Figure 3.

Nanoscopic properties of the clusters fibrillar α-Syn polymorphs form on primary neuron plasma membrane. (A) Super-resolution images rendered with a pixel size of 10 nm (red, top row) show the binding and clustering of fibrillar α-Syn-ATTO-647N polymorphs on neurons after 60 min exposure to the fibrillar polymorphs fibrils, ribbons, and fibrils-91 (50 nM). Bottom row (white) shows fibrillar α-Syn-ATTO-647N clusters identified through the DBSCAN analysis (see Material and Methods). (B) The density (detections per μm²) of single-molecule events on neurons (clustered and nonclustered) in (A) is given, showing differential binding of the distinct fibrillar α-Syn polymorphs. (C) The proportion of single-molecule detections within clusters is given, showing predominantly clustered (90%) binding of the distinct fibrillar α-Syn-ATTO-647N polymorphs. (D) Measurement of area of clusters (μm²) is shown as a cumulative distribution plot and box plot (inset, n = fibrils: 7681, ribbons: 4600, fibrils-91: 8672). The distribution shows that the fibrillar polymorph fibrils-91 populates clusters >200,000 nm². The cumulative plot shows that the polymorph ribbons populates the smallest clusters (<500 nm²). (E) A representative example is given, showing single-molecule trajectories (colored) obtained using SPT-STORM of the α-Syn-Photoactivable-646 fibrils-91 polymorph at two magnifications. Trajectories were analyzed by Bayesian treatment of hidden Markov models (see Materials and Methods), which revealed three diffusive states of α-Syn polymorphs. (F) State 1 represents free, fast-diffusing molecules; state 2 exhibits intermediate diffusion velocity, representing small complexes; and state 3 represents a clustered fraction with very slow diffusion velocity. (G–I) Plots showing averaged diffusion coefficient values (G) and dwell time within each diffusive state (H) and occupancy (I) for each polymorph are given. Note the ribbons polymorph exhibits characteristic different diffusive behavior. Each dot represents the averaged value of thousands of trajectories for a given imaging field. The dot plots in (B) and (C) represent averaged value per image, n = 9 cells from three independent experiments. The box plot in (D) shows median, interquartile range, and 10–90% distribution. The dot plots in (G) and (H) show averaged value per recording (thousands of trajectories) from three independent experiments. A Mann-Whitney test was performed. ^∗p < 0.05, ^∗∗∗p < 0.001; ns, not significant. Scale bars, 1 μm.