Deciphering the Structure, Growth and Assembly of
Amyloid-Like Fibrils Using High-Speed Atomic ForceMicroscopy
Pierre-Emmanuel Milhiet1,2*., Daisuke Yamamoto3., Olivia Berthoumieu1,2, Patrice Dosset1,2, Christian
Le Grimellec1,2, Jean-Michel Verdier4, Stéphane Marchal4, Toshio Ando3
1 Inserm, U 554, Montpellier, France, 2 Université de Montpellier, CNRS, UMR 5048, Centre de Biochimie Structurale, Montpellier, France, 3 Department of Physics,
Graduate School of Science and Technology, Kanazawa University, Kanazawa, Japan, 4 Inserm, U710, Montpellier, France; Université de Montpellier 2, Montpellier, France;
EPHE, Paris, France

Abstract
Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various
neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We havepreviously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer’s disease andpresent in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation.
In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine
protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very highvelocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing azipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils
can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril toassociate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues
about fibrillization mechanism of amyloid proteins.
Citation: Milhiet P-E, Yamamoto D, Berthoumieu O, Dosset P, Le Grimellec C, et al. (2010) Deciphering the Structure, Growth and Assembly of Amyloid-Like Fibrils
Using High-Speed Atomic Force Microscopy. PLoS ONE 5(10): e13240. doi:10.1371/journal.pone.0013240
Editor: Mark R. Cookson, National Institutes of Health, United States of America
Received August 18, 2009; Accepted September 15, 2010; Published October 8, 2010
Copyright: ß 2010 Milhiet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Agence Nationale de la Recherche (ANR), Physique et Chimie du Vivant (PCV) program, grant #AFM-MB-PROT and#HS-Bio-AFM, and by Core Research for Evolutional Science and Technology/Japan Science and Technology Agency)(CREST/JST, DY and TA). The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: pem@cbs.cnrs.fr. These authors contributed equally to this work.

backbone involved in molecular interactions without extended
structural changes of the globular portion of the protein. Directstacking interactions [13], cross-ß spine patterns [4], or 3D
domain swapping [14,15] governs fibril elongation in thesesystems.
Among the proteins classified in the latter model, lithosthatine(also named Reg-1) is a protein of 144 amino acids that is
produced by pancreas acinar cells and secreted into pancreatic
juice. The protein tightly binds calcium carbonate crystals andmay control their formation preventing clogging of the ducts[16–19]. In chronic calcifying pancreatitis, the protein forms
deposits in pancreatic ducts [20]. Lithostathine is also expressedin other tissues including gastric cells [21] and the proteinfunctions as a mitogenic and/or antiapoptotic factor in thedevelopment of early gastric cancer [22]. More recently,
potential role in normal and neoplastic germ cell proliferation
has been described [23]. Lithostathine is very susceptible to selfproteolysis under specific pH conditions and cleavage produces
a soluble N-terminal undecapeptide and a C-terminal form of
133 amino acids. This processed protein, called S1, precipitatesand forms protease-K-resistant fibrils at physiological pH [24]that deposit in the brain of patients with Creutzfeldt-Jakob,

Introduction
Protein aggregation and fibril formation are molecular events
that have been related to the emergence of more than 20 humandiseases called conformational diseases [1] or proteopathies [2]and these diseases develop with the misfolding of normally-soluble
proteins. Over the past decade, significant progress has been madein understanding the structural arrangements of amyloid fibrils.
Despite large differences in size, native structure and functions ofproteins, numerous molecular models of amyloid fibrils display
similarities [3,4]. From a mechanistic point of view, a broad
pathway of fibril formation could be schematized by one of threestraightforward models: i) the ‘‘refolding’’ model, in which selfpropagating conformational rearrangements must occur in orderto adopt a structure competent for self-assembly into fibrils. That isthe case for insulin [5], the SH3 domain of bovine PI3K [6],myoglobin [7] and prion proteins [8]; ii) the ‘‘natively disordered’’model, composed of proteins or peptides whose native structure
was mainly disordered such as polyglutamine proteins [9],huntingtin [10], ataxins, yeast prions or the amyloid beta (Aß)peptide of amyloid plaques [11,12]; iii) the ‘‘gain-of-interaction’’model that concerns proteins having only a part of the peptide
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Lithostathine HS-AFM Imaging

Gertsmann-Straüssler-Scheinker diseases [25] and AD, especially during the very early stages [26]. On the basis of X-ray
structure of the monomer [27,28], biochemical experiments,
high-resolution electron microscopy (EM), and atomic force
microscopy (AFM), it was proposed that lithostathine is first
assembled via lateral hydrophobic interactions as a tetramer.
Each tetramer can then interact with another tetramer throughelectrostatic interactions to form helical structure named
protofibril [26]. Structural assembly of these protofibrils canform a two-dimensional network. Recent works also suggest that
juxtaposed protein units exchange a mobile loop (domain
swapping) to form highly ordered fibrillar structures [25] and nocross-ß pattern was observed for fibrillar lithosthathine.
Similarly to PrPsc, lithostathine deposits were resistant to thedrastic proteinase K treatment [25].
In this paper, we present real time visualization of lithostathine
fibrillization using high-speed Atomic Force Microscopy (HSAFM) that allows for high imaging rate with good lateral andvertical resolution [29]. Under these conditions, we were able tovisualize elongation of lithostathine protofibrils and their assembly
into fibrils. Dynamics and remodelling of these assemblies werealso observed.

Results
AFM imaging of the lithostathine S1 form in air
The S1 form of lithostathine was generated in solution bytrypsin digestion of full-length protein that mimics native automaturation of the protein [24], coated on mica, and imaged using
tapping mode AFM in air. As expected from a previous study [26],a network of protofibrils was observed (Fig. 1). Protofibril lengths
ranged from a few tens of nanometers to several micrometers andtheir density was variable (see a high density in A inset). They
tended to laterally associate forming bundles containing up to 10
protofibrils (white arrows in A inset). The apparent diameter ofprotofibril (8.761.1 nm) was evaluated by measuring the top-totop distance between two adjacent protofibrils because thisdistance is not affected by tip convolution (see sections in
Fig. 1D). This value is in the same range than that obtained by
EM and molecular modeling of lithostathine tetramer [26]. The
height of most of the filamentous structures as compared to mica
was estimated to be 5.9760.71 nm. However a lower height(3.0460.52 nm), half of the first value, can be clearly identified inseveral cases (see white arrowheads in Fig. 1A and section in
Fig. 1C). This result suggests that two protofibrils could be

Figure 1. AFM height images of lithostathine in air. A and B (zoom of A) are height AFM images showing the S1 form of lithostathine forming
protofibrils, isolated or laterally associated. C and D are profiles of B indicated by thin colored lines. The profile C measures the two different heightsthat were observed along protofibrils (white arrowheads in A) and section D the distance between two laterally associated protofibrils (top-to-top
distance). The inset in A illustrates propensity of lithostathine protofibrils to form bundles on mica (white arrows). The black arrows indicate whatlooks like a branching point of protofibrils whereas the black arrowheads indicate overlapping filaments. The scale bars are 200 nm (A) and 100 nm(B) and the height color scale is 20 nm.
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elongating. Alternatively, direction could be influenced by thesubstrate. Due to the size of fibrils and protofibrils that mostly
exceeds the scanning area, it was difficult in this study to analyze
their polarization.
In addition to the lateral association of protofibrils, the stackingof protofibrils and fibrils was also observed (Fig. 3B, Fig. 4 andMovie S2), a result consistent with two different heights observedin air. Interestingly, as suggested by real time imaging of theirassociation shown in Movie S1 and in time lapse in Fig. 3B,
protofibrils have a high propensity to interact with each otheralong their main axis via a zipper-like mechanism. Even rarely,
elongation of a single protofibril over a fibril was also observed. In
the time lapse in Fig. 4, a single protofibril with a 12 nm apparent
diameter (grey arrowhead) is stacked on top of a fibril composed of
2 laterally associated protofibrils (see the profile shown in Fig. 4E).
Real time imaging of the elongation of a protofibril at the top of abundle of protofibrils is shown in Movie S2. During theseexperiments no more than two protofibrils or fibrils were stacked.
Structures suggesting branching of protofibrils or fibrils was also
observed in liquid (white arrowhead in Fig. 2C and Movie S1) but
HS-AFM experiments clearly indicated that these structure in factcorresponds to interaction between the extremities of growing
protofibril or fibril with the side of another and not to formation of
two protofibrils from a parental structure (Movie S1). These
structures were nevertheless not so often observed as compared toexperiments in air. This can be explained by the fact that AFM
imaging in air was performed with mature lithostathine whereas
this maturation was performed in the buffer bathing the tip with
HS-AFM. In addition angle measured between the main axis of
two protofibrils or fibrils was often around 70u. Also expected fromresults in air, overlapping of fibers was observed in liquid (Fig. 2C).
This phenomenon increased as a function of time upon trypsin
incubation and ultimately resulted in a complex network or tangleof fibrils (Fig. 2D). Overlapping most probably results fromimaging a network of fibers in 2 dimensions.
In addition to isolated protofibrils and bundles of laterally
associated protofibrils, a new type of lithosthatine assemblies wasobserved using HS-AFM imaging in liquid, namely helical fibrils(Fig. 5 and Movie S3). These structures were formed from two lefthanded winded protofibrils as supported by their apparent
diameter of 24.260.8 nm as well as the height above the micaof 14.660.7 nm (Fig. 5B) (height of a single protofibril ascompared to the mica is 7.861.7 nm). Helix periodicity was
52.7611 nm ranging from 44 to 74 nm (Fig. 5C). Some helicalfibrils appeared to be symmetric with blunt ends (Fig. 5A) whereas
others had one of the protofibril longer than the other, forming a
stiff protofibril (Fig. 5D). This observation suggests that the two
protofibrils had different lengths or that their winding took place
after their formation. Similar partial winding has also been
observed in aggregating Aß peptide [30]. During our experiments,
slight helical unwinding was sometimes observed (Movie S3) but
we never imaged any winding process or any formation of such
helical structure, strongly suggesting that helical fibril oflithostathine could only be formed in bulk without any interaction
with mica. No higher order helical structures were observed (datanot shown). It is important to differentiate these helical fibrils fromhelical protofibrils that have been described in a previous
publication and correspond to stacking of lithostathine tetramers[26].

vertically stacked on top of each other and the height of one
protofibril imaged in air could be roughly estimated to be 3 nm.
AFM pictures also suggested protofibril branching (black arrows in
Fig. 1A) but, under these conditions, it was difficult to discriminate
between a real branching and an interaction between two
protofibrils. In addition overlapping of fibrillar structures wereclearly delineated by the AFM tip (black arrowheads in Fig. 1A
and 1B). Interestingly globular structures were observed on mica
with a size ranging from 6 to a few tens of nanometers with anapparent height similar to that measured for a protofibril.

Structure and dynamics of lithostathine using high-speed
AFM
In order to avoid damaging of the protofibrils, we then decided
to perform AFM imaging in buffer, a physiological condition thatminimizes the force applied by the tip. Taking advantage of thedevelopment of HS-AFM allowing data acquisition at video rates[29], we also investigated the dynamics of protofibril and fibril
formation. Starting from intact lithostathine, protofibril formation
was monitored in real time after addition of trypsin to the medium
bathing the tip. Even though the scanning area of such a
microscope is well below standard AFM, the high-speed scanner
was sufficient to visualize most of the structures described above.
After 15 min incubation in presence of trypsin, globular
structures were imaged having an apparent diameter ranging
from 15 to 28 nm that compared well with our results in air (seeFig. 2, 3 and 4). These structures protruding from 6.6 to 22 nm
above the mica could correspond to lithostathine oligomers(lithostathine tetramer is about 9 nm in diameter and 2.5 nm inthickness) [26]. After 30 min incubation, filamentous structures
were imaged at one image per sec allowing their association as wellas their elongation to be observed (see Movie S1 and snapshots in
Fig. 2). The width of these structures varied largely and thethinnest filaments most probably correspond to single protofibrils(white arrows in Fig. 2A) since their apparent width measured at
mid-height was 15.564.4 nm (Fig. 2B, red profile), a slightly
overestimated value due to tip convolution as compared to the11.7 nm obtained with EM [26]. Similarly to AFM imaging in air,lateral association of protofibrils (up to 8) was observed (Fig. 2A
and D, Fig. 3A and Movie S1) and more accurate width value wasobtained by measuring top-to-top distance between adjacent
protofibrils (9.962.8 nm)(Fig. 2B, blue profile). It was sometimes
difficult to visualize the groove between two adjacent protofibrils
suggesting that they strongly interact along their main axis.
Thanks to HS-AFM, elongation of filaments was observed in realtime (see Movie S1). Both protofibrils (first part of Movie S1 andtime lapse in Fig. 3) and fibrils were elongated with a velocity
ranging from 27 to 52 nm/s (values correspond to velocity
measured for at least 4 frames)(see Fig. S1, which corresponds tothe plot of the growth distance as a function of time). The way
fibers elongated in Movie S1 lead us to consider the possibility that
we might be imaging a sliding process of fiber on mica rather thana real growth. However this hypothesis can be ruled out by the factthat two fibers that laterally associated can grow independently
from each other (time lapse in Fig. 3A). Alternatively we might
expect the tip scanning to influence a putative sliding process but
such a phenomenon was never observed. Another interesting
feature of elongating protofibril was their ability to connect toanother pre-existing protofibril. As shown in Fig. 3 (blackarrowheads in B3 and B4), the edge of the elongated protofibril
fused with another to form a continuous structure (at least at thisscale). In this time lapse, preferential direction of growth of the
protofibril can be explained by the fact that it was interacting withanother fibrillar structure that could influence the way it was
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Discussion
In this work we carried out HS-AFM experiments underphysiological conditions allowing further structural characteriza3

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Figure 2. Height images of lithostathine protofibrils and fibrils
using high-speed AFM. A is the height image of two partiallyassociated protofibrils. The profile in B corresponding to the thincolored lines in A shows mid-height diameter of 16.7 nm for a single
protofibril (in red, delineated by arrowheads) and a top-to-top distanceof 9.7 nm for two laterally associated protofibrils (in blue). Associationsof the end of one fibril with the edge of another (white arrowhead in C
and Movie S1) as well as overlapping (black arrowheads in C and D) offibrils or protofibrils were easily identified. Long incubation time withtrypsin led to a complex network of protofibrils and fibrils (D) andbundles of lithosthathine protofibrils are frequently observed (greyarrow in D). The scale bars are 30 nm and the height color scale isautomatically adjusted during imaging.
doi:10.1371/journal.pone.0013240.g002

tion of protofibrils and fibrils of lithostathine, a protein involved in
Alzheimer’s disease, as well as real-time visualization of formationof these assemblies. Topography obtained using HS-AFM in liquid
is in good agreement with that obtained in air with a classical setup
or with EM experiments ([26] and Fig. S2) in terms of protofibril
diameter and ability of protofibrils to laterally associate to form
bundles plated on the substrate (Fig. 1). In addition, our dataclearly indicate that one lithostathine protofibril can be stacked on
top of another (we can exclude a sedimentation process since HSAFM imaging was performed with the sample above the tip, seeMaterials and Methods section). Such behavior highlights thepropensity of lithostathine protofibrils to associate along their main
axis and real time observation elucidates the manner in which a
protofibril associate (vertically or laterally) to another protofibril orto a fibril. The association propagated along their main axis
(Fig. 3), strongly suggesting a zipper-like mechanism that could be
related to the model of fibril formation through swapping domains
proposed for lithostathine (see introduction and [25]). We can
exclude that these bundles were induced by a strong interaction
with the mica because this feature seems to be singular to a few
amyloid-like proteins such as transthyretin and human calcitonin[31–33] and, to our knowledge, not observed with amyloid ß
peptides. In addition similar organization was observed when
lithostathine S1 form was deposited on highly ordered pyrolytic
graphite and imaged with AFM [26] or on carbon-coated grid andobserved with EM (see Fig. S2). It is noteworthy to mention that,in both EM and AFM in air experiments, the S1 form was formed
in solution before the coating and therefore a random depositionof protofibrils could not generate such bundles.
We especially provide new insights into the dynamics ofelongation of lithostathine protofibrils and fibrils on a relatively
short time scale (1 image/s). Elongation process appears to be very
rapid (a few tens of nanometers per sec). However it cannot
explain the difference observed with what had been calculated
from AFM experiments with amylin peptides (1.1 nm/min) [34]or with Aß1–40 protofibrils (1.3 nm/min) [35]. Interestingly it
compares well with recent results obtained for the elongation of
Aß25–35 fibrils on mica (ranging from 5 to 100 nm/s) using
scanning-force kymography, a high temporal resolution method
also performed using fibrils coated on mica [36]. So huge
difference in the elongation process between different publications
remains to be clarified but it is tempting to speculate that thisphenomenon largely depends on the protein and on the molecularmechanism of its elongation. HS-AFM also indicates thatlithostathine elongation seems to oscillate between two states, a
blocked state where lithostathine elongation is frozen, and aprocessing state during which the velocity is roughly constant (seean example of the two states in Movie S1). At this time it is difficult
to discriminate if the former is due to strong interaction with thesubstrate or if it really corresponds to native mechanism. The
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latter most probably corresponds to addition of pre-assembled
lithostathine tetramers to protofibril end, but spatial resolution of
our setup was certainly too low to visualize such details ofassembly. Nonetheless, it should have been sufficient to observe
addition of a pre-formed protofibrils to the end of another, andthis was never observed. From a mechanistic point of view, the factthat protofibril elongation was only observed for long incubation
time with trypsin suggests that a critical concentration of processedlithosthathine is required for the formation of tetramer or for
tetramer annealing to the protofibril end.
The shape of lithostathine protofibrils and fibrils were mainly
straight and very similar to the ura2p protein, a saccharomyces
cerevisiae protein with prion properties [37], and to TTR105–115, a
short peptide that serves as a model system for amyloid fibrils [32].
It could correspond to the type I of Aß fibrillar assemblies
according to a recently published classification, namely straightand rigid fibers with a diameter of 10–15 nm [38]. Despite thisapparent stiffness, lithostathine protofibrils could nevertheless twist
to form helical fibrils, always composed of two paired protofibrils.
Similar organization has been observed for several amyloid
proteins such as ura2p mentioned above [39], Aß peptides, anda-synuclein [40] and it was suggested that winding/unwinding
process of a-synuclein was essential for its growth process [30]. As
described with Aß(1–40) peptides [30,35,41], pitch in paired
helical filaments or axial cross-over spacing was variable indicatingthat lithostathine can have different forms even when incubatedunder the same conditions. Lithostathine helical structure also
compares well with paired helical filaments described for the
microtubule-associated protein Tau expressed in neurofibrillarytangles, an intracellular structure present in AD [42]. A niceexample of AFM imaging of Tau in liquid has been recently
published [43]. It is important to notice that no elongation of
protofibrils was observed for helical structure of lithostathine. At
the opposite, we have been able to observe elongation of straightprotofibrils, even when they were interacting with anotherprotofibril or fibril, laterally associated with an existing fibril orstacked on top of it (Figures 3 and 4). Similar behavior has beensuggested for the Aß25–35 peptide [36]. Interestingly the velocityof elongation of a fiber stacked on top of another was in the samerange as that observed for a fibril growing on the mica surface, alsosuggesting that lithostathine-mica interaction was loose under our
experimental conditions. This interpretation is strengthened by thefact that lithostathine assemblies were sensitive to the tip scanningthat sometimes swept away protofibrils or fibrils (data not shown).
Branching of Aß fibrils has been previously observed using AFM
and EM and is proposed to be essential for the post-nucleation
growth process [30,44]. Similarly glucagon fibrils can generate
new fibril ends by continuously branching into new fibrils [45]. In

Figure 3. Time lapse of protofibril association and elongation.
Elongation of single protofibril (white arrows) is shown in time lapses
A1–A4 and B1–B4. In both cases, protofibrils are laterally associatedwith another, at least for one part of their length. One protofibril cangrow while the position of the associated fiber is constant (whitearrowheads in time lapse A). Interestingly the edge of elongating
protofibril can associate with that of a pre-existing protofibril forming acontinuous structure (black arrowheads in B3 and B4). Real time verticalassociation of 2 protofibrils is also observed in time lapse B. They
partially interact in B1 (grey arrow) and are mostly stacked on top ofeach other in B4. A globular structure is also imaged and can be used as
fiducial mark. The time resolution is 2 sec, the scale bar is 30 nm, andthe height scale is automatically adjusted.
doi:10.1371/journal.pone.0013240.g003

Figure 4. Time lapse of the protofibril elongation stacked on top of a fibril. The grey arrowheads indicate the edge of the elongating
protofibril on top of a fibril. The profile in E corresponding to the white line in B indicates that only two fibers are stacked on top of each other (height
differences between blue and red arrows are 6.7 and 8 nm, respectively). The underlying fibril most probably corresponds to two laterally associatedprotofibrils. Micrographs correspond to successive images separated by 1 s. The scale bar is 30 nm and the height color scale is automaticallyadjusted during imaging.
doi:10.1371/journal.pone.0013240.g004

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Lithostathine HS-AFM Imaging

Figure 5. Height images of helical fibrils of lithostathine. A andD) Winding of two protofibrils can form helical fibrils (highlighted in theinset in A that corresponds to the filtered image). The edge of this helix
can be blunt (A) but one of the protofibril composing this helical fibril
can be lonely observed (D), protruding from the helix. B) Profile
delineated in A by the black line. Vertical and horizontal distances are
13.6 nm between grey arrowheads and 20.1 nm between blackarrowheads, respectively. C) Longitudinal profile of helical fibril shownin the A inset (the distance between the black arrowheads is 44.3 nm).
The scale bar is 30 nm and the height scale automatically adjusted.
doi:10.1371/journal.pone.0013240.g005

this case new fibrils mostly grew in the forward direction of theparent fibril with a preferential angle. AFM imaging in air (thisstudy and [26]) suggested lithostathine protofibril branching but
our HS-AFM experiments clearly indicate that these structures aredifferent from those observed with glucagon and Aß proteins andwas therefore generated by the end of elongating protofibrils or
fibrils interacting with the edge of a second fibril. It did notcorrespond to formation of a new structure from a parent fiber.
Because these structures were also observed using AFM in air and
EM (Fig. S2) in which the different structures were formed in thebulk before coating on mica or on the grid, we could hypothesizethat the elongated protofibril has a good affinity for the edge ofpreformed protofibrils. Interestingly, as observed with glucagon[45], a preferred angle was observed between two branchedlithostathine fibers. At this time it is however difficult to elucidate aprecise mechanism. It could be due to the fact that, when the angle
is smaller than the preferred angle, two fibers have a tendency tolaterally associate or to a preferential orientation of fibrils due tointeraction with mica as previously described with amyloid
proteins such as a-synuclein [46] or Aß peptide [47]. This point
remains to be clarified.
Two distinct stages have been proposed for ß-amyloid
fibrillization, namely nucleation and elongation (reviewed in[48]). Spherulitic structures with radial growth of fibrils havebeen observed with Aß(1–40) fibrils [38] and nuclei of Ure2p
proteins have been identified as precursors of Ure2p fibrils [49].
Despite the fact that we have observed globular structures
immobilized on mica that could correspond to nuclei oflithostathine tetramers, no nucleation areas were clearly identified.
Such a result could be explained by the fact that, once in a
growing state, elongation velocity is high and consequently theprobability to observe such an event is low. It also strongly suggests
a different mechanism of fibrillization for ß amyloid peptides ascompared to lithostathine, since no spherulitic structures wereobserved using AFM in air or in liquid.
Taken together, our results provide new insights into lithostathine protofibril elongation and assembly. Lithostathine sharessome features with Aß proteins such its ability to form helicalstructures but mechanism of lithostathine protofibrils formation
appears to be different from that of Aß proteins or glucagon. It is
important to mention that several properties of lithostathine could
not have been determined using a longer timescale and HS-AFM
thus appears as a powerful technique for the analysis of amyloid
proteins.

Methods
Lithostathine Purification
Recombinant lithostathine was produced from Chinese ovary
cells and purified on immunoaffinity column as described
previously [24]. Protein samples were then frozen in liquid
nitrogen and stored at 280uC until use.
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Lithostathine HS-AFM Imaging

well as globular structures can be observed. The movie shows thegrowth of two different fibrils that are blocked by the edge ofanother fibril. In the last part of the movie, the association of one
fibril on top of another is clearly observed. Most of the fibrils seemto be composed of several laterally associated protofibrils (seeFig. 2B). The scan size is 300 nm6300 nm (1286128 pixels) andthe height scale is automatically adjusted during scanning. The
scanning rate is 1 image/s and the movie is accelerated five times.
Found at: doi:10.1371/journal.pone.0013240.s003 (10.38 MB
AVI)

AFM imaging in air
AFM was performed in air with a Nanoscope IIIA (Veeco,
Dourdan, France) using FMR Nanosensors or AC160 TS
Olympus cantilevers. S1 form of lithostathine was coated in
15 mM Tris pH 7.5 at a concentration of 30 mg/ml on freshly
cleaved mica (Goodfellow, Lille, France), rinsed with water, dried
with nitrogen and kept in a dessicator until imaging. The scan rate
was between 0.2 to 1 Hz.

High speed AFM in liquid
The HS-AFM apparatus used in this study is basically the sameas that previously reported by Ando et al [50]. Images wereacquired at a 1 image/s rate using very sharp cantilevers with a
200 mN/m spring constant and a resonance frequency in water of
1.2 MHz (Olympus). Experiments were performed with intact
protein at a concentration of 40 mg/ml that was then processedusing a 1% trypsin solution, directly added in the AFM fluid cell,to generate the S1 form. Trypsin cleavage as well as AFM imaging
was performed in 10 mM Tris, 100 mM NaCl, pH 8 buffer.

Movie S2 High Speed AFM imaging of lithostathine protofibril
elongation stacked on the top of laterally associated protofibrils.
The movie shows the growth of one protofibril at the top of abundle of laterally associated protofibrils. The scan size is
300 nm6300 nm (1286128 pixels) and the height scale isautomatically adjusted during scanning. The scanning rate is 1
image/s and the movie is accelerated five times.
Found at: doi:10.1371/journal.pone.0013240.s004 (0.25 MB AVI)
Movie S3 High Speed AFM imaging of helical fibrils. Twosequences of helical fibril images are shown in this movie. First
sequence: the slight helical unwinding is shown. A single protofibril
is also swept away by the tip. Second sequence: real time imaging
of one single protofibril protruding from a long paired helicalfilament. The coating of one protofibril on mica is also observed.
The scan size is 300 nm6300 nm (1286128 pixels) and the heightscale is automatically adjusted during scanning. The scanning rateis 1 image/s and the movie is accelerated five times.
Found at: doi:10.1371/journal.pone.0013240.s005 (9.59 MB AVI)

Supporting Information
Figure S1 Elongation velocity of fibrillar lithostathine. The
graph represents the growth distance of fibrils or protofibrils as afunction of time. The zero values are origins of measurement intime and position. The bar errors correspond to S.D. values.
Found at: doi:10.1371/journal.pone.0013240.s001 (0.05 MB TIF)
Figure S2 Electron microscopy micrographs of the lithostathine
S1 form. The S1 form was coated on a Formvar-coated copper
grid for 60 s, dried with a filter paper and stained with uranyl
acetate as previously described (Gregoire C, et al. (2001) EMBO J
20: 3313–3321). Specimens were then observed with a Jeol 1220
transmission electron microscope. Lateral association of lithostathine fibrils was clearly observed in A and B. White arrowheads
highlight overlapping of fibrils whereas the black arrowhead
indicates the association of the end of two laterally associatedprotofibrils with the edge of another fibril. The scale bar is
250 nm.
Found at: doi:10.1371/journal.pone.0013240.s002 (3.80 MB TIF)

Acknowledgments
We are grateful to Mat Ferguson and Catherine A. Royer for carefulreading of the manuscript.

Author Contributions
Conceived and designed the experiments: PEM CLG JMV SM TA.
Performed the experiments: PEM DY OB SM. Analyzed the data: PEM
PD. Contributed reagents/materials/analysis tools: PEM DY JMV SM
TA. Wrote the paper: PEM.

High Speed AFM imaging of lithostathine. After
30 min trypsin incubation of the full-length lithostathine, fibrils as

Movie S1

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