Kinetic analysis reveals that independent nucleation events determine the progression of polyglutamine aggregation in C. elegans

Abstract

Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer's, Parkinson's, and Huntington's diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms. Here, we take advantage of the unique properties of a Caenorhabditis elegans model expressing a fluorescently tagged polyglutamine (polyQ) protein, which aggregates into discrete micrometer-sized inclusions that can be directly visualized in real time. We provide a quantitative analysis of protein aggregation in this system and show that the data are described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h^-1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic molecular stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results highlight how independent, stochastic nucleation events in individual cells control the overall progression of polyQ aggregation in a living animal. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

Keywords: C. elegans; amyloid; chemical kinetics; polyglutamine; protein aggregation.

Fig. 1.

Q40-YFP expressed in C. elegans body wall muscle cells displays concentration-dependent, amyloid-like aggregation kinetics with each cell acquiring one inclusion on average. (A) Confocal images of C. elegans strain AM141, progressively displaying bright inclusions of a relatively uniform size and shape in the body wall muscle cells. Lower panels are magnifications of the boxed areas in the Upper panels. (Scale bars: Upper, 50 µm; Lower, 10 µm.) (B) Transmission electron micrographs of embedded sections of 96-h-old animals showing the subcellular localization of the inclusions (yellow outlines) below the muscle sarcomere (red outlines). Right shows a higher magnification image corresponding to the boxed region in the Upper Left panel, displaying a meshwork of fibrils with a typical width on the order of 10 nm. (Scale bars: Left, 1 µm; Right, 100 nm.) (C) Average number of inclusions per animal over time for age-synchronized populations of C. elegans expressing Q40-YFP in body wall muscle cells for homozygous (+/+) and heterozygous (+/−) animals. The plateau value for the number of inclusions approximates the number of cells in which the protein is expressed, indicated by the dashed lines at 95 for the body wall muscle cells and 104 for the total number of muscle cells in which Q40-YFP is observed. n = 12 to 20 animals for each timepoint; error bars indicate the SD. (D) Confocal image of an animal stained with phalloidin around the midpoint of aggregation (66 h) to reveal the muscle filaments. Dashed lines indicate the approximate boundaries between muscle cells, revealing that some have acquired an inclusion by this time, whereas in other cells visible aggregation has not yet taken place. (Scale bar, 20 µm.) (E) Panels i and ii: close-up confocal images of the muscle tissue in strain AM141 followed for 40 min at around the midpoint of aggregation (64 h) (SI Appendix, Fig. S1B). Yellow arrows point to inclusions that are observed to grow during this time, whereas arrowheads indicate mature inclusions that do not change in size. Note that diffuse signal of soluble Q40-YFP is depleted around the mature inclusions. (Scale bar, 10 µm.)

Fig. 2.

Q40-YFP aggregation occurs stochastically in individual muscle cells. (A) Illustration showing the distribution of the 95 body wall muscle cells in adult C. elegans. Four bundles of muscle cells run along the posterior–anterior axis. The animals typically crawl on their left or right side on solid media, resulting in a superposition of the two dorsal and the two ventral bundles (Middle Left). A schematic view from the anterior side shows the localization of the four bundles in the different quadrants (Middle Right). Cell shapes were drawn based on http://wormatlas.org. (B) The 32-h-old animals display their first inclusion at any position along the posterior–anterior axis. The asterisks mark the anterior of the animals; inclusions are highlighted by yellow spheres for better visualization. (C) The 32-h-old animals with two or more inclusions support the notion that nucleation is initiated stochastically in individual cells, in the absence of a pattern of spatial propagation from the first to subsequent inclusions. (D) Cartoon showing the occurrence of aggregation events (yellow stars) in bulk, nanodroplets, and in C. elegans muscle cells. In a typical test tube reaction (Top), all aggregation events take place within the same continuous volume. In nanodroplets (Middle) and C. elegans muscle tissue (Bottom, only one bundle of muscle cells is shown for clarity), the total volume is divided over multiple small volumes, in which the probability of nucleation is low. As a consequence, aggregation will be initiated in different droplets or cells at different points in time. Three aggregation events are shown in each system for clarity, but in reality the number of nucleation events in a given period of time is proportional to the size of the reaction vessel.

Fig. 3.

Determining the mechanisms of Q40-YFP aggregation in vivo by global fitting of aggregation time courses at multiple protein concentrations. (A) Global fit (solid lines) of the average number of inclusions per animal, assuming a constant nucleation rate over time and no cooperativity. The two global free parameters are the nucleation rate and the reaction order. (B) Global fit (solid lines) of the same dataset shown in A, but using a model that forces significant cooperativity. Cooperativity is not spatially restricted in this model and can occur between any of the cells. The two global free parameters are the nucleation rate and the reaction order. (C) Prediction of the total aggregate amount based on the fit in A and assuming fast inclusion growth on a timescale of hours (solid lines), compared to the observed integrated fluorescence over the inclusions per animal. (D) Prediction as in C, but assuming slow inclusion growth on a timescale of days. Data are representative of two independent experiments. The dashed lines connecting the datapoints in A–D are to guide the eye, and error bars indicate the SEM. n = 20 animals per strain and timepoint.

Fig. 4.

The stochasticity of nucleation sets a lower boundary for the SD of the inclusion numbers in a population of animals. (A–D) Prediction (solid line) and experimental datapoints of the SDs for (A) line A, (B), line B, (C) line C, (D) line D. The predictions are based on the nucleation rate for each strain as determined from global fitting as shown in Fig. 3A. n = 20 animals for each strain and timepoint; data are representative of two independent experiments.