Cryo-EM of amyloid fibrils and cellular aggregates

Abstract

Neurodegenerative and other protein misfolding diseases are associated with the aggregation of a protein, which may be mutated in genetic forms of disease, or the wild type form in late onset sporadic disease. A wide variety of proteins and peptides can be involved, with aggregation originating from a natively folded or a natively unstructured species. Large deposits of amyloid fibrils are typically associated with cell death in late stage pathology. In this review, we illustrate the contributions of cryo-EM and related methods to the structure determination of amyloid fibrils extracted post mortem from patient brains or formed in vitro. We also discuss cell models of protein aggregation and the contributions of electron tomography to understanding the cellular context of aggregation.

Figure 1. Cross-β structure and tau deposits in Alzheimer’s disease brain.

From left to right, the upper panels show a schematic of the β-strand arrangement in the cross-β core of an amyloid fibril; the AD brain used for cryo-EM; and a Thioflavin-S stained light microscopy image showing abundant neurofibrillary tangles in temporal cortex. Lower panels, an electron micrograph of negatively stained filaments with a blue arrow indicating a paired helical filament (PHF) and a green arrow indicating a straight filament (SF); cryo-EM reconstructions of PHFs (blue) and SFs (green) with detailed cross sections; and de novo atomic models of filaments showing C-shaped subunits stacked to form each protofilament, with protofilaments paired into twisted polymorphic fibrils (Tau panels adapted from [15**]).

Figure 2. Atomic structures of amyloid fibrils, viewed down the fibril axis.

(a) The cross-β structure, (b) β-helices, (c) a combination of cross-β and β-helical structural motifs. (a) cross-β packing between laminated β-sheets is generic and can be formed by any amino acid sequence (open circles denoting any sidechain). Overlay of the backbones of cross-β structures from Alzheimer’s disease tau filaments [15**], Pick’s disease tau filaments [20**] and fibrils formed by a peptide fragment [13] all overlap better than 1.5 Å rmsd. (b) β-helices require a specific pattern of hydrophobic residues (green-filled circles) on the inside, polar residues (blue-filled circles) on the outside, and a pivotal β-arch-forming glycine (pink-filled circle) closing the triangular motif. Overlay of the backbones of the β-helix in Alzheimer’s disease tau filaments and HET-s β-helices formed by the [HET-s] prion [17] agree within 1.3 Å rmsd. (c) Schematic view of the C-shaped protofilament core formed by the tau protein in human Alzheimer’s disease brain. Each C consists of a β-helix region, where three β-sheets are arranged in a triangular fashion, and two regions with a cross-β architecture, where pairs of β-sheets pack anti-parallel to each other [15**].

Figure 3. Aggregation pathway of Huntingtin exon 1 in cells and in vitro.

(a) Huntingtin exon 1 aggregates (43Q) in the course of conversion from reversible, liquid phase droplets to irreversible fibrous aggregates in a HEK293 cell (reproduced from [48*]. (b,c) The in vitro counterpart of this conversion, showing the start and end points of conversion from liquid droplets to fibrous outgrowth, consumes all the fluorescence from the original droplets [48*]. The in vitro experiment was done with 25Q, with its phase separation induced by addition of dextran, because aggregation occurs too rapidly to follow with pathogenic glutamine repeat lengths.

Figure 4. Huntingtin aggregates in a cell model.

(a) Tomogram section and (b) corresponding rendered view of a cryo tomogram of a Huntingtin exon 1 aggregate from a FIB-milled lamella of a Hela cell (reproduced from [50*]). Fibrils, blue; ribosomes, green, ER, red; vesicles, white. Scale bar, 400 nm.