Direct three-dimensional visualization of membrane disruption by amyloid fibrils

Abstract

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from β(2)-microglobulin (β(2)m). Using cryoelectron tomography, we demonstrate that fragmented β(2)m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.

Fig. 1.

Cryo-EM overviews of liposomes and β₂m fibrils. Low-magnification images of (A) liposomes, (B) short β₂m fibrils, (C) liposomes plus short fibrils, and (D) liposomes plus long fibrils. (Scale bar for A–D main images: 1 μm.) (E and F) Higher magnification views of liposomes with short (E) or long (F) fibrils. Examples of distorted liposomes are indicated by white arrowheads in E. The inset in A shows a higher magnification view of liposomes. (Scale bar for E and F: 200 nm.)

Fig. 2.

Fluorescence microscopy showing liposome damage by short β₂m fibrils. (A) Liposomes plus buffer and (B) liposomes plus short fibrils. Green, NBD-PE labeled giant vesicles; red, TMR-β₂m fibrils. Yellow regions indicate colocalization of lipids and fibrils. In B, the vesicles are either completely disintegrated by fibrillar aggregates or show membrane damage with visible lipid extraction by the fibrils.

Fig. 3.

Cryoelectron tomography of liposome–fibril interactions. (A–C) Sections of tomograms showing liposomes clustered and distorted by short fibrils. (D) A rendered 3D model of a distorted liposome, surrounding fibrils and adjacent small vesicles from C. (Scale bar: 50 nm.)

Fig. 4.

Distortions of liposomes in the vicinity of fibril ends. (A–C) Examples and cartoons of the different types of fibril–liposome interactions observed, with the percentage of each type measured by counting examples in four tomograms. (D–F) Sections of tomograms taken closer to focus, showing examples of the disruption of the lipid bilayer in the region of fibril ends, including (D) formation of a sharp point, (E) a break in the outer leaflet of the membrane, and (F) a bubble forming in the outer leaflet, with corresponding cartoons showing membranes as black lines and fibrils as gray lines. (Scale bar: 50 nm.)