Fig. 1: Yeast polar lipid extract bilayers exhibit lipid phase separation and septin filaments exclusively polymerize on the L_d phase.

HS-AFM frames of yeast polar lipid SLBs showing two (a) or three (c) phases, depending on imaging area. b, d Height analysis of (a) and (c): L_o (liquid-ordered) is 1.2 ± 0.3 nm (n = 57) thicker than L_d (liquid-disordered), and L_β (gel) is 0.7 ± 0.2 nm (n = 76) thicker than L_o. Insets: Section profiles along dashed lines in (a) and (c). e, g HS-AFM frames (Supplementary Movie 1) of septin filaments on a L_d and L_o phase separated membrane before (7 frame average (e)) and after (single frame (g)) a HS-AFM force-sweep experiment. f, h Section profiles along dashed lines in (e) and (g). Septin filaments peak ~5 nm above L_d surface, derived from section profiles in (f) and (h), dashed line in (h). HS-AFM frames of membrane areas with three phases L_d, L_o and L_β, with (i) and without (k) septin filaments. A membrane defect in (k) allows L_d thickness determination to a common baseline, mica (arrow). j, l Section profiles along dashed lines in (i) and (k). The L_d phase has 3.4 ± 0.5 nm (n = 43) thickness. Membrane regions are labelled according to the assigned phases, L_d, L_o and L_β, along with areas covered by septin, or membrane defects. Yeast SLBs were generally formed and imaged in 20 mM HEPES, pH 7.5, 150 mM KCl before septin addition, e.g., k, but were stable across a broad range of conditions e.g., 20 mM HEPES, pH 6, 50 mM KCl (a), or 20 mM HEPES, pH 7.5, 300 mM KCl, 2 mM MgCl₂ (c). Septin filaments on yeast SLBs (e), (g), and (i) were imaged in 20 mM HEPES, pH 6, 50 mM KCl, optimized to minimize tip-sample interaction. Septin filament images, (e) and (i), are representative of 26 analyzed images from 8 biological replicates. For bare membrane statistics, see Table 1.