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
Department
of Pathology, Fukui Medical University and CREST, Japan Science and
Technology Corporation, Matsuoka, Fukui 910-1193, Japan
Received for publication, February 3, 2003, and in revised form, March 18, 2003
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ABSTRACT |
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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 There is an increasing body of evidence showing that many proteins
including the Alzheimer's amyloid Amyloid fibril formation is considered to be a
nucleation-dependent process in which non-native precursor
proteins slowly associate to form the nuclei (8). This process is
followed by an extension reaction, where the nucleus grows by
sequential incorporation of more precursor protein molecules. This
model has been validated by the observation that fibril extension
kinetics is accelerated by the addition of preformed fibrils,
i.e. by a seeding effect. However, the mechanism of fibril
formation by individual polypeptide chains is not completely
understood, and there are several variations of the
nucleation-dependent model (9, 10). To address the
mechanism of amyloid fibril formation, it is important to observe the
process at the single-fibril level. Recently, 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. Although 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. On the other hand, the strong interaction of amyloid proteins and the mica
surface used in AFM measurements resulted in the formation of fibrils
morphologically different from the intact amyloid fibrils (13).
ThT is known to bind rapidly to amyloid fibrils accompanied by a
dramatic increase of fluorescence at around 485 nm, when excited at 455 nm (14). This makes ThT one of the most useful probes to detect the
formation of amyloid fibrils. Fluorescence at around 485 nm becomes
useful in fluorescence microscopic studies, which make use of lasers
for the incident beam of excitation. On the other hand, TIRFM has been
developed to monitor single molecules (15, 16) by effectively reducing
the background fluorescence under the evanescent field formed on the
surface of slide glass (Fig. 1). By
combining amyloid fibril-specific 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 Proteins--
Recombinant human Fluorescence Microscopy--
The fluorescence microscopic system
used to observe individual amyloid fibrils was developed based on an
inverted microscope (IX70; Olympus, Tokyo, Japan) as described
previously (16). The ThT molecule was excited using an argon laser
(model 185F02-ADM; Spectra Physics, Mountain View, CA). The fluorescent
image was filtered with a bandpass filter (D490/30 Omega Optical,
Brattleboro, VT) and visualized using an image intensifier (model
VS4-1845; Video Scope International, Sterling, VA) coupled with a
SIT camera (C2400-08; Hamamatsu Photonics, Shizuoka, Japan).
Direct Observation of Amyloid Fibrils--
A Time-lapse Observation of Amyloid Fibrils--
In the case of
ThT Observation of
We first examined the
Intriguingly, the length range of the detected fibrils is similar to
that observed with electron microscopy (EM) (8, 17) or AFM (18). This
indicates that the evanescent field is very useful for determining the
length of amyloid fibrils. To obtain the exact length of fibrils by
conventional epifluorescence microscopy, one has to analyze the image
by three-dimensional reconstruction because the orientation of fibrils
relative to slide glass is not always parallel to the glass surface. In
contrast, since the penetration depth of the evanescent field formed by
the total internal reflection of laser light is quite shallow (~150
nm for laser light at 455 nm) in comparison with the width of amyloid fibrils (~15 nm from EM), TIRFM selectively monitors long fibrils lying along the slide glass (Fig. 1). Consequently, the length of
observed fibrils is close to the exact length. On the other hand, the
apparent width of the fibrils observed by fluorescence was larger than
the exact width because the observed emission fields extend the dye localization.
We then determined whether Kinetics of Fibril Extension--
We monitored the
seed-dependent extension reaction (Fig. 2, E-H;
also see movie b2m.mov, which is published in the Supplemental Material). At time 0, we identified the location of seeds. As we
increased the incubation time, we could clearly follow the extension of
fibrils: the extension ended at around 2 h under the conditions
used. This fact constitutes direct evidence that the fibril formation
by
Unidirectional fibril formation was first observed using Sup35, a yeast
prion determinant, by epifluorescence microscopy (11). However, another
group reported the bidirectional elongation of Sup35 based on the
observations with EM in conjunction with selective staining by gold
particles (9). Although we cannot exclude the possibility that the
interaction with the glass surface was responsible for the
unidirectional extension, the unidirectional picture is likely to hold
for the fibril formation of
The rate of extension of individual amyloid fibrils was analyzed by
plotting the length of fibrils as a function of time (Fig. 3a). For the respective
fibrils, the extension reaction could be well fitted to a single
exponential curve, consistent with a previous observation of the
seed-dependent extension reaction in test tubes (8, 17,
18). Importantly, the rates of fibril extension, however, varied
significantly depending on the fibrils, although the rate for each
fibril remained constant. The initial fibril growth rate showed a wide
distribution with a mean value of 47.4 ± 15.0 nm
min Medin Fragment and A
Another example is A
In conclusion, we reported a new method to visualize the amyloid
fibrils at the single fibril level. The method makes use of the
specific ThT binding to amyloid fibrils and TIRFM. Since ThT binding is
common to all amyloid fibrils, the present method will have general
applicability for the analysis of amyloid fibrils. One of the
advantages of TIRFM is that only amyloid fibrils lying in parallel with
the slide glass surface were observed, so that we can obtain the exact
length of fibrils. Consequently, the method will be particularly
important for following the rapid kinetics of fibril formation, which
is paramount to elucidating the mechanism of amyloid fibril formation
and little accessible by other approaches.
2-microgobulin (
2-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
2-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
-peptide.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-peptide
(A
),1 prion protein,
transthyretin, and
2-microgobulin (
2-m) tend to misfold and
aggregate into amyloid fibrils (1-3). Moreover, several proteins not
known to be involved in disease and various polyamino acids have been
shown to form amyloid fibrils in vitro under carefully
selected conditions (4, 5). Although no sequence or structural
similarity between the amyloid precursor proteins has been found,
amyloid fibrils share several common structural and spectroscopic
properties (6). 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. Making use of an NMR technique in
combination with hydrogen/deuterium exchange of amide protons and
dissolution of amyloid fibrils by dimethyl sulfoxide, we have shown
that the amyloid fibrils of
2-m are stabilized by a hydrogen-bond
network which is more extensive than that in the native state (7).
2-m amyloid
fibrils.
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Fig. 1.
Schematic representation of amyloid fibrils
by total internal reflection fluorescence microscopy. The
penetration depth of the evanescent field formed by the total internal
reflection of laser light is ~150 nm for laser light at 455 nm, so
that only amyloid fibrils lying in parallel with the slide glass
surface were observed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-m with four additional
residues at the N-terminal
(Glu
4-Ala
3-Tyr
2-Vla
1-Ile1)
was expressed and purified as described (17, 18). Synthesized medC
fragment (NFGSVQFV) and A
-(1-40) were purchased from Peptide Institute, Inc. (Osaka, Japan). The purities of the peptides were >95% according to elution patterns of high performance liquid chromatography.
2-m amyloid
fibrils were prepared as described previously (17, 18). Seed fibrils
prepared by the fragmentation of amyloid fibrils were mixed with 25 µM monomeric
2-m in polymerization buffer (50 mM sodium citrate, pH 2.5, and 100 mM KCl) at
37 °C. After 6-h incubation, the sample solution was diluted 10-fold with polymerization buffer, and 100 µM ThT was added at
the final concentration of 5 µM. An aliquot (14 µl) of
sample solution was deposited on quartz slide glass, and the fibril
image was obtained with TIRFM. All images were recorded on digital
videotape and analyzed using Image-pro Plus (Media Cybernetics, Silver
Spring, MD).
-(1-40) amyloid fibrils were prepared from synthetic A
-(1-40)
(19). To obtain seed, preformed fibrils were fragmented by sonication
as described above. The seeds were added at a final concentration of 10 µg/ml to 50 µM monomeric A
-(1-40) in 50 mM sodium phosphate buffer at pH 7.5 and 100 mM
NaCl. After 3-h incubation at 37 °C in the test tube, the solution
was diluted 5-fold, and ThT was added at a final concentration of 5 µM. The fibril formation of
2-m or A
-(1-40) on the
slide glass was also examined.
2-m, seed fibrils were mixed with 50 µM monomeric
2-m in polymerization buffer (50 mM sodium citrate at pH 2.5 and 100 mM KCl). After ThT was added at 5 µM, the solution was deposited on quartz slide glass, and
the growth of individual fibrils was observed every 15 min under a
microscope at 37 °C. For medC, seed fibrils were prepared by
incubating the monomeric peptide at 1 mM in 10 mM sodium phosphate buffer, pH 7.0, at 37 °C for 24 h. The fibrils were fragmented by a sonicator as described above. An
aliquot (1 µl) of the seed solution was mixed with 1 mM
medC monomer in the same buffer containing 5 µM ThT and
deposited on quartz slide glass at 37 °C for visualization with
TIRFM. The images of amyloid fibrils grown under TIRFM recorded on
digital video tape were captured on a personal computer and the lengths of the fibrils were calculated using Image-pro Plus.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-m Amyloid Fibrils--
2-m, a
99-residue protein with a typical immunoglobulin domain fold (20), is
the light chain of the major histocompatibility complex class I
antigen. However, it is also found as a major component of amyloid
fibrils deposited in dialysis-related amyloidosis, a common and serious
complication in patients receiving hemodialysis for more than 10 years
(8, 21). Although the exact mechanism of the
2-m amyloid fibril
deposition in vivo is still unknown, amyloid fibrils are
easily formed in vitro by a seed-dependent extension reaction at pH 2.5, in which acid-unfolded monomeric
2-m
is added to seed fibrils taken from patients (8, 17, 18).
2-m amyloid fibrils already extended in test
tubes (Fig. 2, A and
B). TIRFM images indicated the presence of 1-5-µm-long
fibrils in the presence of ThT. No such fibrillar structures
were found either in the absence of ThT or in the absence of fibrils.
This indicated that amyloid-specific fluorescence from ThT enables one
to visualize the
2-m amyloid fibrils.
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Fig. 2.
Images of 2-m
amyloid fibrils observed by ThT fluorescence and TIRFM. Amyloid
fibrils were prepared in a test tube (A and B)
and on glass slides (C and D). E-H,
growth processes of amyloid fibrils. Incubation times are 0 (E), 30 (F), 60 (G), and 90 (H) min. In H, ThT fluorescence at time 0 was
overlaid in red to identify the locations of seed fibrils. The
scale bars are 10 µm.
2-m amyloid fibrils also formed on the
slide glass. Goldsbury et al. (13) reported using synthetic human amylin that amylin fibrils that assembled on a mica surface for
AFM measurement exhibited distinct morphological features. The seeds,
i.e. fragmented fibrils, were mixed with monomeric
2-m
and immediately the solution was deposited on quartz slide glass. As
can be seen, the amyloid fibrils extended on the slide glass with an
incubation time of 6 h (Fig. 2, C and D)
were similar to those prepared in the test tube (Fig. 2,
A and B).
2-m is a seed-dependent process, as suggested for
other amyloid fibrils (9-11, 13). A majority of extended
2-m
fibrils exhibited unidirectional elongation from the seeds which were
marked with red (Fig. 2H). Moreover, when the fibrils with
bidirectional elongation were observed, the superposition of the seeds
was suggested. Therefore, we can conclude that the elongation is mostly unidirectional.
2-m. The unidirectional elongation was
also dominant in the formation of fibrils by medC (see below).
1 (Fig. 3b), which cannot be explained by
the statistical distribution of the fibril growth rate. Taking into
account the fact that the extension rate for each fibril is constant,
the diversity in the rate may be related to the difference in the
structure of individual fibrils. Recently, the direct observation of
fibril formation by AFM indicated that the fibril-forming region of
Sup35 forms a diverse population of fibrils that could be distinguished
on the basis of their kinetic properties, including polarity and elongation rate (10). Furthermore, another study with NMR (22) indicated that amyloid fibrils formed by the A
-(25-35) peptide exhibit a heterogeneity in the kinetics of their hydrogen/deuterium exchange behavior for each amide group. Thus, current data obtained for
2-m as well as the results discussed for Sup35 and A
peptide suggest that heterogeneity of structure is a common characteristic of
amyloid fibrils. This could be partly consistent with the idea that the
rate of crystal growth may be affected by bonding topology at the
crystal surface (23).
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Fig. 3.
Kinetics of 2-m
amyloid fibril growth. a, fibril length
versus incubation time. Lines show the fitting to
the pseudo first-order kinetics. b, histogram for the
distribution of the initial fibril growth rate.
-(1-40)--
To confirm the overall
applicability of this method, we examined two other amyloid fibrils.
One is medC corresponding to the C-terminal octapeptide of medin (24).
Medin, a 50 amino acid internal cleavage peptide of lactadherin, is a
component of the very common age-related amyloidosis deposited on the
aortic wall. It has been shown that the C-terminal 8-amino acid peptide
NFGSVQFV from human medin is associated with amyloid fibrils at neutral pH, 37 °C (24). We first prepared the seed fibrils. In the case of
medC, it was difficult to prepare extensively fragmented seeds by
sonication. This might be related to the very rigid and sharp morphology of the medC fibrils. The extension reaction was monitored every 5 min under microscopic conditions (Fig.
4, A and B). We observed the extension of the fibrils, which was mostly unidirectional as was the case for
2-m. Analysis of the extension rate also indicated significant heterogeneity of the extension rate (data not
shown).
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Fig. 4.
Applicability of the ThT method to other
amyloid fibrils. A and B,
seed-dependent fibril growth of medC. A -(1-40) amyloid
fibrils were prepared in a test tube (C) or on a glass slide
(D). In B, the locations of seed fibrils are
indicated by red.
(Fig. 4, C and D). The
intracerebral accumulation of A
as senile plaques or vascular
amyloid is one of the dominant characteristics in the pathogenesis of
Alzheimer's disease (19). A
-(1-40) fibrils prepared in test tubes
and on slide glass, both by the extension reaction, were compared.
Fibrillar structures specifically stained by ThT were formed both in
the test tube (Fig. 4C) and on the slide glass (Fig.
4D). On the slide glass, we often observed clustered
aggregates even in the presence of seeds.
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ACKNOWLEDGEMENTS |
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We thank T. Wazawa and A. Fernández for valuable discussions.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Science, Culture and Sports.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this journal (available at
http://www.jbc.org) contains a movie file (b2m.mov): the growth
processes of amyloid fibrils monitored by ThT fluorescence with total
internal reflection fluorescence microscopy, as shown in Fig. 2,
E-H.
§ These authors contributed equally.
¶ Supported by Japan Society for Promotion of Science Research Fellowships for Young Scientists. Present address: Dept. of Developmental Infectious Diseases, Research Inst. and Osaka Medical Center for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1011, Japan.
** To whom correspondence should be addressed: Inst. for Protein Research, Osaka University 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8614; Fax: 81-6-6879-8616; E-mail: ygoto@protein.osaka-u.ac.jp.
Published, JBC Papers in Press, March 18, 2003, DOI 10.1074/jbc.C300049200
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ABBREVIATIONS |
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The abbreviations used are:
A, amyloid
-peptide;
2-m,
2-microgobulin;
AFM, atomic force
microscopy;
TIRFM, total internal reflection fluorescence microscopy;
medC, C terminus derived from human medin;
EM, electron
microscopy.
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