Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence.
Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.
Ban
Tadato
T
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Hamada
Daizo
D
Hasegawa
Kazuhiro
K
Naiki
Hironobu
H
Goto
Yuji
Y
D016229:Amyloid beta-Peptides / Q000378:metabolism / Q000648:ultrastructure*; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence; D058225:Plaque, Amyloid / Q000378:metabolism / Q000648:ultrastructure; D013844:Thiazoles
D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T