Direct Observation of Amyloid Fibril Growth Monitored by Thioflavin T Fluorescence

Tadato Ban,Daizo Hamada,Kazuhiro Hasegawa,Hironobu Naiki,Yuji Goto

doi:10.1074/jbc.c300049200

Abstract

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.

Highlights

From the ‡Institute for Protein Research, Osaka University and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Cooperation, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan and the ʈDepartment of Pathology, Fukui Medical University and CREST, Japan Science and Technology Corporation, Matsuoka, Fukui 910-1193, Japan
Irrespective of the protein species, electron microscopy and x-ray fiber diffraction indicate that the amyloid fibrils have relatively rigid structures with diameters of 10 –15 nm consisting of cross-␤strands
Gobulin; AFM, atomic force microscopy; TIRFM, total internal reflection fluorescence microscopy; medC, C terminus derived from human medin; EM, electron microscopy

Summary

Accelerated Publication

Epifluorescence with a newly introduced fluorescent dye [11] and atomic force microscopy (AFM) [12, 13] have been utilized for the direct observation of individual amyloid fibrils These techniques are quite useful in providing information on the mode of fibril growth, the need to introduce the fluorescence probe prevents their general application. By combining amyloid fibrilspecific ThT fluorescence and TIRFM, it would be possible to observe the amyloid fibrils and their formation process without introducing any fluorescence reagent covalently bound to the protein molecule. We examined this possibility using the ␤2-m amyloid fibrils. Gobulin; AFM, atomic force microscopy; TIRFM, total internal reflection fluorescence microscopy; medC, C terminus derived from human medin; EM, electron microscopy

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Direct Observation of Amyloid Fibril Growth