Figure 1.
Lipid structures and characterization of β2m fibrils using AFM.
(A) Structure of anionic lipids, POPS, POPG and BMP. (B) Fibril length distributions and representative AFM images of (i) unfragmented (1.30±0.05 µm) and (ii) fragmented (0.30±0.01 µm) β2m fibrils. Scale bar 1 µm.
Figure 2.
Dye release upon addition of β2m to POPC/cholesterol or POPG/cholesterol LUVs.
Dye release from LUVs (5 µM lipid) comprising of (A) 75 mol % POPC: 25 mol % cholesterol and (B) 75 mol % POPG: 25 mol % cholesterol measured continuously over 20 min following addition of 6 µM β2m samples in Buffer A at pH 7.4, 37°C. Error bars represent 1 standard deviation (S.D.) from three replicates for each β2m species. Dye release from LUVs (5 µM lipid) comprising (C) 75 mol % POPC: 25 mol % cholesterol and (D) 75 mol % POPG: 25 mol % cholesterol measured discontinuously over 20 min following addition of 6 µM β2m samples in Buffer B at pH 4.5, 37°C. Error bars represent 1 S.D. from three replicates for each β2m species at each time point. β2m monomer (circle, blue), fragmented fibrils (square, red) and unfragmented fibrils (triangle, green).
Figure 3.
Dye release from LUVs comprised of complex anionic lipid mixtures upon addition of β2m samples at pH 4.5–7.4.
Dye release was measured 10 min after the addition of 6 µM (monomer equivalent concentration) of (A) β2m monomers, (B) fragmented fibrils or (C) unfragmented fibrils added to CF-loaded LUVs (5 µM lipid) in Assay Buffer pH 4.5–7.4, 37°C. LUVs are comprised of 36 POPC: 20 POPE: 7 SM: 25 cholesterol (mol/mol) doped with 0 mol % (diamond, black), 12 mol % (open symbols) or 50 mol % (solid symbols) anionic lipid, POPS (circle, blue), POPG (diamond, green) or BMP (square, red). Corresponding bar graph of dye release at pH 4.5 in 0 mol % (black), 12 mol % (open bar) or 50 mol % (solid bar) POPG, POPS or BMP are shown alongside. Error bars represent 1 standard error (S.E.) from three independent repeats, each of three replicates.
Figure 4.
Dye release from LUVs comprised of 12 mol % BMP at pH 4.5, varying β2m: lipid concentration ratio and cholesterol content.
(A) Dye release measured for LUVs (5 µM lipid) at different monomer equivalent concentrations of β2m. (B) Dye release measured for varying lipid equivalent concentrations of LUVs at 6 µM monomer equivalent concentration of β2m. (C) Dye release from LUVs (5 µM lipid) comprising 36 POPC: 20 POPE: 7 SM (mol/mol), 12 mol % BMP plus varying concentrations of cholesterol at 6 µM monomer equivalent concentration of β2m. Dye release was measured 10 min after addition β2m monomer (circle, blue), fragmented fibrils (square, red) and unfragmented fibrils (triangle, green) in Assay Buffer pH 4.5, 37°C. Error bars represent 1 S.D. from three replicates. Open symbols corresponds to dye release measured for 6 µM monomer equivalent β2m samples with 5 µM lipid equivalent concentration of LUVs containing 25 mol % cholesterol, as in Fig. 3.
Figure 5.
Change in tryptophan fluorescence quenching for β2m in the presence of LUVs. Δ
KSV for 6 µM monomer equivalent concentration β2m monomer, fragmented or unfragmented fibrils 10 min after addition of LUVs (5 µM lipid) at 37°C in (A) Assay buffer at pH 7.4 or (B) Assay buffer at pH 4.5. Lipid mixes comprise 36 POPC: 20 POPE: 7 SM: 25 cholesterol (mol/mol) doped with 0 mol % (blue), 12 mol % (red) or 50 mol % (green) BMP. Error bars represent 1 S.E. from linear regression.
Figure 6.
Confocal fluorescence microscopy of GUVs upon addition of fragmented β2m fibrils.
(A) Confocal images were collected in two different focal planes. The upper plane contains mainly non-vesicular lipid while dense, intact sucrose-loaded GUVs are observed mostly in the lower focal plane. Confocal images of TMR-labeled β2m fragmented fibrils incubated with DiD-labeled GUVs for 15 min at ambient temperature in (B) Assay Buffer at pH 7.4 or (C) Assay Buffer at pH 6.5. (L-R) TMR-labeled β2m fibrils (red), DiD-labeled GUVs (green), superimposition of the TMR and DiD channels. Soluble CF added to the vesicle exterior is shown in blue (lower focal plane only). Representative images when viewed in the upper focal plane and lower focal plane are shown for GUVs comprised of (i) 100 mol % DOPC or (ii) 80 mol % DOPC plus 20 mol % BMP. White arrows highlight areas of lipids bound to fibril aggregates. Scale bar 10 µm.
Figure 7.
Dye release normalized to the change in tryptophan fluorescence quenching measured for β2m with BMP-containing LUVs at pH 4.5.
The ratio of % dye release per membrane interaction detected via a change in tryptophan quenching observed in the absence or presence of lipid. β2m monomers, fragmented and unfragmented fibrils (6 µM monomer equivalent concentration) were incubated in Assay Buffer pH 4.5 with LUVs (5 µM lipid) comprising 36 POPC: 20 POPE: 7 SM: 25 cholesterol (mol/mol) plus 0 mol % (blue), 12 mol % (red) or 50 mol % (green) BMP for 10 min at 37°C. Error bar represent 1 S.E.