Determination of Microscopic Parameters of Amyloid Aggregation by Monitoring Real-Time Growth using TIRF Microscopy

Aggregation of amyloid proteins is involved in the pathology of several diseases. In vitro, growth of the amyloid fibrils has been shown to occur via multiple competing pathways such as primary and secondary nucleation, and fragmentation. There are several ensemble measurements, most commonly the thioflavin-T (ThT) fluorescence assay, to estimate the kinetics. On the other hand, imaging tools are used to report mostly the morphology of the amyloid aggregates. Here, we have used TIRF microscopy to estimate the rate constants of aggregation of amyloid-β (Aβ) peptides by real-time monitoring of the growth of the individual amyloid fibrils using ThT as a fluorescence marker. The real-time TIRF images show appearance of fibrils and its elongation in real-time enabling us to measure the rate constants of nucleation (k n) and elongation (k e). The aggregation experiments are performed on supported lipid bilayers prepared on glass coverslips to mimic the surface of cellular membranes. Comparison of Aβ40 and Aβ42 indicate that the elongation rate constants are quite similar between the alloforms but the rate of nucleation is considerably faster in case of Aβ42. Furthermore, comparison of the growth of the fibrils under different solution conditions show considerable effects on the kinetics and the morphology of the fibrils. Taken together our results suggest that real-time TIRF microscopy is a powerful method to delineate the mechanism of growth of the amyloid fibrils