Fig. 3: ANXA11 condensation induces coupled changes in the phase state of lipids in GUV membranes.
From: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes
a Log(dose) response curves fitted for AF647-labelled ANXA11 FL and ARD binding to GUV membranes at 100 µM Ca2+. Mean ± SD, n = 3. b Fluorescence micrographs of 25 µM recombinant AF647-ANXA11 FL alone or with unlabelled 25 µM LCD or 25 µM Halo. Scale bar–5 µm. Corresponding quantification for condensate area is plotted alongside. Mean ± SD, Kruskal-Wallis with Dunn’s multiple comparison, ****p < 0.0001, ns–not significant (p > 0.05), n = 3 repeats (1171–1439 condensates). c Representative images of ATTO488 GUVs coated in either 0.5 µM ANXA11 FL or 0.5 µM ARD at 100 µM Ca2+ (left). On the right, the same GUVs coincubated with 25 µM AF647-labelled LCD. Scale bar–5 µm. d FRAP recovery curves of 0.5 µM AF647-labelled ANXA11 FL or ARD on the surface of GUVs at 100 µM Ca2+, with and without 25 µM LCD. The corresponding quantification of the % fluorescence recovery after 15 s is plotted alongside. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, ***p = 0.001, ns–not significant (p > 0.05), n = 4 GUVs. e Fluorescence images of ATTO647 GUVs labelled with 5 µM PK dye to extract the relative order (φ) of membrane lipids. GUVs were incubated with 100 µM Ca2+ and either 0.5 µM ANXA11 or ARD in the presence of 25 µM LCD. Scale bar–5 µm. f Quantification of the relative lipid order (φ) of GUVs as shown in (e). Mean ± SD. Unpaired two-tailed with Welch’s correction, ****p < 0.0001, n = 5 repeats (44–126 GUVs). g A schematic illustrating HIS-LCD conjugation to DGS-NTA(Ni) lipids. Below are images showing 25 µM AF647-labelled HIS-LCD or HIS-Halo binding to NTA(Ni) GUVs. h Fluorescence images of ATTO647 GUVs labelled with 5 µM PK dye to extract the relative order (φ) of membrane lipids. NTA(Ni) GUVs were incubated with either 25 µM HIS-LCD or HIS-Halo. Scale bar–5 µm. i Quantification of the relative lipid order of GUVs as shown in (h). Mean ± SD. One-way ANOVA with Dunnett’s multiple comparison, ****p < 0.0001, ns–not significant (p > 0.05), n = 5 repeats (83–114 GUVs).
