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Methods 53 (2011) 275–284
Contents lists available at ScienceDirect
Methods
journal homepage: www.elsevier.com/locate/ymeth
Assessing mutant huntingtin fragment and polyglutamine aggregation
by atomic force microscopy
Kathleen A. Burke a, Jordan Godbey a, Justin Legleiter a,b,c,⇑
a
The C. Eugene Bennett Department of Chemistry, West Virginia University, 217 Clark Hall, P.O. Box 6045, Morgantown, WV 26506, USA
WVnano Initiative, West Virginia University, 217 Clark Hall, P.O. Box 6045, Morgantown, WV 26506, USA
c
The Center for Neurosciences, West Virginia University, 217 Clark Hall, P.O. Box 6045, Morgantown, WV 26506, USA
b
a r t i c l e
i n f o
Article history:
Available online 25 December 2010
Keywords:
Huntington’s disease
Atomic force microscopy
Polyglutamine
Neurodegenerative disease
Protein aggregation
a b s t r a c t
Huntington disease (HD), a neurodegenerative disorder, is caused by an expansion of more than 35–40
polyglutamine (polyQ) repeats located near the N-terminus of the huntingtin (htt) protein. The expansion
of the polyQ domain results in the ordered assembly of htt fragments into fibrillar aggregates that are the
main constituents of inclusion bodies, which are a hallmark of the disease. This paper describes protocols
for studying the aggregation of mutant htt fragments and synthetic polyQ peptides with atomic force
microscopy (AFM). Ex situ AFM is used to characterize aggregate formation in protein incubation as a
function of time. Methods to quickly and unambiguously distinguish specific aggregate species from
complex, heterogeneous aggregation reactions based on simple morphological features are presented.
Finally, the application of time lapse atomic force microscopy in solution is presented for studying synthetic model polyQ peptides, which allows for tracking the formation and fate of individual aggregates on
surfaces over time. This ability allows for dynamic studies of the aggregation process and direct observation of the interplay between different types of aggregates.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
1.1. Neurodegenerative disease related protein aggregation
A number of systemic and neurodegenerative disorders, including Alzheimer’s disease (AD), Huntington’s disease (HD) and Parkinson’s disease (PD), are characterized by the accumulation of
nanoscale protein aggregates within tissue or cellular compartments [1–4]. These diseases are defined by the misfolding of proteins from their native structure, promoting the formation of
proteinaceous, fibrillar structures rich in b-sheet content. Initially,
it was postulated that neurodegeneration was directly mediated by
fibril-containing lesions; however, increasing evidence suggests
that, rather than being pathogenic, fibrillar aggregates are inert
or potentially protective [5]. The assembly of misfolded protein
into fibrils can follow a complex aggregation pathway, resulting
Abbreviations: AD, alzheimer’s disease; AFM, atomic force microscopy; EPCG,
(2)-epigallocatechin-3-gallate; GST, glutathione S-transferase; htt, huntingtin; HD,
huntington disease; N17, the first 17 amino acids at N-terminus of htt; PD, parkinson’s disease; PBS, phosphate buffer saline; polyQ, polyglutamine; SDS-AGE,
sodium dodecyl sulfate agarose gel electrophoresis.
⇑ Corresponding author at: West Virginia University, The C. Eugene Bennett
Department of Chemistry, 217 Clark Hall, P.O. Box 6045, Morgantown, WV 26506,
USA. Fax: +1 304 293 4904.
E-mail address: [email protected] (J. Legleiter).
1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2010.12.028
in several metastable, intermediate species forming, such as oligomers (Fig. 1). However, these metastable species may be off-pathway to fibril formation. Several studies support the notion that
these small, potentially diffusible assemblies, rather than mature
amyloid fibrils, may be the primary culprits in neuronal dysfunction and death [5–8]. The elusive toxic species (whether monomeric or higher-order) and the mechanisms by which they
trigger neuronal dysfunction remain controversial.
1.2. Huntington’s disease
An expansion of a polyglutamine (polyQ) repeat near the N-terminus in the huntingtin (htt) protein causes Huntington’s disease
(HD), a fatal neurodegenerative disorder [9]. Expansion of the
polyQ domain leads directly to aggregation of htt and the deposition of cytoplasmic and intranuclear inclusion bodies comprised
of fibrillar material [10]. Furthermore, at least nine neurodegenerative disorders are associated with polyQ expansions, including the
spinocerebellar ataxias [11]. Intriguingly, the age of onset and
severity of polyQ-associated diseases are tightly correlated with
the number of repeat units of the polyQ stretch [12,13]. For instance in HD, repeat lengths shorter than 35 glutamines are not
associated with disease, 35–39 glutamines may or may not cause
disease, 40–60 glutamines result in adult onset HD, and more than
60 glutamines elicits juvenile forms of HD [12,13].
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Fig. 1. A potential pathway for neurodegenerative protein aggregation. Neurodegenerative protein aggregation begins with a protein adopting an abnormal conformation
that promotes aggregation via intermediate stages characterized by distinct morphologies (i.e., oligomers, protofibrils, and annular structures) into mature fibrils and
eventually large inclusions. The representative AFM images are of aggregates formed by a htt exon1 construct with a polyQ domain length of 53 repeat units.
Several lines of evidence point to the necessity of htt aggregation in HD related toxicity [14,15], however, this necessity appears
to be intimately linked with the specific nature of the aggregates
present [5,16,17]. As a result, a large effort has been focused on
understanding the aggregation of model synthetic peptides and
mutant htt fragments containing various length polyQ expansions.
Synthetic polyQ peptides with expanded polyQ repeats form amyloid-like fibrils, with synthetic peptides with glutamine stretches
shorter than the disease-causing range readily forming fibrils
[18]. Purified mutant htt fragments containing polyQ domains with
more than 39Q formed detergent-insoluble protein aggregates in a
polyQ length-dependent manner [18–20], and polyQ length dependent formation of intranuclear and cytoplasmic inclusion bodies by
mutant htt fragments was demonstrated in cell culture [17,21,22].
Collectively, these studies indicate that the underlying kinetics of
fibril and inclusion body formation is directly correlated to polyQ
length. Mutant htt fragments and other expanded polyQ proteins
also assemble into a variety of spherical and annular oligomeric
structures [23–25].
1.3. Atomic force microscopy
Due to the progression of protein aggregation from globular
precursors to mature fibrils and the active debate concerning
which aggregate type represents the most disease relevant form,
techniques that allow for distinguishing and characterizing distinct morphological features of aggregates within heterogeneous
aggregation reactions are of enormous benefit. In recent years,
atomic force microscopy (AFM) has emerged as a particularly
useful technique in studying disease-related protein aggregation
[25–36].
Since its invention in 1986 [37], AFM has become an important
technique for imaging and exploring biological systems in three
dimensions with nanoscale spatial resolution. In AFM, the vertical
displacement of a cantilever affixed with a sharp tip that physically
interacts with surfaces is measured, most often by an optical lever
produced by reflecting a laser beam off the back of a flexible cantilever onto a position-sensitive photo detector. In contact mode,
cantilever deflection is monitored. In tapping and non-contact
mode, the cantilever is harmonically driven near its resonance frequency, and its oscillation amplitude is monitored. Three-dimen-
sional topography maps of sample are obtained by raster
scanning the probe across a surface while maintaining cantilever
deflection or amplitude at a constant value by vertically displacing
either the tip or sample. In comparison to other high resolution
imaging techniques such as TEM and SEM, the three dimensional
imaging capabilities of AFM allows for the measurement of the
height and volume of nanoscale objects. Another striking advantage of AFM is that it can be operated synergistically with other
optical microscopic techniques [38,39]. Unlike other high resolution imaging techniques, AFM is operable in fluids, providing the
opportunity to directly image important biological processes
dynamically under near physiological conditions, i.e., appropriate
pH and ionic strength. This ability allows AFM to dynamically track
the fates of individual, nanoscale structures [27,28,30], providing
information that is simply not possible by other available imaging
methods. More recently, AFM experiments of protein aggregation
have moved to more biologically relevant surfaces such as lipid
membranes [40–42,32], collagen VI-coated surfaces [43], and intact collagen fibers [44]. AFM has also been used to verify that morphologies of protofibrils and fibrils of a-synuclein did not change
when aggregation occurred in the presence of various concentration of polyethylene glycol to model molecular crowding in cells
[45].
AFM is a technique for imaging surfaces, and as such, its application is limited to processes occurring on these surfaces. For
example, AFM has been used extensively to image the surface of
cells [46–48]; however, it cannot directly image processes occurring within the cell. Rather, large imaging forces can be employed
to push deeper into the cell to ascertain some information of the
internal cellular structure [49,50]. Another potentially limiting factor for AFM experiments with complex mixtures of similarly sized
components is that, aside from using a chemically-functionalized
probe, there is not a straight forward manner for identifying and
distinguishing these disparate components from each other. Additionally, The finite shape and size of the AFM tip leads to exaggerated measurements in the lateral direction, and can lead to other
image artifacts; however, there are methods to correct some of
these issues [51,52]. As will be discussed in more detail later, the
physical interaction of the AFM tip with samples can lead to artifacts, as the process of imaging can alter the morphology of the
sample.
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2. Materials and methods
2.3. Deposition of protein samples for ex situ AFM imaging
2.1. Purification of GST-htt-exon1 fusion proteins
Using a brightly colored marker pen, the backside of a rectangular piece of mica (1 inch by 0.5 inch) (Ted Pella Inc, Redding, CA)
was marked with a small dot near one end of the substrate. This
mark acted as a reference point for aligning the AFM probe over
the deposited sample for later imaging. A colored marker is preferred because it is easier to distinguish from other spurious features on the mica surface from an overhead optical microscope
view during alignment of the cantilever. The mica was cleaved,
exposing a clean surface for deposition. An aliquot of 3 lL from
incubation of HD51Q or HD53Q (which has an additional myctag at the N-terminus in comparison with HD51Q) at specific time
points was deposited on the mica surface directly above the dot
(actual marking was on the backside of the mica and not on the
side where the sample was deposited). The deposited droplet
was left on the substrate for 1 min. Next, the substrate was
washed with 200 lL of ultrapure water to remove excess salts
and unbound peptide. To prevent damage to any delicate aggregates, the substrate was tilted 45°, and the wash was applied
above the sample, allowing it to gently flow over the deposited
sample. The bottom edge of the mica was placed on a Kimwipe
to absorb the excess wash and prevent water from flowing back
onto the deposited sample. The sample was dried with a gentle
stream of nitrogen. Freshly prepared samples were attached to metal substrate supports using double sided tape or a similar adhesive, and the excess mica was trimmed away.
Glutathione S-transferase(GST)–HD exon1 fusion proteins were
purified as described elsewhere (Fig. 2) [53]. Briefly, expression of
GST-htt fusion proteins was induced in Escherichia coli by isopropyl
b-D-thiogalactoside (4 h at 30 °C). The cells were lysed by addition
of 0.5 mg/ml of lysozyme. The fusion proteins were purified from
lysate by liquid chromatography (LPLC, BioRad or FPLC, GE Pharmacia) using a GST affinity column. Alternatively, GST-beads can
be used for purification. Gel electrophoresis was used to verify
the relevant fractions and determine purity. Cleavage of the GST
moiety by PreScission Protease (GE Healthcare, Piscataway, NJ) or
Factor Xa (Promega, Madison, WI) initiates aggregation. Fresh
GST-HD fusion proteins were used for each experiment. For experiments presented here, two different htt exon1 fragments were
used (Fig. 2): (1) HD51Q, which contains 51 repeat glutamines
and (2) HD53Q, which contains 53 repeat glutamines and an additional N-terminal myc-tag that is not present in the first construct.
Solutions with all fusion proteins were centrifuged at 20,000g for
30 min at 4 °C to remove preexisting aggregates prior to the addition of the cleaving agent, and all experiments were carried out in
buffer A (50 mM Tris–HCl, pH 7.0, 150 mM NaCl, 1 mM DTT). Solutions of HD51Q and HD53Q were prepared to a final concentration
of 20 lM in Eppendorf tubes and incubated at 37 °C and shaken at
1000 rpm. At 0, 1, 3, and 5 h after initiation of aggregation, the
incubations were sampled as described below.
2.4. Ex situ AFM imaging conditions
2.2. Peptide preparation
The peptide KK-Q35-KK was obtained via custom synthesis
(Keck Biotechnology Resource Laboratory, New Haven, Connecticut). The role of the flanking lysines on each terminus was to
aid in solubility. Disaggregation and solubilization of polyQ peptides was accomplished based on protocols described elsewhere
[54]. In short, crude peptide was dissolved for three hours in a
1:1 mixture of trifluoroacetic acid (Acros Organics) and hexafluoroisopropanol (Acros Organics) to a concentration of 0.5 mg/mL.
After rigourous vortexing, the solvent was then evaporated off
in a Vacufuge concentrator (Eppendorf, Hauppauge, NY), producing a thin peptide film. The remaining peptide residue was resuspended in ultrapure water adjusted to pH 3 with trifluoroacetic
acid to a concentration of 2.0 mg/ml. This stock solution was subsequently diluted with Phosphate-buffered saline (PBS) (Fisher
Scientific) to a final concentration of 20 lM at a pH of 7.3 prior
to any experiment.
Ex situ experiments were performed with a Nanoscope V MultiMode scanning probe microscope (Veeco, Santa Barbara, CA)
equipped with a closed-loop vertical engage J-scanner. Images
were taken with diving board shaped oxide-sharpened silicon cantilever with a nominal spring constant of 40 N/m and resonance
frequency of 300 kHz. Scan rates were set at 1–2 Hz with cantilever drive frequencies 10% below resonance. At 0hr, 1 h, 3 h, and 5 h
samples of HD51Q and HD53Q were deposited onto freshly cleaved
mica, washed with ultrapure water to remove salt, and dried under
a stream of N2 gas. The mica was then placed onto metal pucks and
imaged in tapping mode AFM in air.
2.5. In situ AFM imaging conditions
In situ AFM experiments were performed with a Nanoscope V
MultiMode scanning probe microscope (Veeco, Santa Barbara,
CA) equipped with a sealable fluid cell and a closed-loop vertical
Fig. 2. A schematic depiction (not to scale) is shown of the GST-htt exon1 fusion proteins used (HD53Q and HD51Q). The fusion proteins contain a cleavage location
(PreScission protease site or Factor Xa site) between GST and the respective htt fragment, which upon cleavage initiates the protein aggregation reaction.
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engage J-scanner. Images were taken with V-shaped oxide-sharpened silicon nitride cantilever with a nominal spring constant of
0.5 N/m (Veeco, Santa Barbara, CA) or with diving board shaped silicon cantilevers with a nominal spring constant of 0.1 N/m (Vistaprobes, Phoenix, AZ). Scan rates were set at 1–2 Hz with cantilever
drive frequencies ranging from 8 to 10 kHz. Synthetic peptides
were prepared as previously described to a final concentration of
20 lM in PBS at a pH 7–8 and injected directly into a fluid cell positioned above a freshly cleaved mica substrate. The surface was
continually imaged once the peptide was injected.
2.6. Image processing
AFM images were analyzed with Matlab equipped with the image processing toolbox (Mathworks, Natick, MA). Physical dimensions of aggregates were measured automatically in the
following way: (1) Images were imported into Matlab. (2) A flattening algorithm was applied to correct for curvature due to the
imaging process. (3) Binary maps of aggregate location were created from the flattened images by using a height threshold. This
was accomplished by assigning values of 0 or 1 to any pixel of
the image that represented a height below or above the allocated
threshold, respectively. (4) Discrete aggregates were located by
applying pattern recognition algorithms to the binary map. (5)
Once a discrete aggregate was located, physical properties (including height, volume, average diameter, width, length, aspect ratio,
position within the image, etc.) were measured automatically. Each
aggregate was also assigned an individual number so that
aggregates chosen based on specific measured properties cold be
located, allowing us to verify that chosen dimensions correspondent to specific aggregate types.
3. Specific experiments
3.1. Tracking the aggregation of incubated htt exon1 fragments
The first 17 amino acids of the N-terminus (N17) of htt have
been shown to function as a membrane targeting domain [55]
and in trafficking htt to the ER and vesicles [56]. As N17 is directly
adjacent to the polyQ region of htt, it has been proposed that this
flanking sequence also modulates the conformation of the polyQ
stretch and, as a result, htt aggregation. Recent reports using a variety of polyQ peptides with flanking sequences taken directly from
htt have demonstrated that N17 has a profound effect on aggregation kinetics [57,58]. In particular, Thakur et al. found that the N17
structure was altered by the polyQ domain and that this flanking
region facilitated the formation of globular oligomeric aggregates,
which were precursors of fibrils.
To assess the ability N17 to modulate aggregation, we used two
mutant htt fragments with similar polyQ repeat lengths (HD53Q
and HD51Q) that were purified from E. coli as a fusion to GST
(Fig. 2) [53]. After purification and high speed centrifugation to remove small aggregates that might serve as seeds, GST-HD fusion
proteins appeared to be non-aggregated by AFM analysis. Cleavage
of the GST moiety with a site-specific protease (PreScission protease for HD53Q and FactorXa for HD51Q) released the intact mutant
htt fragments, initiating aggregation in a time dependent manner
as reported [20,25,30,31]. Importantly, the HD53Q has a 10 amino
acid long myc-tag preceding the N-terminus of the htt exon1 construct; HD51Q does not have this tag. We hypothesized that this
exogenous tag would interfere with the N17 domains ability to
promote the initial formation of oligomers.
To test this hypothesis, HD53Q and HD51Q were incubated at
37 °C and 1000 rpm at a concentration of 20 lM. Samples were taken at 0, 1, 3, and 5 h after the initiation of aggregation (addition of
protease), deposited on mica, and imaged with AFM operated in
air. Approximately 10 images from each time point and multiple
depositions of each construct were obtained for later data analysis.
Aggregates from both constructs displayed increasing morphological complexity over the course of the incubation as a variety of
aggregate forms were observed for both constructs (Fig. 3). In these
incubations, the prominent aggregate species formed by the two
constructs were oligomers and fibrils. For both oligomers and fibrils, there were no distinguishable morphological features when
comparing aggregates of HD51Q with those formed by HD53Q.
To quantify the relationship between oligomers and fibrils for
both constructs, the number of each aggregate type per square micron can be determined as a function of time; however, this requires strict criteria for distinguishing between aggregate species.
Criteria can be established based on characteristic morphological
features of different aggregate types observed in AFM images
(Fig. 4). For our purposes oligomers were defined as 2–15 nm in
height with an aspect ratio (defined as Ra = r1/r2, with r1 being
Fig. 3. Representative ex situ AFM images demonstrate the aggregation of mutant htt fragments with polyQ repeats of 51Q and 53Q. Incubations with protein concentrations
of 20 lM HD51Q and HD53Q (which contains an N-terminal myc tag) were imaged at different time points after the addition of their respective proteases. Both observed
heterogeneous mixtures of aggregate types over a period of 5 h. At later times, fibrils gradually became more abundant for both constructs. Insets for each image zoom into 1
by 1 lm areas to highlight morphological detail of aggregates. Examples of oligomers, annular aggregates, and fibrils are indicated by yellow, green, and blue arrows,
respectively.
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Fig. 4. Representative AFM images and aggregate profiles that compare basic dimensions of oligomers, fibrils, annular aggregates, and fibril bundles are presented. Height
profiles under each image are indicated by colored lines. By using a combination of morphological features, such as height and aspect ratio, relative populations of aggregates
types can be distinguished and quantified in a heterogeneous mixture.
the largest and r2 being the shortest distances across an aggregate)
less than 2.5, indicating a globular structure. Fibrils were defined
as aggregates greater than 2.0 nm in height that had an aspect ratio
greater than 2.5, which corresponds to an elongated fibrillar structure. These criteria were established by manual measurement of
dimensions associated with representative aggregates of each type.
Other commonly observed aggregate types are annular ring structures and fibril bundles. Due to the small number of these aggregate types observed in these experiments, their abundance was
not quantified.
In order to obtain a sufficient count of individual aggregates
to have a meaningful analysis, hundreds of particles over several
images must be analyzed. This task requires the use of software
that automates size analysis of objects contained in AFM images
(Fig. 5). The software locates individual objects in an AFM image
by creating a binary map based on a height threshold. If the
height of an individual point within an image is larger than
the established threshold height, its value is set to one, and if
it is below, the value is set to zero. This creates a binary map
of the image where aggregates are represented by regions of
adjacent values of one. Using pattern recognition algorithms,
the location of individual aggregates can be determined from
the binary map, and by referencing these locations, specific
properties of aggregates can be measured automatically. These
properties include volume, height, average diameter, aspect ratio,
and other geometrical characteristics. This process facilitates
quick analysis of thousands of individual aggregates, organizing
individual measurements into large data sets that can be filtered
base on specific aggregate features. As the two primary aggregate types observed in this study were oligomers and fibrils,
we used an aspect ratio filter to determine the relative number
of the two aggregate forms.
Fig. 5. Image analysis facilitates the sorting of complex heterogeneous mixtures of aggregate species of htt. An AFM image (A) can be analyzed by a height threshold filter that
creates a binary map which highlights the location of discrete aggregates (B). Once this has been accomplished, the binary map can be used as a reference to locate each
aggregate (indicated by asterisks) and measure specific morphological features (C) such as aspect ratio (D). By taking advantage of unique morphological characteristics of
different aggregate types (the aspect ratio in this example), specific types of aggregates can be located and counted, i.e., oligomers (E) and fibrils (F) as indicated by asterisks.
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Fig. 6. The formation of htt oligomers is attenuated by the presence of an Nterminal myc tag. Quantification of the number of oligomers (A) and fibrils (B) per
unit area detected by AFM images of HD51Q and HD53Q at 20 lM as a function of
time. While the number of fibrils per unit area was indistinguishable between the
two constructs, the presence of the N-terminal myc tag appeared to reduce the
number of oligomers formed by HD53Q in comparison with HD51Q. ⁄indicates
p < 0.5. (C–D) Based on corrected volume measurements and the molecular mass of
each htt exon1 fragment, the numbers of molecules per aggregate observed for
20 lM incubations of HD51Q (C) and HD53Q (D) were calculated for each time
point. The darker colors represent a greater population of oligomers composed of
that number of molecules. Black arrows indicate where 400 kDa oligomers would
be observed. Based on this analysis, both fragments formed similar sized oligomers
despite the relative difference in their number. These oligomers were a heterogenous mixture with a predominate size of 400 kDa.
Quantification of distinct aggregate types per unit area (Fig. 6
A–B) demonstrated that aggregation resulted in a complex mixture
of aggregate forms at any given time for both HD53Q and HD51Q.
The addition of the myc tag to HD53Q appeared to inhibit the formation of oligomers, as starting at time point 1 h there were significantly (p < 0.05) more oligomers observed in incubation of
HD51Q. While the number of oligomers increased with time for
both constructs, the rate of oligomer formation appeared much larger for HD51Q. As the extra myc tag may (potentially via steric hindrance) interfere with interpeptide interactions mediate by N17,
the ability of the myc tag to inhibit oligomer formation is consistent with reports that N17 promotes oligomerization [57]. Despite
the increased number of oligomers observed in incubation of
HD51Q, there did not appear to be any difference in the number
of fibrils observed in comparison between the two constructs.
To determine if the oligomers formed by both constructs
were similar in their composition and size, we used the volume
distributions of oligomers (obtained via the aspect ratio filter) to
estimate the number of protein molecules per aggregate and the
approximate molecular weight of oligomers for both HD51Q and
HD53Q (Fig. 6 C–D). Due to the finite size and shape of the AFM
probe tip, the observed volume of any aggregate is exaggerated.
Therefore, estimating this contribution and correcting for it is
essential for this analysis. In order to compensate for these contributions, a partial correction based on simple geometric models of
the interaction of a spherical tip imaging particles of defined
dimensions was developed and verified via simulation [51]. In
short, by assuming an ellipsoidal shape of the oligomers, correlation plots of the effective diameter vs height of individual oligomers can be constructed. The intercept of the linear least squares
fit of such a plot represents the average increase in the effective
diameter of these aggregates due to tip dilation. When using the
intercept of this plot to correct volume measurements of individual
aggregates, the intercept was subtracted from the original measured effective diameter of the oligomer, and the corrected volume
of each oligomer was calculated based on its height and newly corrected diameter. The volume of an individual HD51Q or HD53Q
protein was estimated based on the molecular weight of each construct and the average density of proteins [59,60]. By dividing the
observed corrected volume of each individual aggregate by the
estimated volume of a single monomer, the number of molecules
Fig. 7. Time lapse sequence of in situ AFM images demonstrate the ability to track the aggregation of synthetic polyQ peptides. (A) 20 lM solution of KK-Q35-KK was injected
into the fluid cell, and the same area was imaged over time. This allows for the tracking the fate of individual aggregates. Insets for each image zoom into the same 1 by 1 lm
areas in the sequence of images to highlight morphological changes of aggregates over time. The blue arrow indicates a fibril that displays variable width and height along its
contour. A branching point is specified by the green arrow. A stable oligomer is pointed out in two successive images by yellow arrows. (B) The surface area occupied by
individual fibril or network of fibrils is measured as a function of time, demonstrating the ability to track the fate and rate of aggregation of discrete aggregates.
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per each oligomer was calculated. This calculation assumes perfect
packing of individual monomers within the oligomer and that the
density of the proteins is the same in aggregated and unaggregated
forms. Despite the larger number of oligomers observed in incubations of HD51Q, the number of fibrils formed for both constructs
appeared to be indistinguishable at each time point (Fig. 6 C–D).
This analysis indicated that oligomeric species observed for both
constructs have a broad size distribution, are highly heterogeneous, and change in subunit composition with time. Furthermore,
the relative size of these oligomers are comparable to those observed to form by similar sized htt exon1 fragments in cellular
and animal models as assessed by Sodium dodecyl sulfate agarose
gel electrophoresis (SDS-AGE) gels [31].
3.2. Tracking aggregation of synthetic polyQ peptides in solution
In situ tapping-mode AFM under liquids offers the ability to
study peptide aggregation and fibril formation under near physiological conditions in a time-dependent manner. By continuously
recording images of the same area during aggregation process, it
is possible to identify specific aggregates, determine their morphology, and monitor their individual growth. When freshly solubilized preparations of the KK-Q35-KK peptide were imaged in
solution on a mica surface (Fig. 7), small oligomeric and short putative fibrillar structures appeared on the surface within a few minutes, however, fibrils were the dominate aggregate form at all time
points. For higher resolution AFM images of KK-Q35-KK aggregates
see the insets within Fig. 7. With time, more aggregates appeared
on the surface, and fibrils continually increased in length. Fibrils often exhibited branching, and there were variable widths along an
individual fibril’s contour, indicating that there may be structural
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variability contained within fibrils. While very few oligomers were
observed, there were instances of stable oligomers persisting for
several minutes. However, most oligomers that did appear either
initiated fibril growth or were quickly incorporated into an adjacent growing fibrillar aggregate. While most fibrils appeared without an obvious oligomeric precursor, it cannot be ruled out that an
oligomer in solution converted into a fibrillar structure prior to
depositing on the surface because only aggregates that are on the
mica surface can be imaged by AFM. Importantly, these time lapse
image obtained from in situ AFM can be analyzed with the same
imaging processing software previously described. Here we show
an analysis of the rate of growth of individual fibrillar aggregates,
by identification and measurement of surface coverage of the same
aggregate in sequential images. We chose to use surface coverage
as opposed to length of fibrils for this analysis due to the excessive
branching of many of the observed fibrils. This analysis demonstrates that the rate of growth of individual aggregates can vary
greatly.
For these types of experiments, care must be taken that the continuous imaging process itself does not influence the observed
aggregation (Fig. 8). Typically there are two types of potential issues associated with this: (1) sweeping away or machining apart
aggregates as they form or (2) promoting protein deposition under
the influence of the AFM tip. An easily implemented control for
these issues is to intermittently offset to a different location on
the surface that has not previously been imaged to compare aggregate formation and morphology to the area that has been continuously imaged. Alternatively, one could increase the scan size to
confirm that the aggregates continuously imaged by the AFM tip
are not significantly different than those observed around the
periphery of the scanning area (Fig. 8). Another consideration is
Fig. 8. An example of a tip induced artifact during an in situ AFM experiment on HD53Q. (A) In a series of time lapse AFM images, it appears that small oligomers (examples
indicated by various colored arrows) appear on the surface that gradually swell into very large globular structures. (B) However upon doubling the scan size, it becomes
apparent that these large globular species only appear where the probe was continuously scanning (indicated by the dashed box), indicating that the imaging process was
inducing the formation of these structures.
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that the solid/liquid interface required for these experiments can
also influence the aggregation kinetic and aggregate morphology
of fibril forming peptides as has been observed for other proteins
[28,61].
4. Conclusions
Owing to its unique ability to image, interact, and manipulate
biologically relevant samples at the nanoscale under near physiological conditions, atomic force microscopy has proven particularly
useful in studying the processes of pathological peptide assembly
associated with a variety of diseases. Due to its unique abilities,
AFM, in conjunction with other complimentary technique, has potential to address several currently unresolved issues concerning
the aggregation of proteins and peptides containing expanded polyQ domains, especially at liquid/solid interfaces.
Understanding the kinetics and aggregation pathways of polyQ
containing proteins can provide invaluable insights into potential
toxic mechanisms associated with aggregates. Several aggregation
pathways have been proposed for the formation of fibrillar structures of polyQ peptides. Two of the more prominent aggregation
schemes are: (1) re-arrangement of a monomer to a thermodynamically unfavorable conformation that directly nucleates fibril
formation and (2) the formation of amorphous, soluble oligomeric
intermediates that slowly undergo structural re-arrangement into
a b-sheet rich structure that leads to fibrils. While model polyQ
peptide fibrils have been shown to share many of the classical features associated with amyloid [18,62], initial reports proposed the
nucleation-elongation model for the formation of these fibrils [63].
More recent studies have re-evaluated theses studies quantitatively and demonstrated the data was insufficient to rule out other
potential mechanisms [64]. Subsequent studies have further demonstrated that smaller aggregates of polyQ proteins appear prior to
nucleation of fibril formation [65], and small oligomers, displaying
various degrees of stability, of model polyQ peptides with glutamine lengths ranging from 7 to 32 have been observed by AFM
[31]. Many oligomers observed in these studies directly initiated
the formation of fibrils [31]; however, it was simultaneously observed that many fibrils appeared to form without any obvious
oligomeric precursors [31]. These observations suggest that these
aggregation pathways are not mutually exclusive, adding to the
complexity of polyQ aggregation reactions. While it is clear that
proteins (such as htt) with expanded polyQ tracts assemble into fibrils, these proteins form spherical and annular oligomeric structures, as evidenced by TEM and AFM studies [23–25]. Further
studies using AFM to study a variety of fragments of htt exon1 have
shown that aggregation reactions result in heterogeneous mixtures
of distinct aggregate morphologies in a polyQ length- and concentration-dependent manner [31]. These studies point to the complex nature of polyQ aggregation, necessitating the use of
techniques capable of extracting quantitative data concerning relative amounts of specific aggregates forms, as demonstrated here
using AFM.
The importance of protein context on polyQ aggregation has
been shown in several studies and systems [66–70]. More broadly,
such studies suggest the critical importance of flanking sequences
on polyQ structure and aggregation. Studies on synthetic peptides
revealed that the addition of a 10-residue polyP sequence to the Cterminus of a polyQ peptide altered both aggregation kinetics and
conformational properties of the polyQ tract [71]. Flanking polyP
sequences can also inhibit the formation of b-sheet structure in
polyQ peptides by inducing PPII-like helix structure, extending
the length of the polyQ domain necessary to induce fibril formation
[72]. Flanking sequences on htt exon1 fragments with various polyQ domain lengths modulate toxicity in yeast models not only in
cis, but also in trans during aggregation [73,74]. Interestingly, the
proline-rich regions of htt exon1 reduced polyQ-related toxicity
in these studies [73,74]. The first 17 aa of htt has been shown to
facilitate the formation of globular, oligomeric aggregates [57].
Such findings underscore the critical importance of protein context
to polyQ aggregation and aggregate stability. AFM has the potential
to further address issues concerning the role of flanking sequences
on polyQ aggregation by its ability to directly visualize the aggregation process of carefully designed polyQ peptides with a variety
of flanking sequences. However, caution must be used, as recent
theoretical and experimental results have demonstrated that the
addition of residues, such as flanking lysines, to aid in solublizing
peptides, can also have a potentially large impact on aggregation
kinetics [58].
Beyond protein context and polyQ length, it has become
increasingly evident that several other factors can affect the aggregation of polyQ containing proteins and peptides (for a comprehensive review see [75]). For example, htt undergoes a variety of
posttranslational modifications. A major modification of htt exon1
is acetylation and phosphorylation of threonine-3, which greatly
impacts the aggregation of htt in cells and in vivo [76]. The first
17 amino acids of htt can be both phosphorylated and SUMOylated,
both of which impact htt aggregation and cellular localization
[77,78]. Furthermore, SUMOylation appears to control the transport of htt from the ER to the nucleus [55,79]. With proper chemical treatments of purified htt constructs, AFM has the potential to
explore with great detail the kinetics of posttranslational modifications on htt exon1 aggregation and the morphological characteristics of the resulting aggregates. While the htt protein experiences
several other posttranslational modifications (i.e., S421 by Akt
and SGK [80,81]), the applicability of AFM to address the impact
of these modifications on htt aggregation is currently limited due
to difficulties in expressing and purifying longer fragments of htt
in sufficient yield.
It has been well documented that cellular localization (i.e., ER,
nucleus, and mitochondria) attenuates the aggregation and toxicity
of htt [17,55,56,79,82,83]. Furthermore, a variety of cellular components (i.e., cytoskeletal components [84], single-stranded oligonucleotides [85], lipids [83,86–88], molecular chaperones
[2,53,89]) and other exogenous factors (i.e., antibodies [90–93])
have been shown to impact polyQ related aggregation and toxicity
in a variety of models. With careful examination of aggregate size
and morphology, AFM can also be used to help assess the ability of
these and other exogenous factors to modulate htt and polyQ
aggregate formation and stability. For example, AFM was used to
demonstrate that hsp40 and hsp70 attenuate the formation of oligomeric and annular aggregates without affecting formation of mature fibrils [25]. AFM was also employed in studies that revealed
the ability of green tea polyphenol (2)-epigallocatechin-3-gallate
(EGCG) to potently inhibits the aggregation of mutant htt exon1
proteins [94]. Several studies have used AFM in attempts to understand the effect of a variety of antibodies and intrabodies on htt
aggregation [30,95,96]. Similar AFM have been performed with
other amyloid forming proteins to understand the impact of lipids
[40–42,32], antibodies/intrabodies [29,97,98], small molecules and
peptides [99–101], and cytoskeletal components [43,44] on aggregation. One of the implicit goals of such efforts is the identification
of potential preventative or therapeutic agents. However, without
precise knowledge of what constitutes a toxic species in vivo, steps
to modulate aggregation or alter aggregate stability could potentially be counterproductive. Therefore, understanding of the aggregation process and what constitutes primary toxic species is vital.
Studies of other amyloid forming peptides have illustrated the
ability of such proteins to from polymorphic aggregate structures
that can often be attributed to variations in sample preparation
[102,103]. In this respect, it has become increasingly clear that
Author's personal copy
K.A. Burke et al. / Methods 53 (2011) 275–284
htt can form a variety of aggregate structures in vitro [31]. However, several important questions remain: Do aggregates observed
by in vitro techniques such as AFM correspond to those that actually occur in cellular and mouse models of HD and, more importantly, patients? What aggregates forms are present in inclusion
bodies within cells? Significant contributions to address the second
question have been made that demonstrated that N-terminal htt
forms neuronal intranuclear inclusion bodies that are comprised
of heterogenous mixtures of fibrillar and oligomeric aggregate
structures [10]. AFM has begun to show its potential applicability
in further addressing both questions, particularly for oligomeric
species. For example, oligomers of recombinant htt exon1 fragments formed in vitro and characterized by AFM were shown to
be similar in lateral dimensions to oligomers observed in cortical
neurons from human HD brain as determined separately by EM
analysis [31]. Using AFM, it was shown that globular oligomeric
aggregates purified from R6/2 and HdhQ150 knock-in mice were
remarkably similar in size and morphology to those generated by
htt exon1 fragments in vitro [104]. In comparison with oligomers
of htt exon1 fragments formed in an immortalized striatal cell line
and in brain homogenates from a mouse model of HD, mutant htt
fragments formed similar oligomers in vitro as assessed by AFM
[31]. One of the major obstacles in determining the most important
toxic species of htt (and other polyQ-containing proteins) is
obtaining homogenous populations of often metastable aggregates.
AFM analysis may prove to be an invaluable tool to quickly assess
the success of efforts to obtain (and stabilize) homogenous populations of specific aggregate forms of htt fragments for cellular toxicity studies. In this regard, it has been recently demonstrated that
size exclusion chromatography (SEC) can be used to separate subpopulations of oligomeric aggregates formed by htt exon1 fragments [105]. These subpopulations were shown to be highly
homogenous in size as demonstrated by AFM analysis of samples
taken directly from the SEC column [105]. Perhaps, similar approaches can be employed to obtain subpopulations of htt aggregates for microinjection studies aimed at elucidating the relative
toxicity of distinct aggregate forms.
Acknowledgment
Support from West Virginia University (start-up package, JL) is
gratefully acknowledged.
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