Fig. 4: ALG2 and CALC modulate phase coupling within ANXA11-GUV assemblies.
From: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes
a Fluorescence micrographs of 25 µM recombinant AF647-ANXA11 FL in the presence of unlabelled 0.1 mM ALG2, 1 mM CALC or 1 mM Halo. The concentrations of the modulators were determined from molar ratios matching their relative abundance in cells (1[ANXA11]:4[ALG2]:40[CALC]). Scale bar–20 µm. b Representative fluorescence images of ATTO488 GUVs incubated with 0.5 µM AF647-ANXA11 FL at 100 µM Ca2+ co-incubated with either 2 µM ALG2 or 20 µM CALC. Scale bar–5 µm. c Quantification of the fluorescence intensity of AF647-ANXA11 FL recruited to GUVs as in (b). Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ***p = 0.0002, ****p < 0.0001, n = 3 repeats (35–55 GUVs). d Quantification of the fluorescence intensity of 25 µM AF647-labelled HIS-LCD recruited to NTA(Ni)-GUVs co-incubated with either 0.1 mM ALG2 or 1 mM CALC. Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ****p < 0.0001, n = 3 repeats (117–281 GUVs). e A FRAP recovery profile of 0.5 µM AF647-ANXA11 FL on the surface of GUVs in the presence or absence of 2 µM ALG2 or 20 µM CALC at 100 µM Ca2+. The corresponding quantification of the % fluorescence recovery after 15 s is plotted alongside. Mean ± SD. One-way ANOVA with Dunnett’s multiple comparison ***p = 0.0004, **p = 0.0011, n = 3 GUVs (FL;FL + ALG2) 5 GUVs (FL + CALC). f 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 0.5 µM ANXA11 FL co-incubated with either 2 µM ALG2 or 20 µM CALC. Scale bar–5 µm. g Quantification of the relative lipid order (φ) of GUVs shown in (f). Mean ± SD. One-way ANOVA with Dunnett’s multiple comparison, ***p = 0.0006, ****p < 0.0001, n = 5 repeats (58–134 GUVs). h Quantification of the relative lipid order (φ) of NTA(Ni)-GUVs labelled with 5 µM PK dye. NTA(Ni) GUVs were incubated with 25 µM HIS-LCD and with either 0.1 mM ALG2 or 1 mM CALC. Mean ± SD. One-way ANOVA with Dunnett’s multiple comparison, *p = 0.0318, **p = 0.0045, n = 4 repeats (28–94 GUVs).
