imaging, atomic force microscopy,
111 and nanotechnology approaches on *in vitro* reconstituted systems and yeast cells to show
112 that membrane-diffusive septins organize into filament-based sub-micrometric patches.

113 These structures, in coexistence with high-order septin structures, directly modulate the
114 lateral organization of membranes. We provide experimental evidence supporting that two
115 membrane-interacting interfaces of septins, the polybasic motif of Cdc11 and amphipathic
116 helix of Cdc12, are involved in this process.

117
118 **RESULTS**

119 **Septins regulate the lateral organization of the budding yeast membrane during the**
120 **cell cycle**

121 In many eukaryotic cells, one of the central and most studied functions of septins is to
122 promote cytokinesis, i.e. the last step of cell division (48). In budding yeast, the cytokinetic
123 function of septins is linked to their unique ring-like organization at the bud neck and their
124 interaction with the plasma membrane (28). To investigate the role of septins in membrane
125 organization at the bud neck of S. cerevisiae, we monitored the general polarization (GP) of
126 the lipophilic probe Laurdan as a readout of the lipid packing of the plasma membrane
127 (Figure 1). Briefly, the GP quantitatively reports the shift in the emission spectrum of the
128 probe depending on the polarity of its immediate lipid environment. Thus, an increase in the
129 environment polarity is typically associated with liquid-disordered (Ld) phases, being more
130 fluid and less packed, as compared to liquid-ordered (Lo) phases. The relative GP index,
131 between -1 and 1, is inversely proportional to the membrane polarity and consequently, it
132 numerically increases with lipid packing or order (49, 50). We used yeast cells expressing
133 Cdc3 endogenously tagged with mCherry at its N-terminus (mCherry-Cdc3) to correlate GP
134 values with septin localization in early G1, when the septin signal is absent from the
135 presumptive bud site/bud neck, and during the rest of the cell cycle, where septins are bound
136 to the plasma membrane at the bud neck and form a collar (Figure 1B). We observed a
137 heterogenous lateral organization of GP values at the plasma membrane of yeast cells, as
138 denoted by the presence of patches with high GP values (GP > 0), irrespective of the cell
139 cycle phase (Figure 1B). The segmentation of the cell cortex from transmitted light images
140 indicated, however, that the overall GP values at the yeast plasma membrane was relatively
141 constant throughout the entire cell cycle (Figure 1C). The angular cross-section analysis
142 (Figure 1D) of the mCherry-Cdc3 signal and GP values at the cortex of mother cells showed
143 that the presence of septins at the bud neck correlates with a local increase in the Laurdan
144 GP index (~ΔGP ~ 0.3-0.5), as compared to septin-lacking regions either at early G1
145 (unbudded cells lacking a septin ring) or during the rest of the cell cycle (budded cells)
(Figure 1E). We observed that while the septin signal spanned a region of ~1 µm, in agreement with the expected bud neck diameter (26), reorganization of GP values expanded over larger distances, suggesting that septins might affect the lateral membrane fluidity over a long-range. To assess that the effect was not due to actin cables and patches that also populate the bud neck (51), we treated cells with Latrunculin A (LatA), which inhibits actin polymerization (Figure 1C-E). Under these conditions, we observed the same trend in the local increase of GP values overlapping with the septin signal at the division site without affecting the global fluidity of plasma membrane, suggesting that the observed effects are independent of the actin cytoskeleton organization and likely ascribable directly to septins.

Septin architecture and membrane remodeling are intimately connected during the cell cycle (28). Consequently, we asked if specific septin architectures may correlate with the observed effect on the lateral organization of the yeast plasma membrane. To this end, we performed STED microscopy on yeast cells endogenously expressing Cdc10-yeGFP and immunolabeled with an anti-GFP nanobody Atto 647N (52). Under our spatial-resolution (i.e., ~ 45 nm), we could detect an accumulation of the Cdc10-yeGFP signal at the presumptive bud site at early stages of septin assembly that turned into a filament-like organization upon septin-hourglass formation (Figure 1F), in line with a previous report (52). Interestingly, we also identified the presence of discrete sub-micrometric septin patches that were particularly visible at the expected contour of the cell and, often, in proximity to high-order septin structures (Figure 1F and Figure 1-figure supplement 1), which shows the negative control, i.e. yeast cells not expressing Cdc10-yeGFP). These observations suggest that high-order and sub-micrometric septin structures at the plasma membrane might potentially contribute to septin-mediated lateral membrane organization.
<figure><img src="image_2.png" /><figcaption>Figure 1. Septin architecture and phase behavior of the yeast plasma membrane during the cell cycle. A)</figcaption></figure>

Schematic representation of the lipophilic probe Laurdan (blue) and its partitioning in Lo (green) and Ld phases (red). **B)** Transmitted light (TL) and confocal images of yeast cells expressing mCherry-Cdc3 (gray) and stained with the Laurdan probe after 10 min incubation at 30°C. Color-coded GP images (fire LUT) of z-stacks maximum projections of unbudded or budded cells, either untreated (DMSO, -LatA) or treated with 0.1 mM of LatrunculinA (+LatA). Scale bar, 2 µm. **C)** Quantification of the global GP index at the plasma membrane of unbudded or
178 budded cells, either untreated (DMSO, -LatA) or treated with 0.1 mM of LatrunculinA (+LatA). Number of cells 179 analyzed, n = 12, 12 and 12, respectively, from at three biological replicates. Error bars represent s.d.; Mann-180 Whitney test: n.s. P > 0.05. D) Schematics showing the angular cross-section analysis (green-dashed line) of the 181 plasma membrane (orange) starting for mother cells at the opposite pole of the bud neck or, for unbudded cells, 182 at an arbitrary position, 0°, following a counterclockwise direction. E) Average cross-section analysis (n = 6 cells) 183 showing the mCherry-Cdc3 signal (gray) and GP index values (orange) at different angular positions, as detailed 184 in D from three biological replicates. Error bars represent s.d. F) STED images of yeast cells expressing Cdc10-185 yeGFP revealed with an anti-GFP nanobody Atto647N during early septin assembly at the presumptive bud site 186 and early hourglass assembly at the bud neck. Scale bar, 1 µm. Distribution of the apparent area of septin 187 patches (µm²) at the mother cell. N = 10 cells, number of patches, n = 421 from two biological replicates. 188

189 Assembly of septin patches on membranes is a diffusion-driven process.

190 To get insights into the mechanisms leading to septin patch formation in yeast cells, we set 191 out to investigate *in vitro* the contribution of the lipid membrane, as this feature was 192 previously shown to modulate septin architecture (17, 37). First, we determined by liposome 193 floatation assays the binding affinity of septins for membrane lipids at physiological ionic 194 strength (i.e. 150 mM NaCl). To this end, we used purified recombinant yeast septin 195 octamers (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11), as previously reported 196 (53), and we allowed their binding to small unilamellar vesicles (SUVs) made of either 100% 197 mol phosphatidylcholine, as a neutral lipid mixture, or 25% of total negatively charged lipids, 198 including phosphatidylinositol (PI) or equivalent % mol of PI(4,5)P₂, phosphatidic acid (PA), 199 or phosphatidylserine (PS) at the expenses of PI, as detailed in Table S1. We determined 200 septin binding through western-blot analysis by comparing the signal of Cdc11 at the top 201 (SUV-bound protein) and bottom (unbound protein) of a sucrose gradient (54) (Figure 2A). In 202 the absence of SUVs, septins were collected in the bottom fraction (CTRL, Figure 2A). In the 203 presence of SUVs, yeast septins bound to SUVs of almost all tested negatively charged lipid 204 compositions, except for PS, with an increased affinity for PI(4,5)P₂, in agreement with 205 previous studies (17, 37–39). Noteworthy, septins also bound the neutral PC lipid, pointing 206 out that they not only display an affinity for anionic lipids.
We then characterized by sub-diffraction Airyscan microscopy the contribution of the lipid composition to the spatial organization of septins (Figure 2C). To this end, 5 nM of GFP-tagged septin octamers were allowed to bind to supported lipid bilayers (SLBs) made with the same composition as that used in liposome floatation assays, with the addition of trace amounts (0.2%) of Atto647N-labeled DOPE (A647-DOPE) to visualize the bilayer surface (Figure 2-figure supplement 1). Airyscan imaging showed that septins organized into sub-micrometric patches randomly distributed over the bilayer surface for all tested lipid compositions (Figure 2C). The formation of septin patches was, however, more evident on PI(4,5)P₂-containing SLBs.

To get further insights into the formation of septin patches, we focused on SLBs containing 2.5% mol PI(4,5)P₂, as it was the lipid composition displaying the highest septin association with membranes (Figure 2A). Furthermore, PI(4,5)P₂ is predominantly localized at the plasma membrane, where many of the septin's functions have been reported to take place (29, 30). We estimated the apparent area of septin patches on PI(4,5)P₂-containing SLBs to be between 0.05 to 2 µm² (Figure 2D), in agreement with the same range of magnitudes observed in yeast cells (Figure 1F). The quantification of the density of membrane-bound GFP-tagged septin octamers (d), as described in (55, 56), showed an increased density of septins in larger patches (Figure 2D). However, we also detected a substantial population of sub-micrometric septin patches displaying high septin densities. This observation suggests that septin patches might contain layers of septin octamers, typically occurring in a dense septin meshwork, in agreement with a recent report (39).

Next, we evaluated the contribution of septin concentration by monitoring septin patch formation upon addition of 1 nM, 2 nM, 5 nM, or 10 nM of GFP-tagged septin octamers on SLBs made with 2.5% mol PI(4,5)P₂ (Figure 2F-G). We observed that the apparent area of septin patches increased with septin concentration up to 10 nM, where septin patches started to connect into a large and single structure. Evaluation of the density of septin octamers forming patches relative to the density of bulk membrane-bound septins ($d_{patch}/d_{bulk}$) showed a monotonic decrease with increasing septin concentrations (Figure 2G). Therefore,
235 membrane-bound septin octamers preferentially assemble into patches rather than diffusing
236 in the membrane bulk at low septin concentration.

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<figure><img src="image_3.png" /><figcaption>**Figure 2.** Binding and organization of septins on lipid membranes. A) 1 µM of Septin-GFP hetero-octamers were incubated without liposomes (-, CTRL) and with liposomes (2 mM) made of egg-phosphatidylcholine (PC) or with different lipid mixtures containing 25% total negative charge: Liver-phosphatidylinositol (PI), Brain-PI(4,5)P₂ (PI(4,5)P₂), Egg-phosphatidic acid (PA), Brain-phosphatidylserine (PS). The suspension was subjected to liposome floatation assays. The composition of each liposome suspension was: PI(4,5)P₂: 80% mol PC, 15% mol PI, 5% mol PI(4,5)P₂; PI: 75% mol PC, 25% mol PI; PA: 75% mol PC, 15% mol PI, 10% mol PA; PS: 75% mol PC, 15% mol PI, 10% mol PS; PC: 100% mol PC. 1 µM of Septin-GFP hetero-octamers were polymerized in</figcaption></figure>

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247 septin polymerization buffer and the liposome-bound (Top, T) or free (Bottom, B) septin Cdc11 was detected by
248 western blotting with anti-Cdc11 antibody. B) Quantification of septin-GFP binding from liposome floatation
249 assays (right panel): septin-GFP binding = Top/(Top+Bottom) x 100. Results represent the mean ± s.d. from three
250 biological replicates. C) Airyscan images showing the association of 5 nM septin-GFP in septin buffer with SLBs
251 made with the same lipid composition as in A. Scale bar, 1 µm. D) Representation of the area of septin patches
252 (µm²) as a function of the septin density (d, µm⁻²) on 2.5% mol PI(4,5)P₂-SLBs upon addition of 5 nM of septin-
253 GFP heterocomplexes (n = 477 septin patches, from three biological replicates). E) Airyscan images showing the
254 association of 1 nM, 2 nM, 5 nM and 10 nM of septin-GFP with SLBs made of 2.5% mol PI(4,5)P₂. Scale bar, 2
255 µm. F) Representation of the area of septin patches (µm²) as a function of septin concentration (nM). G) Ratio of
256 the membrane-bound septin density (d, µm⁻²) on patches (dₚatch) versus at the membrane bulk (dₐulk) as a function
257 of septin concentration (nM). Results in F and G represent the mean ± s.d (n = number of patches from two
258 biological replicates, n = 615, 351, 477, 1036, respectively).

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261 The formation of elongated septin filaments and high-order structures has been proposed to
262 stem from a diffusion-driven motion of septins in the presence of membranes (57). The
263 results above indicate that septin patch formation might also originate from a 2D diffusion of
264 septin octamers on membranes. To get further insights into the contribution of this process,
265 we monitored the fluorescence recovery of GFP-tagged septin signal on PI(4,5)P₂-containing
266 SLBs upon the addition of 5 nM of septins and formation of septin patches (Figure 3A-C).
267 Upon septin patch bleaching, as indicated by the cross-section analysis in Figure 3B (orange
268 dashed line in Figure 3A), we observed a mild fluorescence recovery of the septins-GFP
269 signal after ≥ 7 min on septin patches (Figure 3C). Indeed, we obtained that the recovery
270 time of the fluorescent signal, τ, was τ = (77 ± 12)s. We can then estimate the mobile
271 fraction φ<sub>m</sub> = (I<sub>∞</sub> - I<sub>0</sub>) / I<sub>i</sub> = 0.16 % and the immobile fraction φ<sub>imm</sub> = 1 - φ<sub>m</sub> = 0.84 %. High
272 immobile fraction indicates a high effective viscosity in the septin patch. An effective diffusion
273 constant can be estimated by D<sub>eff</sub> = ω<sup>2</sup> / (4τ) ~ (0.99 ± 0.16) 10<sup>-14</sup> m<sup>2</sup>/s indicating a slow two-
274 dimensional septin dynamics (Figure 3-figure supplement 1). This value was still more than a
275 decade lower than the isotropic diffusion constant ($D_i$) of a single septin-octamer center of mass at long time (58):

277 $D_i = \frac{k_B T \ln(\frac{L}{d})}{3\pi\eta L} \sim 0.113 \ 10^{-12} m^2/S$,

278 where $L \sim 32$ nm is the septin octamer length, $d \sim 4$ nm is the rod diameter, $\eta \sim 1$ Pa.s is the membrane viscosity (1000 times the water viscosity), $k_B = 1.38 \ 10^{-23} J/K$ is the Boltzmann constant and $T \sim 300 K$ is the system temperature (in Kelvin units). We confirmed that this trend was independent of the SLB fluidity by evaluating the fluorescence recovery of the lipid membrane (Figure 3-figure supplement 2). The cross-section analysis of the septin signal at $\ge 7$ min post-bleaching showed a moderate increase in the septin intensity at the bleached region relative to the same region at the moment of bleaching (Figure 3D), suggesting that the diffusion of septin octamers in the membrane bulk may contribute to the formation of the septin patch. To confirm the involvement of a diffusion-driven process in septin-patch assembly, we depleted the system from membrane-unbound septins by washing out three times the imaging chamber. Under these conditions, we could not detect a decrease in the membrane-bound septin-GFP signal both in the bilayer bulk and septin patch, suggesting that depletion of septins in solution do not affect the behavior of septin patches once the equilibrium is reached (Figure 3-figure supplement 3). As expected, replenishment of the system with 5 nM of GFP-tagged septin complexes was accompanied by a rise in the global septin intensity that led, after 7 min of incubation, to a significant increase of the intensity at the bleached and non-bleached region of the septin patch. This observation suggests that the assembly of septin octamers into a patch-like structure might be driven by septin diffusion in the membrane plane and the addition of septin layers, possibly through the association of septins from the bulk solution.

298 To get further insight on the structural organization of septins on PI(4,5)P$_2$-containing SLBs, we used correlative light and atomic force microscopy (CLAFM) (Figure 3E-G). Addition of 5 nM of GFP-tagged septin octamers to SLBs lead to the formation, as expected, of septin patches that we first identified using fluorescence microscopy and then characterized
through AFM topography imaging (Figure 3E). In agreement with previous AFM studies, we also observed the appearance of lipid blobs associated with yeast septins (47, 53), possibly resulting from bacterial lipids co-purifying with septins, as previously reported for other lipid-binding proteins (59). The cross-section analysis of the contact point map, which corresponds to the sample's topography acquired at the minimal loading force (i.e., at the tip-sample contact point), showed that septin patches mostly protrude by ~ 4 nm above the lipids (Figure 3F). This height is consistent with the thickness of yeast septin octamers (60), suggesting that septins are likely laying as a monolayer on top of the membrane. Furthermore, the presence of some peaks with an 8 nm height suggests that septins can also be present as two layers on top of each other, in agreement with previous studies with *Drosophila* septins (39). Closer inspection of septin patches showed that they are formed of randomly organized short septin filaments of a length between 60 to 150 nm (Figure 3G), which corresponds to 2 to 5 multiples of octamers (60). Collectively, these observations suggest that yeast septin hetero-octamers assemble as filament-based patches on PI(4,5)P₂-containing membranes.

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<figure><img src="image_14.png" /><figcaption>Figure 3. Dynamics and structural organization of septins on PI(4,5)P₂-containing SLBs.</figcaption></figure>

A) Confocal images of septin-GFP bleaching and fluorescence recovery experiment after 10 min binding of 5 nM of septin-GFP on 2.5% mol PI(4,5)P₂-containing SLBs at different time points: before bleaching (T = -30 s), at the bleaching moment (T = 0 s), after bleaching (T = 420 s), after the solution wash out (T = 420 s), and after replenishment with 5 nM of septin-GFP (T = 1250 s). The FRAP ROI is indicated in yellow. Scale bar, 2 µm.
B) Cross-section of the septin-GFP signal along the orange line in A before bleaching (blue) and at the bleaching
moment (gray). The different features of the septin signal on SLBs are highlighted (septins at the bulk membrane and bleached and non-bleached septin patch). C) Representation of the septin-GFP intensity as a function of time (in seconds) at the FRAP region. Graph represents the mean ± s.d (n = 8, ROIs from two independent experiments). D) Cross-section of the septin-GFP signal along the orange line in A at the bleaching moment (gray), after bleaching (blue), and after replenishment with 5 nM of septin-GFP (orange). E) Correlative fluorescence (left) and AFM topography (right) images of septin-GFP patches on 2.5% mol PI(4,5)P₂-SLBs. Scale bar, 1 µm. False-color scale in AFM images, 17 nm. F) Magnified AFM topography images corresponding to the white box in E (scale bar, 250 nm and 50 nm for the magnified image. False-color scale, 7 nm and 20 nm). G) Contact point image of the topography image in F (scale bar, 250 nm. False-color scale, 7 nm). Cross-section along the white line showing the height of septin-GFP hetero-octamers and the membrane relative to the glass substrate. H) Length distribution of septin filaments (n = 73 from two independent AFM experiments).

341

## Septin assemblies modulate the microscopic organization of membranes *in vitro*

Next, we set out to investigate if filament-based septin assemblies observed on PI(4,5)P₂ membranes might also impact lipid organization *in vitro*. To this end, we monitored the binding of 5 nM GFP-tagged yeast septin octamers onto 2.5% mol PI(4,5)P₂-containing SLBs doped with 0.2% mol of two distinct lipid dyes: Atto647N-labeled DOPE (A647-DOPE) and Texas Red-labeled PI(4,5)P₂ (TF-TMR-PI(4,5)P₂) (Figure 4A-B). Sub-diffraction Airyscan microscopy images showed that before septins addition, SLBs displayed a homogenous distribution of both lipid dyes. As expected, addition of 5 nM of septins led to the immediate formation of septin domains that was accompanied by a marked time-dependent redistribution of the A647-DOPE dye (Figure 4A and Figure 4-figure supplement 1). Cross-section analysis showed that A647-DOPE-depleted regions co-localize with the occurrence of septin patches (Figure 4B). Conversely, at the same regions, we did not detect an apparent depletion of the TF-TMR-PI(4,5)P₂ dye. The kymograph analysis of septin binding also showed that nucleation and growth of septin patches immediately engage a selective depletion of the DOPE dye with respect to the PI(4,5)P₂ dye (Figure 4C). The analysis of the relationship between the apparent size of septin patches as a function of the average intensity of either the A647-DOPE or TF-TMR-PI(4,5)P₂ signal showed that the intensity of the PI(4,5)P₂ dye appeared steady and thus, independent of the septin patch size (Figure
360 4D). Conversely, depletion of the DOPE signal showed a size-dependency, with an almost
361 total signal depletion with septin patches larger than 0.1 µm². We found that depletion of the
362 A647-DOPE signal induced by the septin patches was not due to fluorescence quenching
363 (Figure 4-figure supplement 2), as we did not detect an increase of the Atto647N signal upon
364 bleaching GFP-tagged yeast septins. Furthermore, A647-DOPE depletion did not depend on
365 the wavelength and type of the fluorescent dye of the DOPE derivative (Figure 4-figure
366 supplement 3). We also confirmed that the effect on A647-DOPE partitioning was specific to
367 septins by monitoring the lipid organization upon addition of 1 µM of the PH domain of
368 PLCδ1 (PH<sup>PLCδ1</sup>), which selectively interacts with PI(4,5)P₂ (61), fused to GFP (GFP-PH<sup>PLCδ1</sup>)
369 on 2.5% mol PI(4,5)P₂-containing SLBs (Figure 4-figure supplement 4). We found that
370 addition of GFP-PH<sup>PLCδ1</sup> led to its immediate membrane association. However, this was not
371 mirrored by any significant lateral redistribution of the A647-DOPE or TF-TMR-PI(4,5)P₂ lipid
372 dyes (Figure 4-figure supplement 4). Collectively, these data suggest a direct effect of
373 septins on the lateral distribution of lipids. This feature appears to be selective for the A647-
374 DOPE reporter and moderate or undetectable for other lipid derivatives, such as PI(4,5)P₂ or
375 phosphatidylcholine (PC) (Figure 4-figure supplement 3C-D). This septin-mediated selective
376 exclusion of A647-DOPE molecules in the membrane plane might be related to the
377 decreased affinity of septins for phosphatidylethanolamine (PE) observed in liposome
378 floatation assays at different % mol of PE (Figure 4-figure supplement 5).

379 Next, we assessed if septins can directly modify the macroscopic organization of liquid-
380 ordered (Lo) and liquid-disordered (Ld) phases using a model ternary mixture consisting of
381 unsaturated DOPC, saturated DPPC and cholesterol that is typically used to mimic the
382 plasma membrane of eukaryotic cells (9, 49). We monitored the dynamic interaction of 5 nM
383 septin-GFP complexes on mica-supported lipid membranes made of 2.5% mol Brain-
384 PI(4,5)P₂, 2.5% mol DOPC, 30% mol DPPC, 44.8% mol cholesterol, 20% mol Liver-PI and
385 trace amounts (0.2% mol) of the Lo marker DSPE-PEG-ASR (49) (Figure 4E-G). After mica-
386 SLB preparation, samples were equilibrated at room temperature for one hour and placed
387 under the confocal/Airyscan microscope at a temperature of ~ 25 °C. Under these conditions,
the ternary mixture containing 2.5% mol PI(4,5)P₂ displayed a mixed phase, as the Lo marker did not appear to display preferential partitioning (Figure 4E). The addition of septin complexes led to their immediate association with membranes and the formation of septin patches with a larger size as compared to SLBs lacking cholesterol (Figure 4H). We observed that cholesterol-containing SLBs septin domains expanded over time and eventually collided, reaching micrometric sizes after ≥ 5 min. The appearance of septin patches was mirrored by a marked reorganization of the Lo marker that appeared excluded from the septin-GFP signal (Figure 4G), suggesting a direct effect of septins on the behavior of Lo/Ld phases *in vitro*. Collectively, these observations point out that formation of septin patches directly modulate the phase behavior and macroscopic re-organization of lipids *in vitro*.

399
<figure><img src="image_15.png" /><figcaption>**Figure 4.** Microscopic organization of *in vitro* membranes upon septin binding. **A)** Airyscan images showing the organization of 5 nM septin-GFP hetero-octamers (in blue) injected in septin buffer onto SLBs containing 2.5%</figcaption></figure>
403 mol Brain-PI(4,5)P₂ and doped with 0.2% mol fluorescent TF-TMR-PI(4,5)P₂ (in gray) or 0.2% mol fluorescent A647-DOPE (in magenta) at T = 0 and T = 10 min. Scale bar, 5 µm. **B**) Cross-section analysis along the dashed line in A. **C**) Kymograph analysis along the blue arrow in A of septin-GFP signal (blue), A647-DOPE (magenta) and TF-TMR-PI(4,5)P₂ (gray) from T = 0 to 8 min. **D**) Intensity distributions of A647-DOPE (magenta) and TF-TMR-PI(4,5)P₂ (gray) relative to the size of septin patches (µm²) from three biological replicates. **E**) Airyscan images showing the dynamics of septin-GFP hetero-octamers (in blue) binding to mica-supported SLBs made of 2.5% mol Brain-PI(4,5)P₂, 2.5% mol DOPC, 30% mol DPPC, 44.8% mol cholesterol, 20% mol Liver-PI, Lo marker (0.2% mol fluorescent DSPE-PEG-ASR, in orange) and TF-TMR-PI(4,5)P₂ (in gray). Scale bar, 5 µm. **F**) Kymograph analysis along the yellow line box in E of the septin-GFP (blue), DSPE-PEG-ASR (Lo marker, orange) and TF-TMR-PI(4,5)P₂ (gray) signal from T= 0 min to 5 min. **G**) Cross-section analysis along the dashed line in the kymograph. **H**) Quantification of the size of septin domains on membranes containing 0% mol or 48% mol cholesterol from Airyscan images. Domains analyzed, n = 6693 and 163, respectively from ≥ three replicates. Error bars represent s.d.; Mann-Whitney test: **** P < 0.0001.

416

417 The polybasic domain of Cdc11 and amphipathic helix of Cdc12 support the
418 membrane reorganization and curvature-sensing properties of septins.

419 Septin interaction with membranes is thought to be mediated by a polybasic (PB) motif, 420 adjacent to the GTP-binding motif at the N-terminus, and by an amphipathic helix (AH) 421 downstream to the septin-unique element (SUE) (17, 37, 40–42, 47, 62). While the PB motif 422 has been primarily reported to mediate septin interaction with anionic lipids (17, 37, 38, 45), 423 the AH was shown to mediate both membrane association and micron-scale curvature 424 sensing of septins *in vitro* and *in cellulo* (40, 41, 43). To determine the contribution of the PB 425 and AH domains on the lipid reorganization-mediated by septins, we generated and purified 426 recombinant GFP-tagged septin octamers carrying a deletion of the PB motif of Cdc11 427 (residues 2-18; ΔPB-Cdc11) or a C-terminal deletion of Cdc12 that eliminates part of the AH 428 (residues 390-407; ΔAH-Cdc12), or the complete deletion of the C-terminal extension (CTE) 429 of Cdc12 (residues 339-407; ΔCTE-Cdc12) (Figure 5A-E and Figure 5-figure supplement 1). 430 These mutants were designed on the basis of previously published septin mutants (17, 41, 431 432 430
monolayers (Figure 5A). We observed that under our conditions wild-type septin-GFP complexes annealed end-to-end, forming short single filaments made of two to three multiples of octamers, as well as long paired filaments connected by regular lateral bridges. Under these conditions, all complexes containing ΔPB-Cdc11 or ΔAH-Cdc12 or ΔCTE-Cdc12 mutants kept their ability to associate with lipid monolayers, although they did not organize into long paired filaments. For the ΔPB-Cdc11 mutant this observation is consistent with the known role of the α0 helix of Cdc11, where the PB motif is located to mediate septin polymerization in solution (60). However, while the Cdc12 mutants assembled into short filaments mostly made of four septin hetero-octamers, the ΔPB-Cdc11 complexes stayed mainly as octamers or double octamers (Figure 5C).

Consistent with their ability to associate with membranes, GFP-tagged septin complexes containing ΔPB-Cdc11, ΔAH-Cdc12, or ΔCTE-Cdc12 also organized into septin patches on 2.5% mol PI(4,5)P₂-containing SLBs (Figure 5B). Airyscan imaging showed a similar septin-patch organization and total surface of septin occupancy (% relative to the SLB surface) between wild-type septins and septin hetero-octamers containing the two Cdc12 mutants (Figure 5D-E). In contrast, septin complexes containing ΔPB-Cdc11 assembled into smaller patches, consistent with their reduced capacity to form longer filaments (Figure 5C). Despite this feature, the ΔPB-Cdc11 septin complexes showed a significant increase in the SLB surface occupancy, as compared to wild-type or Cdc12 mutant septin complexes (Figure 5D). Similar to their wild-type counterpart, the assembly of the ΔPB-Cdc11, ΔAH-Cdc12, or ΔCTE-Cdc12 septin complexes on 2.5% mol PI(4,5)P₂-containing membranes led to the appearance of A647-DOPE depleted regions, which however were smaller in size as compared to wild-type septin complexes (Figure 5E). The two Cdc12 mutants showed, however, a similar average size of A647-DOPE depleted areas, suggesting that deletion of the entire CTE does not further compromise the lipid-organizing properties of septins relative to the AH partial deletion.

Septin function at the plasma membrane is usually linked to curvature-associated localizations (30, 63). Therefore, we set out to evaluate the curvature-sensing ability of septin
complexes containing ΔPB-Cdc11 or ΔAH-Cdc12 on 2.5% mol PI(4,5)P₂-containing membranes. To introduce curvature, we engineered silica substrates to display an array of conic-shaped structures with a base diameter of ~ 1.4 µm and a height of ~ 800 nm by soft nano-imprint lithography (soft-NIL) (Figure 5F), as we previously reported (64, 65). Silica conic-shaped substrates were functionalized with 2.5% mol PI(4,5)P₂-containing SLBs doped with 0.2% mol fluorescent TF-TMR-PI(4,5)P₂ (Figure 5G) and the association of wild-type and mutant GFP-tagged septin complexes at the top (radius *r* ~ 0.15 µm), middle (*r* ~ 0.5 µm), and bottom (*r* ~ 0.7 µm) of the structure was characterized by Airyscan microscopy (Figure 5G-H). The enrichment (*E*) of 100 nM wild-type septin hetero-octamers at a given curvature (κ = 1/*r*) of the conic structure (*I*<sub>c</sub>) was obtained from the GFP-tagged septin signal (I<sup>s</sup>) normalized towards the lipid intensity (I<sup>l</sup>) relative their respective signals on the flat SLB, i.e., the surface of silica without cones (I<sub>h</sub>) (*E* = I<sub>c</sub><sup>s</sup>/I<sub>c</sub><sup>l</sup>). Under these experimental conditions, wild-type septins showed a preferential accumulation on positive curvatures, consistent with previous observations using lipid-functionalized silica beads and wavy substrates (40, 42). We found that wild-type septins displayed the highest accumulation at κ ~ 6.6 µm⁻¹, while this was not the case for the ΔAH-Cdc12 mutant that instead did not show preference for any positive curvatures tested. Furthermore, the ΔPB-Cdc11 only accumulated (i.e., enrichment ≥ 1.0) at κ ~ 6.6 µm⁻¹ with a relative enrichment that was ~1.2-times lower than wild-type septins. However, the membrane binding of the ΔPB and ΔAH mutant was 4 to 3.4-times lower than that of wild-type septin complexes, respectively (Figure 5-figure supplement 2), which might explain the effect in the curvature-sensing profile of the ΔPB-Cdc11 and ΔAH-Cdc12 mutants, as curvature recognition was shown to be septin concentration-dependent (40).

484
<figure><img src="image_16.png" /><figcaption>**Figure 5.** Association of wild-type septins and septin mutants with lipid membranes. A) TEM images after negative staining showing the structural organization of 100 nM wild-type septin complexes or ΔPB-Cdc11, ΔAH-</figcaption></figure>
Cdc12, ΔCTE-Cdc12 mutant septin complexes on monolayers made of 77.5% mol Egg-PC, 2.5% mol Brain-PI(4,5)P2, 20% mol Liver-PI. Samples were incubated overnight at 4°C. Scale bar, 100 nm, 50 nm for the inset.

**B)** Airyscan images showing the association of 5 nM wild-type or mutant GFP-septin hetero-octamers with SLBs containing 2.5% mol PI(4,5)P₂ and 0.2% mol fluorescent A647-DOPE at T = 1h. Scale bar, 1 µm.

**C)** filament length (in nm) of wild-type septins and septin mutants obtained from the TEM images in A (number of filaments, n = 48, n = 33, n = 35, n = 50, as in the graph from two independent experiments).

**D)** Total surface (%) of the SLB occupied by septin patches of wild-type or mutant septin-GFP hetero-octamers (n = 10 images analyzed per condition, from three biological replicates).

**E)** Size of A647-DOPE-depleted regions after addition of wild-type and mutant septin-GFP hetero-octamers (n = 4790, 5464, 450, 5425, respectively (as in the graph order) from three biological replicates).

**F)** SEM images of silica substrates displaying an array of cones with a radius r ~ 0.7 µm at their base and a height h ~ 800 nm generated by soft-NIL. Scale bar, 3 µm. Representation of the acquisition of a single plane at the top and bottom of cones to visualize the distribution of lipids and septins.

**G)** Average intensity (AVG projection of n > 5 images) of the surface distribution of fluorescent PI(4,5)P₂ (gray) and the surface distribution of 100 nM wild-type, ΔPB-Cdc11 or ΔAH-Cdc12 septin-GFP complexes (Fire LUT) at the top and bottom of cones functionalized with 2.5% mol PI(4,5)P₂-containing membranes. Scale bar, 1 µm.

**H)** Enrichment of wild-type, ΔPB-Cdc11 or ΔAH-Cdc12 mutants septin-GFP complexes at the top (κ ~ 6.6 µm⁻¹), middle (κ ~ 2 µm⁻¹) and bottom (κ ~ 1.4 µm⁻¹) of nano-cones relative to the flat membrane (κ ~ 0 µm⁻¹). Nano-cones analyzed, n = 265, 277, 342, and 580; n = 269, 279, 376 and 540; n = 266, 275, 315 and 586, for wild-type, ΔPB-Cdc11 or ΔAH-Cdc12 mutants respectively, from three technical replicates. Error bars represent s.d.; One-way ANOVA test: n.s > 0.05, \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.0001.

## The polybasic domain of Cdc11 assists membrane remodeling by septins at the bud neck

The results above suggest that the Cdc11 PB and Cdc12 AH domains might play a role in the membrane remodeling function of septins *in cellulo*. We focused our investigations on the less explored function of the Cdc11 PB domain in cells, compared to the Cdc12 AH (41). To this end, we generated a *cdc11(Δ2-18)* mutant yeast strain that carries a deletion of the PB domain. The *cdc11(Δ2-18)* mutant was viable at 30°C, consistent with previous data (66), but temperature-sensitive at 37°C (Figure 6-figure supplement 1), indicating that the PB domain becomes important for yeast cells to survive at high temperatures. First, we monitored the
recruitment of mCherry-Cdc3 to the bud neck of *cdc11(Δ2-18)* cells at 30°C by time-lapse microscopy (Figure 6A-C). We found that while the kinetics of septin recruitment in mutant cells was comparable to that of the isogenic wild-type strain, 100% of cells carrying a deletion of the PB domain failed to undergo septin ring splitting (Figure 6B). This trend was also detected at 37°C (Figure 6-figure supplement 2), where, in addition, ~ 20% of mutant cells also displayed an absence of septin recruitment at the bud neck at the time of bud emergence (Figure 6B), thus explaining their markedly reduced viability at this temperature (Figure 6-figure supplement 1). Next, we measured septin intensity at the bud neck of *cdc11(Δ2-18)* mutant and *CDC11* wild-type small- and medium-budded cells (Figure 6C). We found a significant increase of mCherry-Cdc3 fluorescence between small- and medium-budded cells in all the conditions tested, consistent with a maturation of the septin collar during cell cycle progression. At 30°C, we observed a significant decrease in the mCherry-Cdc3 intensity at the bud neck of medium-budded *cdc11(Δ2-18)* cells compared to the *CDC11* control. This trend was, however, enhanced at 37°C, where both small- and medium-budded *cdc11(Δ2-18)* mutant cells had significantly decreased septin levels at the bud neck relative to their wild type counterpart. Collectively, these results suggest that the PB domain of Cdc11 plays an important role in septin collar assembly and reorganization during the yeast cell cycle.

Next, we investigated the septin collar architecture in cells lacking the PB domain of Cdc11 by STED imaging. To this end, we used wild-type *CDC11* and *cdc11(Δ2-18)* mutant cells expressing Cdc10-yeGFP that were immunolabeled with an anti-GFP nanobody Atto 647N (Figure 6D). We found that in *cdc11(Δ2-18)* cells septin structures appeared fragmented at the bud neck (Figure 6E). We also detected an increased presence of septin patches along the expected contour of the plasma membrane in mutant cells compared to the *CDC11* strain, in agreement with the pattern observed *in vitro* (Figure 5). Furthermore, the septin collar of cells lacking the PB of Cdc11 appeared less organized than the isogenic wild-type strain, which instead showed the presence of filaments ordered in parallel to the mother-bud axis, in agreement with previous reports (28, 52).
The results above indicate that the PB domain of Cdc11 is crucial for septin architecture at the bud neck of yeast cells (Figure 6B) and/or an effect of the Cdc11 PB domain on the lateral organization of the yeast cell membrane, as observed in our in vitro studies (Figure 5).

Therefore, we set out to monitor the GP of the Laurdan probe incorporated in CDC11 and cdc11(Δ2-18) mutant cells expressing mCherry-Cdc3 (Figure 6F). At 30°C, the cdc11(Δ2-18) mutant did not display a significant change on the global fluidity (GP index) of the plasma membrane compare to wild-type cells (Figure 6-figure supplement 3). However, we observed that the local increase in the Laurdan GP index at the mother cell bud neck overlapping with the mCherry-Cdc3 signal was significantly lower for the cdc11(Δ2-18) mutant cells, with only~20-30 % of cells undergoing a reorganization of high GP values (GP > 1, indicative of Lo phase), as compared to the isogenic wild-type strain (Figure 6G). To determine if other membrane-binding domains also support the effect of septins on the plasma membrane fluidity, we used the cdc12-6 mutant that carries a C-terminal deletion that eliminates part of the AH domain (residues 390-407) (41). In the case of the cdc12-6 mutant, experiments were performed at 25°C, as septins mostly depolymerized at 30°C (67, 68). At 25°C, cells lacking the AH domain of Cdc12 also showed a reduction in the number of cells displaying a local GP reorganization, compared to the CDC12 wild-type strain (Figure 6-figure supplement 3).

This indicates that the AH domain of Cdc12 and PB domain of Cdc11 participate in the plasma membrane-organizing function of septins in yeast cells.
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>Figure 6. Dynamics and organization of the septin collar in wild-type and cdc11(Δ2-18) yeast cells. A)</figcaption></figure>

Transmitted light (TL) and TIRF images of wild-type *CDC11* and *cdc11(Δ2-18)* mutant cells expressing mCherry-

Cdc3. Cells with the indicated genotypes were imaged every 5 min for 6 h at 30 °C. Scale bar, 2µm. B)

Quantification of cells according to their phenotype for what concerns septin ring splitting (purple), unrecruited

566

567

568

569

570
(gray), and recruited (blue) septins at the bud neck of *CDC11* and *cdc11(Δ2-18)* mutant cells expressing mCherry-Cdc3 at 30°C and 37°C. The number of cells analyzed is indicated in the graph, from two biological replicates. C) Septins integrated density (IntDensity) at the neck of *CDC11* and *cdc11(Δ2-18)* budding cells according to the bud size: small (orange) and medium (purple) at 30°C and 37°C. Number of structures analyzed, n = 22, n = 24, n = 28, n = 25, n = 21, n = 24, n = 20, n = 20, respectively from two biological replicates. Error bars represent s.d.; Mann-Whitney test: n.s. > 0.05, * P < 0.05, ** P < 0.01. D) STED images of *CDC11* and *cdc11(Δ2-18)* mutant cells expressing Cdc10-yeGFP revealed with an anti-GFP nanobody Atto647N. Magnified images are identified with the corresponding color. Scale bar, 1 µm. E) Cross-section analysis of the outmost septin signal along the mother-bud axis as indicated in the cartoon. Average cross-section (n = 6 cells) of the anti-GFP nanobody Atto647N signal of *CDC11* and *cdc11(Δ2-18)* mutant cells. Error bars represent s.d. F) TL and confocal images of *cdc11(Δ2-18)* budded cells expressing mCherry-Cdc3 (magenta) and stained with the Laurdan probe. Color-coded GP value images (fire LUT) of z-stacks projections. Scale bar, 2 µm. G) % of *CDC11* and *cdc11(Δ2-18)* mutant cells displaying high GP values (GP > 1, indicative of Lo phases) at the bud neck of the mother cell cortex. Number of images analyzed, n = 13, and n = 11, respectively, from three biological replicates. Error bars represent s.d.; Mann-Whitney test: *** P < 0.0001.
589 DISCUSSION

590 Septins are central regulators of many membrane-associated processes in eukaryotic cells
591 (29, 30). Yet, how septins interact with and organize membranes remains a key question in
592 the field. In this study, we investigated the function of septins as direct regulators of the
593 lateral organization and remodeling of membranes *in cellulo* and *in vitro*. Septin collar
594 formation at the division site of budding yeast and mammalian cells plays an essential role as
595 a scaffold recruiting cytokinetic factors to the division site (69–71). Our results in the budding
596 yeast *S. cerevisiae* show that localization of septins at the division site correlates with a local
597 increase in the GP values of the environment-sensitive Laurdan probe and that this property
598 is independent of the actin cytoskeleton. However, septins do not impact the global fluidity of
599 the yeast plasma membrane elsewhere than at the bud neck. This observation suggests that
600 the lipid packing effect of septins is local and linked to their membrane-bound state.
601 Noteworthy, our results indicate that the PB and AH domains of Cdc11 and Cdc12,
602 respectively, are required in this process.

603 Using STED microscopy, we reported the presence of sub-micrometric septin patches
604 decorating the contour of the yeast plasma membrane coexisting with high-order septin
605 structures during the budding yeast cell cycle, enlarging the repertoire of previously reported
606 septin architectures (28, 52, 72). Our findings using a minimal reconstituted system of septin-
607 filament assembly on fluorescently-labeled lipid membranes show that septins organization
608 into filament-based patches appears to be regulated by the lipid composition and dependent
609 on the functional role of the predicted membrane-interacting interfaces of yeast septins. By
610 eliminating either the AH of Cdc11 or PB domain of Cdc12, we show that septin patch
611 formation relies on the collective assembly of septin arrays made of at least two to five end-
612 to-end hetero-octamers. We show that on PI(4,5)P₂ membranes, sub-micrometric septin
613 patches might originate from the coexistence of two septin pools. First, the membrane-bound
614 fraction of septins might lead to the assembly of larger filament-based structures through the
615 rotational and translational diffusion-driven interaction of octamers, consistent with previous
616 mathematical models and molecular dynamics on rod-shaped colloidal liquids (58, 73, 74).
Indeed, the diffusive behavior of a rod-shaped structure, such as a septin octamer, might involve different states, leading to different slowing-down phenomena, especially once the local density and viscosity of the system increases. We propose that septin patch formation stems from the translational diffusive motion of septins on flat membranes, as previously reported by (75), and the existence of a subdiffusive behavior arising from a rotational, end-over-end, motion, as pointed out by the observation of a non-constant diffusion coefficient.

At higher concentrations, $c \ge 1/L^2$, where L is the length of an octamer, the rotational motion of each octamer will appear severely restricted, as well as the translational motion perpendicular to the octamer axis thus, becoming the lateral interactions of short septin filaments more probable over filament annealing thus, favoring the patch appearance. The contribution of a rotational motion is also compatible with our AFM observations, showing that patches are made of short-filaments of septin arrays with different orientation angles.

Second, the resulting dense septin array might promote the appearance of septin layers from the vertical or in-plane association of the solution septin fraction, consistent with previous reports on fly septins (39).

Our results show that the integrity of septin structures is particularly impaired with $\Delta$PB-Cdc11 mutant complexes, despite their unmodified membrane-binding ability. It is possible that increased $PI(4,5)P_2$ concentrations might compensate for the decreased polymerization that we observe on 2.5% mol $PI(4,5)P_2$ lipid monolayers due to the PB deletion, as septin complexes carrying the same mutation are unable to polymerize in solution but form long filaments at 20% mol $PI(4,5)P_2$ (17, 60).

Interestingly, septin patches have been recently found at the yeast plasma membrane under conditions that induce autophagy (72, 76). These septin patches restrain the membrane diffusion of the autophagic protein Atg9 to modulate the autophagy response. Whether the autophagy-related septin patches arise from sub-micrometric septin patches that we observe by STED remains to be investigated. Our results suggest that septin patches might fulfill two complementary roles by facilitating compartmentalized molecular interactions of septin-interacting partners and/or as reservoirs to build larger high-order septin structures. The
645 identification of different septin architectures coexisting in cells compared to *in vitro* systems
646 reflects the essential role of cellular factors in regulating septin assemblies, such as septin-
647 interacting proteins(70), cytoskeletal forces(18, 25, 26), membrane topology (28), or lipid
648 composition. In this work, we focused on the role of the lipid composition and we found that
649 the size of septin patches grows on SLBs containing cholesterol, suggesting that in living
650 cells, where cholesterol (or ergosterol in budding yeast) is abundant, the formation of larger
651 septin assemblies compatible with their function as micron-scale diffusion barriers might be
652 modulated by the lipid composition.

653 Septin assembly at the division site is essential for forming and stabilizing the actomyosin
654 ring (18, 25, 26). By promoting membrane solidity through local changes in lipid packing,
655 septin assemblies may also facilitate the stabilization of dynamic membrane remodeling
656 processes, such as cell division (28) or cell blebbing (77), as cortex slippage can originate
657 due to the fluid nature of the lipid membrane (78).

658 Using PI(4,5)P₂-functionalized soft-NIL templates displaying micrometric to sub-micrometric
659 positive curvatures, we showed that the PB domain of Cdc11 contributes, although to a
660 minor extent, to the curvature-sensing properties of septins, in addition to the reported role of
661 the AH within the CTE of Cdc12 (40, 41). Previous work showed that the PB domain of
662 septins is important for septin polymerization and phosphoinositide specificity during
663 membrane association (37, 45, 60). Our results with the ΔPB-Cdc11 mutant, which is largely
664 defective in polymerization *in vitro*, suggest that septin filament assembly and membrane
665 association is an important feature of septin localization on membranes displaying positive
666 curvatures. Yeast cells expressing a Cdc11 mutant lacking the PB domain showed defects in
667 septin collar architecture and lack of septin ring splitting. As stated above, this mutant also
668 failed to engage a local membrane reorganization at the bud neck. Thus, in addition to a
669 direct effect on septin structural organization, the PB domain of septins could indirectly
670 impact septin architecture through their interaction with and remodeling of membranes.

671 Finally, our observations show that the existence of multiple curvatures in the same structure
672 facilitates septin accumulation at sub-micrometric diameters over micrometric-scale
673 curvatures previously observed on silica beads (40). Cooperative interactions dictate septin
674 binding to different curvatures and depend on the sum of septin assembly parameters such
675 as the K<sub>on</sub>/K<sub>off</sub> rates, protein diffusion and filament annealing (43). Thus, differences in protein
676 lateral mobility due to geometrically confined membranes (79), an enhanced PI(4,5)P<sub>2</sub>
677 interaction due to lipid defects associated with membrane curvature (80, 81) might modulate
678 septin assembly locally.

679 In conclusion, we have combined *in vitro* and *in cellulo* systems to uncover the molecular
680 mechanisms that place septins as direct regulators of the lateral organization of membranes.

681 Cytoskeleton-driven membrane compartmentalization is essential to support cellular
682 functions, from signal transduction to cell adhesion and migration, and this feature has been
683 largely studied for the actin cytoskeleton (5, 8). Septins fulfill a central role as cytoskeleton
684 scaffolds in many eukaryotic organisms and, unlike actin, directly bind membranes.

685 Therefore, it will be important to explore further the exact functional role of septin-mediated
686 local membrane compartmentalization in living cells.

687
688 MATERIALS AND METHODS

689 Lipids and reagents

690 Natural and synthetic phospholipids, including DOPC, DPPC, cholesterol, POPE, Egg-PC,
691 Liver-PI, Brain-PI(4,5)P₂ and fluorescent TopFluor-TMR-PI(4,5)P₂, TopFluor-TMR-PC,
692 Rhodamine-PE and Laurdan (6-Dodecanoyl-2-dimethylaminonaphthalene) are from Avanti
693 Polar Lipids, Inc. Atto647N-DOPE was from Sigma. DSPE-PEG-ASP was from Abberior.
694 Polyclonal rabbit anti-Cdc11 antibodies were from Santa Cruz (Sc-7170), DyLight 800
695 conjugated anti-rabbit secondary antibody was from Invitrogen (SA5-10044) and Latrunculin
696 A was from Sigma-Aldrich.

697

698 Yeast strains and plasmids

699 All yeast strains and plasmids used in this study are listed in Supplementary Table S2 and
700 S3. Yiplac211 plasmid bearing cdc11(Δ2-18) was generated by Gibson assembly and
701 verified by sequencing. Yiplac211 plasmid bearing CDC11 or cdc11(Δ2-18) were integrated
702 at the ura3 locus of a heterozygous CDC11/cdc11::TRP1 diploid strain after cutting with
703 Apal. Single copy integration was verified by Southern blot. Yeast cdc11::TRP1 haploid
704 strains complemented by the integrated CDC11 or cdc11(Δ2-18) constructs (ySP17533 and
705 ySP17529, respectively) were generated by sporulation and subsequent tetrad dissection. All
706 primers used for the generation of plasmids are listed in Supplementary Table S4.

707

708 Yeast growth conditions

709 Yeast cultures were grown at an appropriate temperature (i.e., 30°C for wild-type and
710 cdc11(Δ2-18) strains, and 25°C for the cdc12-6 mutant strain) overnight in synthetic SD
711 medium containing 2% glucose (live cell imaging) or in YEPD (STED microscopy). For
712 Laurdan experiments, the Laurdan dye was resuspended as a 5mM stock solution in DMSO
713 and used at 5µM. Cells were incubated for 1 hour at controlled temperature under agitation
714 (i.e., 30°C for wild-type and cdc11(Δ2-18) strains, and 25°C in the case of the cdc12-6
715 mutant strain). Latrunculin A (LatA) was prepared as a 10mM stock solution in DMSO and
716 cells were incubated with 0.1mM LatA for 10 minutes at appropriate temperature before
717 Laurdan staining (i.e., 30°C for wild-type and cdc11(Δ2-18) strains, and 25°C in the case of
718 the cdc12-6 mutant strain).

719

720 Protein expression and purification

721 Plasmids encoding either MBP-Cdc12/(6xHis)Cdc3-GFP or Cdc10/Cdc11 (82) were co-
722 transformed into E.coli strain BL21 (DE3) Rosetta cells, then purified by amylose resin affinity
723 and Ni-NTA-agarose beads as described (53). Cdc11-capped septin octamers containing
724 wild-type Cdc11-Cdc12-(6His)-Cdc3-GFP-Cdc10 complex or mutants (Supplementary Table
725 S2) were produced and purified following the same protocol. Septins were stored in storage
726 buffer containing 20 mM Tris pH 8, 300 mM NaCl, 0.2 mM MgCl2, and 2 mM DTT. For in vitro
727 experiments, 5 nM septin complexes were polymerized in septin polymerization buffer
728 containing 20 mM Tris pH 8, 2 mM MgCl2, 400 µM GTP, 150 mM NaCl.

729 Recombinant GST-eGFP-PH-domain (PLCδ1) was purified as described (64).

730

731 Liposome floatation assay

732 Liposomes of various lipid compositions (Table S1) were prepared by mixing stocks in
733 chloroform/methanol. Lipids were placed in a vacuum chamber for 1 h to eliminate solvent.
734 The lipid mixtures were resuspended in 20 mM Tris-HCl pH 8, 150 mM NaCl preheated at
735 appropriate temperature (depending on the mixture, 45°C or 56°C for phosphatidic acid-
736 containing mixtures) to obtain 2 mM lipid suspensions. Lipid suspensions were submitted to
737 six cycles of freezing and thawing using liquid nitrogen and a water bath at 45 °C or 56°C.
738 Lipid suspensions were then extruded 15 times using an Avanti Mini-extruder with 100 nm
739 polycarbonate filters (to give ~100 nm liposomes). Proteins and liposomes (1 µM GFP-tagged
740 septin hetero-octamers and 2 mM total lipids) were incubated 30 min in 50 µl septin buffer at
741 room temperature in 200 µl thick-walled polycarbonate tubes (adapted to a TLA 100
742 Beckman rotor). The protein-lipid suspensions were adjusted to 30% w·vol⁻¹ sucrose by
743 adding and mixing 50 µl of 60% sucrose in septin buffer and overlaid with two cushions: 50 µl
744 of 25% sucrose and 50 µl of septin buffer with no sucrose. The tubes were then centrifuged
745 at 200,000 g for 1 h at 22 °C (TL 100, Beckman). After ultracentrifugation, 30 µl fractions were
746 collected at the top and bottom and GFP-tagged septin hetero-octamers were precipitated
747 with 10% trichloroacetic acid. The fractions were analyzed by conventional western blotting
748 using a polyclonal rabbit anti-Cdc11 antibody and a rabbit Dylight 800. Immunodetection was
749 performed using the Odyssey® imaging system (LI-COR Biosciences).
750

750

751 Supported lipid bilayers

752 Glass coverslips were cleaned by sequential 30 min sonication steps in 1 M NaOH,100%
753 ethanol and, milliQ water, with water rinsing between each step. The lipid mixtures of
754 supported lipid bilayers (SLBs) consisted of: 70-80% Egg-PC, 20-25% Liver-PI and 2.5-5% of
755 Brain-PI(4,5)P₂. In the case of the ternary mixture, the lipid composition of SLBs was: mica-
756 supported lipid membranes made of 2.5% mol Brain-PI(4,5)P₂, 2.5% mol DOPC, 30% mol
757 DPPC, 44.8% mol cholesterol, 20% mol Liver-PI. The amount of total negatively charged
758 lipids was kept to 25% for all the mixtures containing phosphoinositides at the expenses of
759 Liver-PI. Fluorescent lipid analogs were added at 0.2% mol.
760

760 For fluorescence microscopy and AFM experiments, supported lipid bilayers were prepared
761 as follows. Briefly, lipids dissolved in methanol/chloroform were mixed and dried for 1h in a
762 vacuum oven at 60°C. Small unilamellar vesicles (SUVs, diameter ~ 100nm) were obtained
763 by extrusion of multilamellar vesicles in citrate buffer (citrate 20 mM pH 4.6, KCl 50 mM,
764 EGTA 0.5 mM) heated to 44°C. 40 µl of SUVs were deposited on a two-well silicone insert
765 (IBIDI) and incubated 40 min at 40°C. Bilayers were carefully rinsed with wash buffer (20 mM
766 Tris-HCl pH 8, 300 mM NaCl) and then equilibrated with equilibration buffer (20 mM Tris-HCl
767 pH 8, 150 mM NaCl, 1 mg/ml lipid-free BSA). GFP-tagged septin hetero-octamers (wild-type
768 or mutants) were diluted to 10 nM in septin buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 2
769 mM MgCl₂, 400 µM GTP). Septins complexes at a final concentration of 5 nM were added on
770 SLBs.
771

771
772 Silica thin film nanostructuration

773 The master used for the nano-imprint lithography is made of undoped silicon (100) 300 µm
774 thick. First, the substrate is cleaned with Piranha. Then a 100 nm chromium layer is
775 deposited by sputtering (UNIVEX 150), next a negative photoresist (Az2020) is spin-coated
776 on the substrate and a UV lithography is performed through a mask. The mask's design is a
777 periodic nano-holes pattern made in a chromium layer, the nano-holes diameters are 2 µm
778 wide with a pitch of 4 µm. After development (1min in AZ 726) the obtained pattern is an
779 array of nano-disks, with a 2 µm diameter and a 4 µm pitch, of photoresist. These disks are
780 then thinned with an oxygen plasma generated by an Inductively Coupled Plasma – Reactive
781 Ion Etching equipment (ICP RIE Corial 150L) in order to achieve a 700 nm diameter. After
782 the ICP-RIE process, the chromium layer is etched with a chromium-based solution
783 (Chromium etchant standard from Sigma Aldrich 651826) for 5 min revealing a nano-disks
784 pattern (the negative pattern of the mask) on the chromium. These disks are used as
785 hardmask for the plasma etching of the Si substrate. The RIE recipe use CHF3 plasma as
786 etchant and the conditions (Power, Pressure, Flow) are defined to obtain a troncoïdal shape
787 for the pillars. The last step is the sample cleaning with Piranha.

788 Next, SiO₂ vertical cone-shape nanostructures were prepared on conventional borosilicate
789 coverslips with precision thickness No. 1.5 (0.170 ± 0.005 mm) by soft nano-imprint
790 lithography (soft-NIL), as previously reported (64, 65). The dimensions of the silicon master
791 and the resulting printed silica nanostructures were characterized by scanning electron
792 microscopy (SEM).

793

794 Airyscan/confocal microscopy and fluorescence recovery after photobleaching
795 imaging

796 SLBs and septins were imaged on a Zeiss LSM880 Airyscan/confocal microscope. Excitation
797 sources used were: 405 nm diode laser, an Argon laser for 488 nm and 514 nm and a
798 Helium/Neon laser for 633 nm. Acquisitions were performed on a 63x/1.4 objective.
799 Multidimensional acquisitions were acquired via an Airyscan detector (32-channel GaAsP 800 photomultiplier tube (PMT) array detector). For fluorescence recovery after photobleaching 801 (FRAP) measurements, GFP-tagged septin hetero-octamers were photobleached over a 802 circular or square area using 50 iterations of the 488 nm laser with 70% laser power 803 transmission. We recorded n =20 pre-bleaching images every 0.042 s or for long acquisitions 804 (i.e. ≥ 7 min), every 30 s.

805 The half-life (t₁/₂), which is the time at the half-maximal fluorescence intensity from the 806 plateau, and the immobile (IMf) and mobile fraction (Mf) of septins bound to membranes were 807 estimated after normalization of fluorescence intensities and exponential fitting of FRAP 808 curves using Prism GraphPad software. We obtained the % of Mf and IMf by applying the 809 following equations: Mf = I∞ - I₀/Iᵢ - I₀ and IMf = 1 - Mf, where I∞ is the fluorescence 810 intensity at the plateau, I₀ is the minimal intensity after bleaching, and Iᵢ is the maximal 811 intensity before bleaching. 812

813 Atomic Force Microscopy

814 AFM experiments were performed at room temperature on a JPK NanoWizard IV XP 815 microscope (JPK BioAFM, Bruker Nano GmBH, Berlin, Germany) mounted on an inverted 816 Nikon Ti2-U microscope (Nikon Instruments Europe B.V., Amsterdam, The Netherlands) 817 equipped with a standard monochrome CCD camera (ProgRes MFCool, Jenoptik, Jena, 818 Germany). Fluorescence imaging was performed by wide-field illumination (Intensilight Hg 819 lamp, Nikon) with a 100X objective (Nikon CFI Apo VC, 1.4 NA, oil immersion) and the 820 appropriate filter cubes. A software module (DirectOverlay, JPK Bio-AFM, Bruker Nano 821 GmbH, Berlin, Germany) was used to calibrate the tip position with the optical image. AFM 822 images were acquired in Quantitative Imaging (QI™) mode under a setpoint force of 250 pN, 823 based on the previous works (83). Rectangular-shaped BL-AC40TS-C2 cantilevers 824 (Olympus) with a nominal spring constant of 0.09 N·m⁻¹ and a nominal tip radius of 8 nm 825 were used for septin nano-domain characterization and PEAKFORCE-HIRS-F-A cantilevers 826 (Bruker) with a nominal spring constant of 0.35 N·m⁻¹ and a nominal tip radius of 1 nm were
827 used for high resolution imaging. All AFM experiments were operated under liquid conditions.
828 Before each experiment, the cantilever spring constant and sensitivity were determined by
829 the thermal noise method (84). QI mapping was performed at 256 x 256 pixels/line at a scan
830 rate of 9.4 kHz. SLBs were prepared on 1M NaOH treated coverslips following the same
831 experimental procedure detailed in the previous section. After overnight incubation of 5 nM of
832 Septin-GFP filaments, samples were mounted on the AFM imaging holder and imaged in 20
833 mM Tris-HCl pH 8, 2 mM MgCl₂, 150 mM NaCl.

834

835 Transmission Electron Microscopy

836 Samples for transmission electron microscopy (TEM) were prepared by incubating GFP-
837 tagged septin hetero-octamers (wild-type or mutants) with lipid monolayers formed at an air-
838 buffer interface. The lipid solution consisted of 77.5% Egg-PC, 20% Liver-PI and 2.5% of
839 Brain-PI(4,5)P₂ in chloroform. Briefly, each Teflon well was filled with 20 µl of a solution of
840 0.005 mg/ml GFP-tagged septin hetero-octamers in septin buffer (20 mM Tris-HCl pH 8, 2
841 mM MgCl₂, 400 µM GTP, 150 mM NaCl). A drop (~ 0.5 µl) of a 0.5 g/L lipid solution in
842 chloroform was deposited at the air-water interface in each well to form a lipid monolayer.
843 The Teflon block was kept overnight at 4℃ in a humid chamber. Lipid monolayers with
844 adsorbed GFP-tagged septin hetero-octamers were collected by briefly placing hydrophobic
845 grids (Electron Microscopy Sciences, Hatfield, PA, USA) on the surface of the solutions with
846 the carbon-coated side facing the solution. The lipid monolayers were absorbed on a carbon-
847 coated grid on a Formvar carbon-coated grid (Delta Microscopies FCF100-Cu) for 30 sec.
848 The grids were then negatively stained for 1 min using 2% uranyl acetate in water. Data were
849 collected using a Tecnai F20 transmission electron microscope at 120KV and equipped with
850 a Veleta camera.

851

852 Live cell imaging

853 Cells were mounted on 1% agarose pads in SD medium on Fluorodishes (FD35-100 World
854 Precision Instrument) and filmed at controlled temperatures (25 or 30°C) with a 63X 1.4 NA
Oil DIC M27 immersion objective mounted on a Zeiss LSM 980 NLO microscope equipped with Chameleon Ultra-II Coherent LASER (680-1080nm). Excitation sources used were an Argon laser for 514 nm and Chameleon laser for 800 nm. Multidimensional acquisitions were acquired via GaAsP and Multialkali photomultiplier tube (PMT) array detectors. Z-stacks of 5 planes were acquired with a step size of 0.4 µm. Z stacks were max-projected with ImageJ. Selected cells were illuminated with an 800 nm laser and emission read at 420-460 nm (representing the liquid ordered phase) and 490-540 nm (representing the liquid disordered phase). For Laurdan experiments, the Laurdan dye was used at 5 µM. Cells were incubated for 1 hour at appropriate temperature under agitation. Latrunculin A (LatA) was used at 0.1 mM for 10 minutes at appropriate temperature.

## STED imaging

Samples for STED microscopy were prepared by incubating yeGFP-tagged Cdc3 cells (wild-type CDC11 or mutants) with a 1/250 dilution of an anti-GFP nanobody Atto 647N (ChromoTek), as previously detailed in (52). Untagged yeGFP yeast cells were used as a control for the nanobody specificity and background evaluation. After 90min of antibody incubation, cells were washed twice in PBS by gently shaking at room temperature and mounted using Abberior Mount Liquid (Abberior).

Images were acquired on an Expert Line STED microscope (Abberior Instruments GmbH) using a PlanSuperApo 100X oil immersion objective N.A. 1.4 (Olympus). Atto 647N was imaged at 640 nm excitation using a 775nm depletion laser line. Detection was set to 650-750nm. A dwell time of 10µs was used. Images were collected in line accumulation mode (5 lines), the pinhole was set to 1.0 Airy units and a pixel size of 20nm was used for all acquisitions.

## Image processing and quantification

Line profiles of the fluorescence intensities and the kymographs were made using ImageJ.

The semi-automatic analysis of images to determine DOPE-depleted area and septin
883 domains size were done using the macro toolset in ImageJ. Quantifications of septin binding
884 were done using ImageJ. Septin filaments size was measured using FiberApp (85).

885 AFM image processing and profile analysis were performed using the open-source software
886 Gwyddion (86). Membrane stiffness (Young modulus) was obtained based on the Hertzian
887 model for elastic indentations. Young's moduli were calculated at a sample deformations that
888 was < 20% of the bilayer thickness (87), as previously reported (83). Correlative fluorescence
889 and AFM image representation were performed using ImageJ (88) and the BigWarp plugin
890 (89).

891 Generalized Polarization (GP) values were calculated for each pixel of yeast plasma
892 membrane using a GP calculation macro toolset according to the following equation:
893 GP = (I<sub>420</sub> - I<sub>490</sub>) / (I<sub>420</sub> + I<sub>490</sub>) where I<sub>420</sub> and I<sub>490</sub> represent the intensity of pixels in the areas of
894 interest in the image acquired in the ordered and disordered spectral channels, respectively.
895 The cross-sections showing the GP values at the yeast plasma membrane were done with
896 ImageJ.

897

898 **Data representation and statistical analysis**

899 Representation of cross-section analysis and recruitment curves were performed using
900 Origin and Prism GraphPad software. Statistical analysis was performed using the Mann-
901 Whitney two-tailed test or ordinary one-way ANOVA multiple comparisons test, with single
902 pooled variance using Prism GraphPad software. In all statistical, the levels of significance
903 were defined as: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

904

905 **Data availability**

906 All data generated or analyzed during this study are included in the manuscript and
907 supporting file; source data files have been provided.

908

909

910
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1117

1118 **ACKNOWLEDGMENTS**

1119 We acknowledge Erdinc Sezgin for help with the Lo marker DSPE-PEG-ASR. We also thank
1120 Julien Viaud for kindly providing the GFP-PH<sup>PLC5</sup> construct. The authors are grateful to Alenka
1121 Copic and Cyril Favard for helpful discussions. This work has been supported by a grant of
1122 the Agence Nationale pour la Recherche (SEPTORG ANR-18-CE13-0015-01) to S.P. and
1123 L.P. We acknowledge the imaging facility MRI, member of the national infrastructure France-
1124 BioImaging supported by the French National Research Agency (ANR-10-INBS-04,
1125 «Investments for the future»).

1126

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1129

1130 **AUTHOR CONTRIBUTIONS**

1131 Conceptualization: L.P, S.P. and F.E.A. Methodology: F.E.A., S.I., S.L., C.C., D.S, R.D, M-P.B, A.P, S.P. and L.P. Supervision: A.C-G, L.P. and S.P. Funding acquisition: L.P. and S.P.
1132
1133 Writing: L.P and F.E.A. with inputs from authors.

1134

1135 **DECLARATION OF INTERESTS**
1136 The authors declare no competing interests.

1137

SUPPLEMENTARY INFORMATION

<table><thead><tr><th colspan="3">SUVs mixture</th></tr><tr><th></th><th>Lipid</th><th>Mole %</th></tr></thead><tbody><tr><td rowspan="3">Compositon PI(4,5)P<sub>2</sub></td><td>Egg-PC</td><td>80</td></tr><tr><td>Brain-PI(4,5)P<sub>2</sub></td><td>5</td></tr><tr><td>Liver-PI</td><td>15</td></tr><tr><td rowspan="2">Compositon PI</td><td>Egg-PC</td><td>75</td></tr><tr><td>Liver-PI</td><td>25</td></tr><tr><td rowspan="3">Compositon PA</td><td>Egg-PC</td><td>75</td></tr><tr><td>Egg-PA</td><td>10</td></tr><tr><td>Liver-PI</td><td>15</td></tr><tr><td rowspan="3">Compositon PS</td><td>Egg-PC</td><td>75</td></tr><tr><td>Brain-PS</td><td>10</td></tr><tr><td>Liver-PI</td><td>15</td></tr><tr><td>Compositon PC</td><td>Egg-PC</td><td>100</td></tr></tbody></table>

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1139

<caption>Table S1. Composition of the different SUVs lipid mixtures used for the liposome floatation assay.</caption>

1140

<table><thead><tr><th>Name</th><th>Relevant genotype</th></tr></thead><tbody><tr><td>ySP41</td><td>MATA, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3</td></tr><tr><td>ySP15355</td><td>MATalpha, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, bud4::LEU2:: BUD4, cdc3::mCherry-CDC3::URA3</td></tr><tr><td>ySP16736</td><td>MATA, bud4::LEU2::BUD4, cdc3::mCherry-CDC3::URA3</td></tr><tr><td>ySP17147</td><td>MATA, cdc3::mCherry-CDC3::URA3, cdc12-6</td></tr><tr><td>ySP17529</td><td>MATalpha, cdc11::TRP1, ura3:URA3::cdc11(Δ2-18), bud4::LEU2::BUD4, cdc3::mCherry-CDC3::URA3</td></tr><tr><td>ySP17533</td><td>MATalpha, cdc11::TRP1, ura3:URA3::CDC11, bud4::LEU2::BUD4, cdc3::mCherry-CDC3::URA3</td></tr><tr><td>ySP17816</td><td>MATA, cdc11::TRP1, ura3:URA3::CDC11, bud4::LEU2::BUD4, CDC10-yeGFP::KanMX</td></tr><tr><td>ySP17818</td><td>MATA, cdc11::TRP1, ura3:URA3::cdc11(Δ2-18), bud4::LEU2::BUD4, CDC10-yeGFP::KanMX</td></tr></tbody></table>

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1142

Table S2. List of S. cerevisiae strains used in this study
1143

<table><thead><tr><td>Plasmid name</td><td>Relevant genes</td></tr></thead><tbody><tr><td>pFM455</td><td>MBP-CDC12, His6-CDC3-GFP</td></tr><tr><td>FE-1198-Cdc12(ΔCTE)</td><td>MBP-cdc12(Δ339-407), His6-CDC3-GFP</td></tr><tr><td>FE-1198-Cdc12(ΔAH)</td><td>MBP-cdc12(Δ389-407), His6-CDC3-GFP</td></tr><tr><td>pFM453</td><td>CDC10, CDC11</td></tr><tr><td>FE-1235-Cdc11(ΔPB)</td><td>CDC10, cdc11(Δ2-18)</td></tr><tr><td>pSP1831 (pYlplac211-CDC11)</td><td>CDC11p::CDC11cds::CDC11t</td></tr><tr><td>pYlplac211-ΔPBcdc11</td><td>CDC11p::cdc11(Δ2-18)cds::CDC11t</td></tr></tbody></table>

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**Table S3.** List of plasmids used in this study.
<table><thead><tr><th>Plasmid name</th><th>Primers name</th><th>Primers sequence</th></tr></thead><tbody><tr><td rowspan="4">Cdc11-Cdc12<sub>(Δ339-407)</sub>-Cdc10-Cdc3-GFP</td><td>FE-1198-Primer A</td><td>CCGCGTGGATCCATGAGTGCTG</td></tr><tr><td>FE-1198-Primer B (rev)</td><td>CTCATATGTTATTCCTCCTTGAATTCGTGTGACAATTTCCTTGCTGGTGC</td></tr><tr><td>FE-1198-Primer C</td><td>GCACCAGCAAGGAAATTGTCACACGAATTCAAGGAGGAATAACATATGAG</td></tr><tr><td>FE-1198-Primer D</td><td>ACCGCCGTCGACACGTAAAAATCCCTTCTTC</td></tr><tr><td rowspan="2">Cdc11-Cdc12<sub>(Δ389-407)</sub>-Cdc10-Cdc3-GFP</td><td>FE-1198-Primer B' (rev)</td><td>CTCATATGTTATTCCTCCTTGAATTCTACCTTTCCCTTGAATTTCTTCCAAGTCACCGTTCAG</td></tr><tr><td>FE-1198-Primer C'</td><td>CTGAACGGTGACTTGGAAGAAATTCAAGGAAAGGTAGAATTCAAGGAGGAATAACATATGAG</td></tr><tr><td>Cdc11-Cdc12-Cdc10-Cdc3<sub>(Δ440-520)</sub>-GFP</td><td>FE-1198-Primer B'' (rev)</td><td>ATGGCAATGGTCGACTGCCTCATGTAAAGTTTTTTCTTC</td></tr><tr><td rowspan="4">Cdc11<sub>(Δ2-18)</sub>-Cdc12-Cdc10-Cdc3-GFP</td><td>FE-1235-Primer A</td><td>TTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGG</td></tr><tr><td>FE-1235-Primer B (rev)</td><td>CGATCATCACAGTGAAGGTTATACCTCTCATATATGTTATTCCTCCTTGAGCTCACG</td></tr><tr><td>FE-1235-Primer C</td><td>CGTGAGCTCAAGGAGGAATAACATATATGAGAGGTATAACCTTCACTGTGATGATCG</td></tr><tr><td>FE-1235-Primer D (rev)</td><td>AAGCTTGTCGACTCATTCTTCC</td></tr><tr><td rowspan="7">pYlplac211-cdc11-ΔPB</td><td>FE-Insert (Fw)ΔPBcdc11</td><td>GTTGCTAACCACCAGCCATGAGAGGTATAACCTTCACTGTG</td></tr><tr><td>FE-Insert (Rev)ΔPBcdc11</td><td>CTTTTCAACAGCAGTTCTTGCTGAACTCTCTCCTCAAATGC</td></tr><tr><td>FE-Vector (Fw)ΔPBcdc11</td><td>GCATTTGAGGAGAGAGTTCAGC</td></tr><tr><td>FE-Vector (Rev)ΔPBcdc11</td><td>CATGGCTGGTGGTTAGCAAC</td></tr><tr><td>FE-5'UTR-Cdc11 Fw</td><td>GTTATAGCTCCGTTAAACC</td></tr><tr><td>FE-Cdc11 rev</td><td>GTAACAAGATTGTCGTCG</td></tr></tbody></table>

<caption>**Table S4.** List of primers used in this study.</caption>
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<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

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<caption>**Figure 1-figure supplement 1. A-B) Transmitted light, confocal and STED images of wild-type yeast cells (i.e., no expressing yeGFP-tagged proteins) (A) compared to a STED image of yeast cells expressing Cdc10-yeGFP (B). All conditions were immunostained with an anti-GFP nanobody Atto647N. Images were acquired under the same experimental settings. Gray scale of confocal and STED images is displayed in a Fire LUT. Scale bar, 10 µm.**</caption>

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<figure><img src="image_27.png" /><figcaption>Figure 2-figure supplement 1. Airyscan images showing the signal of fluorescent A647-DOPE (0.2% mol) on lipid bilayers (SLBs) made with the SUVs compositions detailed in Table S1 after addition of 5 nM septin-GFP hetero-octamers prepared in septins buffer (150 mM NaCl). Scale bar, 5 µm.</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>Figure 3-figure supplement 1. Fitting of the FRAP analysis.

The fitting function, based on Brownian motion theory, is: $I(t) - I_0 = I_\infty (1 - e^{-t/\tau})$ and gives $I_\infty = (2725 \pm 110) (a.u.); 1/\tau = (0.013 \pm 0.002) s^{-1}$ or also $\tau = (77 \pm 12)s$ for $I_0 = 515 (a.u.)$. The time $\tau$ is the typical recovery time of the fluorescent signal. We can then estimate the mobile fraction $\phi_m = \frac{I_\infty - I_0}{I_0} = 0.16 \%$ and the immobile fraction $\phi_{imm} = 1 - \phi_m = 0.84 \%$. We can associate an effective diffusion constant of the septin octamers in the patch (during the experimental time) to the relaxation time $\tau$, indeed</figcaption></figure>

$$ \tau \approx \frac{\omega^2}{4D_{eff}}, $$

where $\omega = 1.75 \ \mu m$ is the typical radius of the photobleached spot:

$$ D_{eff} \approx \frac{\omega^2}{4\tau} \sim (0.99 \pm 0.16) 10^{-14} m^2/s. $$

<figure><img src="image_29.png" /></figure>
<figure><img src="image_30.png" /><figcaption>**Figure 3-figure supplement 2.** Fluorescence recovery of the A647-DOPE lipid signal of SLBs made of 77.3% mol Egg-PC, 20% mol Liver-PI; 2.5% mol Brain-PI(4,5)P2, and 0.2% mol fluorescent A647-DOPE and incubated with 5 nM of septins-GFP. The FRAP experiment was performed on yellow ROI indicated. Snapshots of the SLBs corresponding to the A647-DOPE channel before, at the moment of bleaching and after bleaching. Representation of the fluorescence intensity of A647-DOPE over time acquired at the FRAP ROI. Scale bar, 1 µm.</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>**Figure 3-figure supplement 3.** Cross-section of the septin-GFP signal along the orange line in Figure 3A after the bleaching process (T = 420s) before (black) and after (red) septin wash out. The different features of the septin signal on SLBs are highlighted (septins at the bulk membrane and bleached and non-bleached septin patch).</figcaption></figure>

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<figure><img src="image_32.png" /><figcaption>**Figure 4-figure supplement 1.** Time-dependent effect on the formation of DOPE-depleted domains by septin nano-domains upon binding on PI(4,5)P₂-containing membranes. Airyscan images showing septin association with lipid bilayers containing 77.4% mol Egg-PC, 20% mol Liver-PI; 2.4% mol Brain-PI(4,5)P₂, 0.2% mol fluorescent TF-TMR-PI(4,5)P₂ and 0.2% mol fluorescent A647-DOPE, at time T = 0 min, 30 min and 60 min. 5 nM septin-GFP hetero-octamers were added in septin buffer (150 mM NaCl). Scale bar, 5 µm.</figcaption></figure>

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<figure><img src="image_33.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_34.png" /></figure>

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**Figure 4-figure supplement 2.** Effect of septin-GFP bleaching on the A647-DOPE lipid signal. The FRAP experiment was performed on the septins-GFP channel after septins were let for 10 min to bind SLBs containing 77.3% mol Egg-PC, 20% mol Liver-PI; 2.5% mol Brain-PI(4,5)P2, and 0.2% mol fluorescent A647-DOPE. **A)** Snapshots of the septin-GFP and A647-DOPE channel before, at the moment of bleaching and after bleaching a ROI of ~ 1 µm diameter on the septin-GFP channel and corresponding to a septin nano-domain. **B)** Fluorescence intensity of septin-GFP and A647-DOPE over time acquired at the FRAP ROI. Scale bar, 1 µm.
<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /><figcaption>**Figure 4-figure supplement 3.** A) Airyscan images and B) cross-section along the red-dashed line showing the septin-GFP hetero-octamers on 2.5% mol Brain-PI(4,5)P₂-containing SLBs doped with 0.2% mol fluorescent A647-DOPE and 0.2% mol fluorescent Rhodamine-DOPE. C) Airyscan images showing the septins-GFP hetero-octamers on 2.5% mol Brain-PI(4,5)P₂-containing SLBs doped with 0.2% mol fluorescent A647-DOPE and 0.2% mol fluorescent TF-TMR-PC. D) Size of lipid-depleted areas calculated on Airyscan images. The number of depleted areas measured for A647-DOPE, Rhodamine-DOPE, and TF-TMR-PC are n = 1024, n = 3749 and n = 1448, respectively. Scale bar, 5 µm.</figcaption></figure>

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<figure><img src="image_39.png" /></figure>

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1221 **Figure 4-figure supplement 4.** Airyscan images showing the binding of 1 µM of GFP-PH<sup>PLCδ</sup> with lipid bilayers containing 77.3% mol Egg-PC, 20% mol Liver-PI; 2.3% mol Brain-PI(4,5)P<sub>2</sub>, 0.2% mol fluorescent TF-TMR-PI(4,5)P<sub>2</sub> and 0.2% mol fluorescent DOPE-A647. Scale bar, 5 µm.

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<figure><img src="image_40.png" /></figure>

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1226 **Figure 4-figure supplement 5.** 1 µM of septin-GFP hetero-octamers were incubated with liposomes (2 mM) made of Egg-PC, Liver-PI, Brain-PI(4,5)P<sub>2</sub> and different % mole of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE) (0% PE, 25% PE, or 50% PE) with 25% total negative charge. The suspension was subjected to liposome flotation assays. Septin-GFP hetero-octamers were polymerized in septin polymerization buffer and the septin Cdc11 was detected by western blotting. Quantification of septin binding to liposomes: Top/(Top+Bottom)x100. Results are presented as mean ± s.d. (n = 3). Error bars represent s.d.; Mann-Whitney test: * P < 0.05, ** P < 0.01.

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<figure><img src="image_41.png" /><figcaption>**Figure 5-figure supplement 1.** Domain representation of wild-type and mutant septins. Coomassie-stained SDS gel showing the purified GFP-tagged septin hetero-octamers used for *in vitro* assays.</figcaption></figure>

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<figure><img src="image_42.png" /><figcaption>1243
1244 **Figure 5-figure supplement 2.** Mean gray value of 100 nM wild-type, ΔPB-Cdc11 or ΔAH-Cdc12 mutants septin-1245 GFP complexes on the flat surface of silica nanostructured substrates functionalized with PI(4,5)P₂-containing 1246 SLBs. Number of images analyzed, n, n = 56, 54 and 66 respectively, from two technical replicates. Error bars 1247 represent s.d..</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>1250
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1252 **Figure 6-figure supplement 1. Viability of the cdc11(Δ2-18) mutant at different temperatures.** Serial dilutions of cells with the indicated genotypes were spotted on YEPD plates and incubated at different temperatures (25°C, 30 °C, 32°C, 34°C, 37°C).</figcaption></figure>

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<figure><img src="image_44.png" /><figcaption>**Figure 6-figure supplement 2.** Transmitted light (TL) and TIRF images of wild-type *CDC11* and *cdc11(Δ2-18)* mutant cells expressing mCherry-Cdc3. Cells with the indicated genotypes were imaged every 5 min for 6 h at 37 °C. Scale bar, 2µm.</figcaption></figure>

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<figure><img src="image_50.png" /><figcaption>**Figure 6-figure supplement 3.** A) Transmitted light (TL) and confocal images of yeast cells expressing mCherry-Cdc3 in *CDC12* and *CDC11* cells stained with the Laurdan probe. Color-coded GP value images (fire LUT) of z-stacks max-projections. Scale bar, 2 µm. B) Quantification of the global GP index at the plasma membrane in *CDC12*, *cdc12-6*, *CDC11* and *cdc11(Δ2-18)* budded cells after staining with the Laurdan probe. Number of cells analyzed for each condition, n = 15, 7, 10, and 12, respectively, from three replicates. Error bars represent s.d.; Mann-Whitney test: n.s. P > 0.05. C) TL and confocal images of *cdc12-6* budded cells expressing mCherry-Cdc3 (magenta) and stained with the Laurdan probe. Color-coded GP value images (fire LUT) of z-stacks projections. Scale bar, 2 µm. % of *CDC12* and *cdc12-6* mutant cells displaying high GP values (GP > 1, indicative of Lo phases) at the bud neck of the mother cell cortex. Number of images analyzed, n = 12, and n = 16, respectively, from two independent experiments. Error bars represent s.d.; Mann-Whitney test: **** P < 0.0001.</figcaption></figure>

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