**1. Introduction**

The relationship between neurodegenerative disease symptoms and the structural dynamics of associated amyloidogenic proteins has been revealed. For amyloid-β (Aβ), different fibril structures and morphologies have been observed in Alzheimer's disease (AD) patients [1,2]. Therefore, the Aβ polymorphism was correlated with pathological phenotypes. Similarly, fibril polymorphism and the associated toxicity/infectivity was identified in other amyloidogenic proteins, such as human tau, α-synuclein, and yeast prion-derived Sup35. Tau protein amyloid fibrils demonstrated different isoforms and structures between AD [3], Pick's disease [4], progressive supranuclear palsy (PSP) [5], corticobasal degeneration (CBD) [5], and chronic traumatic encephalopathy (CTE) in tauopathy [6]. α-synuclein fibrils with different structures exhibited different toxicities [7] and caused different

symptoms in α-synucleinopathy [8,9]. One of the best studied prion model proteins, yeast Sup35, formed fibrils with different structures and mechanical stiffness depending on the temperature during aggregation, which was reflected in prion activities, such as fragmentation and propagation [10–12]. Elucidating the structural dynamics of the amyloid protein is indispensable to uncovering the mechanism underlying disease onset and the action of drug candidates. Examination of the structure could lead to the diagnosis and treatment methods for the progression of the disease in each individual [13].

Amyloidogenic proteins commonly have intrinsically disordered regions in some or all of their monomeric structure, and they change these to form aggregation core structures along the aggregation pathway. The structural dynamics during the aggregation process depend on the surrounding physicochemical conditions in vivo, including the free-in-solution or membrane-bound forms, pH, and electrolytes. Aβ40, a 40 residue Aβ variant cleaved from the amyloid precursor protein (APP), is unstructured [14,15] and can also form a lowly populated 310 helical structure in solution [16]; disordered oligomers were also reported [17]. This variant formed disordered helical structures upon interaction with the lipid membrane [18], and could disrupt the membrane structure via a two-step mechanism consisting of fiber-independent pore formation and fiber-dependent 'detergent-like' membrane fragmentation [19]. Aβ42—a 42 amino acid chain of Aβ variants—showed different structural dynamics from Aβ40 through the interaction with a membrane. Aβ42 oligomerization was accelerated by the lipid membrane [20]. Aβ42 oligomers assembled into the pore-forming oligomers with three distinct pore sizes that functioned as ion channels, while Aβ40 did not form such pores [21]. Upon interaction with a reconstituted membrane, Aβ42 assembled into a β-barrel structure, while Aβ40 formed fibrils [22]. Tau [23], α-synuclein, and other amyloidogenic proteins, including amylin, also formed ion-channel oligomers in the membrane [24]. Cytosolic acidification caused by oxidative stress promoted AD [25] and Parkinson's disease (PD) [26]. A change in pH altered the structural dynamics and aggregation pathways of Aβ [27–30], tau [31], α-synuclein [32–35], and amylin [36]. Some amyloid oligomeric conformers were more toxic than amyloid fibrils [37–39]. Different oligomers showed different structures and toxicities [40,41]. Thus, amyloid fibrils with different structures may reflect variable toxicity, aggregation pathways, and surrounding microenvironments in different patients and symptoms [1,42].

To date, investigations of the structural dynamics of amyloidogenic protein aggregation examined the structure and dynamics separately. X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM) revealed the spatial coordinates of constituent atoms in the protein structure. X-ray crystallography demonstrated the fibril structures of amyloidogenic protein fragments [43,44] and oligomers of Aβ peptides [45–48]. NMR studies reported the high-resolution structural details of membrane-bound amyloid oligomers [41,49,50]. Solution- and solid-state NMR was used to investigate the monomer and transient intermediate structures in Aβ assemblies [16,51–58]. In addition, solid-state NMR studies revealed the in-register parallel β-sheets in Aβ fibril structures [1, 42,59–64]. Cryo-EM studies discovered the Aβ42 fibril structure [65] and individual Aβ40 strain structures from AD patients [2]. However, the structural images were static and average across the applied protein samples. Thus, those methods require structural homogeneity in the analyzed protein samples.

Single molecule observation using fluorescence dyes under optical microscopy was used to visualize the structural dynamics of amyloidogenic protein aggregations. Thioflavin T (ThT), specific for the cross-β structure, [66,67] was used to visualize the fibril elongation of Aβ40 [68], α-synuclein [69], and prion protein (PrP) [70]. Other fluorophores co-assembled or cross-linked to proteins were used to visualize the aggregation dynamics of α-synuclein [71–76] and the fibril growth of Aβ [77] and Sup35 [78]. However, the captured images did not show the protein structure but the spatial distribution of fluorescence spots.

Atomic force microscopy (AFM) visualizes individual molecules at the nanometer spatial resolution in solution, although the temporal resolution is low in conventional AFM [79–84]. AFM was used to capture structural images and measure the nanomechanical properties of individual amyloid aggregates [85–87] in the ongoing heterologous aggregation processes of Aβ [20,88–98], synuclein [90,99–106], and amylin [107,108]. High-speed AFM (HS-AFM) enabled the kinetic measurement of the structural dynamics of biological molecular processes [79–84] including amyloid aggregation [93,108–116]. Here, we show that HS-AFM links structural and dynamics studies, reviewing recent HS-AFM studies and including our findings for Aβ42 [93] and amylin [116], which is associated with not only type II diabetes but also AD [117–123].

#### **2. HS-AFM Observation of A**β**42 Fibril Growth**

#### *2.1. HS-AFM Observation of Self-Replicative A*β*42 Fibril Growth*

Aβ fibril formation has been thought to be similar to the self-replication mechanism for prion proteins. In this mechanism, the soluble conformer assembles and changes its structure to an abnormal form, and some of the abnormal conformers grow into fibrous structures with the incorporation of the soluble conformers [60]. The time-lapse AFM with a lower scanning rate observed Aβ40 protofibrils and mature fibrils growing in a self-replicative manner [89,107]. To assess Aβ42, characterized by faster aggregation, we used HS-AFM.

The sample preparation procedure and the imaging conditions were critical for the HS-AFM observation, as described in Section 8.1. We prepared low molecular weight (LMW) and high molecular weight (HMW) Aβ42 fractions in 10 mM sodium phosphate, with pH 7.4 [93,124]. Aβ42 fibril formation and elongation was observed in LMW Aβ42 within approximately 1 h after the addition of 0.1 M NaCl [93] (Figure 1). The LMW Aβ42 was introduced to the HS-AFM sample chamber (Figure 1a). Sodium chloride was added just before or after the peptide introduction; this was immediately followed by HS-AFM observation. Species in solution were adsorbed to the surface and became detectable by HS-AFM (Figure 1a,b) [93]. The bound aggregates interacted with species in the solution and were able to elongate as shown in Figure 1b.

The HS-AFM images clearly distinguished the three structurally distinct types of fibrils in this condition: (1) the spiral fibrils with a ≈100 nm periodicity in height, (2) the straight fibrils without any structural periodicity, and (3) the hybrid fibrils in which the spiral and the straight parts were mixed (see Figure 2) [93]. This result indicates that the manner of Aβ42 fibril growth followed the prion-like self-replication in the spiral and the straight parts [93]. In addition, HS-AFM also uncovered the structural switch in the hybrid fibril elongation and the presence of spherical oligomers in the sample mixture [93]. A recent solution NMR study identified fibril elongation and the formation of a heterogeneous mixture (fibrils and oligomers) when Aβ monomers were added to sonicated preformed seeds in real time [125]. However, a solid-state NMR investigation is needed to obtain additional high-resolution structural information; whereas, HS-AFM experiments eased the retrieval of the structural switch and fibril polymorphism in real time.

**Figure 1.** High-speed atomic force microscopy (HS-AFM) observation of amyloid-β (Aβ)42 fibril elongation. (**a**) Schematic view of HS-AFM observation of low molecular weight (LMW) Aβ42 incubation. LMW Aβ42 in 10 mM sodium phosphate, at pH 7.4, was introduced with 0.1 M NaCl for aggregation acceleration. Some aggregates in the solution bound to the mica surface and the fibrous aggregates in them were elongated by the incorporation of the LMW Aβ42 in free solution. (**b**) Representative HS-AFM images of LMW Aβ42 incubation at the indicated time after addition of 0.1 M NaCl. The scale bar is 300 nm. Reproduced from [93].

The molecular process of Aβ aggregation was characterized by HS-AFM observation under various physicochemical conditions. The structural switch of the growth mode was also characterized to be an inherent process in the Aβ42 self-replication reaction. To characterize the structural switch, Aβ42 fibril formation and elongation was observed after the addition of 0.1 M sodium chloride or potassium chloride [93]. The interaction between the HS-AFM stage surface and the observed Aβ42 molecules was different in the presence of sodium and potassium ions, as described in Section 8.3. As shown in Figure 3a, the fibril type distribution was different under the sodium and potassium conditions [93]. The spiral and the hybrid types were dominant in the sodium and potassium ion buffers, respectively, with no significant difference in the fibril length between the two conditions (Figure 3a,b,d) [93].

**Figure 2.** Kymographs of spiral, straight, and hybrid types of Aβ42 fibrils from HS-AFM images of the indicated time ranges after addition of 0.1 M NaCl. Arrows indicate the positions at which the growth mode switched from the spiral to the straight at 24 min and from the straight to the spiral at around 25 min. Reproduced from [93].

The appearance frequency of the spiral and the straight growth modes decreased and increased in the potassium buffer, respectively, when compared with the sodium buffer (Figure 3c) [93]. The lengths of the spiral and straight sections in the potassium condition were shorter and longer than in the sodium condition, respectively [93]. These results indicated that the structural switch process could be modulated by changes in the physicochemical conditions, and that the sodium buffer constrained fibril growth to the spiral mode, while the potassium buffer decreased the activation energy and the free energy difference between the spiral and the straight states of the structure switch process [93]. These findings revise the conventional model of the self-templating replication of amyloid fibrils, suggesting that the fibril structure strain can be altered by the external physicochemical environment even after fibril seed formation (nucleation) [93].

**Figure 3.** HS-AFM observation of fibril elongation from LMW Aβ42 incubation on mica surface in 10 mM sodium phosphate, pH 7.4 titrated with 0.1 M NaCl, or KCl. (**a**) HS-AFM images of Aβ 42 fibrils approximately 1 h after the addition of NaCl or KCl as indicated. The spiral and straight parts in the hybrid-type fibrils are indicated by open and closed circles. (**b**–**d**) Distributions of the fibril type (**b**), the growth mode (**c**), and the fibril length (**d**) under NaCl and KCl conditions. The top hatched portion for KCl in (**b**) indicates the fibrils whose structure was not determined due to their length being shorter than the spiral pitch. Reproduced from [93] except for the image in NaCl shown in (**a**).

#### *2.2. Kinetic Analysis of A*β*42 Fibril Growth*

The high spatiotemporal resolution of HS-AFM enables kinetic analysis of fibril elongation according to the fibril structure. Aβ42 fibrils, especially at the fast ends, repeated the pause (dwell) time and the growth phase during their elongation in both the spiral and the straight growth modes (Figure 4a) [93]. The dwell time, step time, and step size showed the single exponential distributions (Figure 4b), suggesting that the transition between the dwell phase and growth phase proceeds with first-order kinetics [93]. The kinetic parameters for this transition were different between the spiral and straight growth modes (Figure 4c) [93]. The step sizes (62 nm and 36 nm of mean values for the spiral and the straight growth modes, respectively) were much longer than the width of the single β-strand (0.47 nm) in amyloid fibrils, which suggests that a number of peptides were taken into the fibrils during the growth phase, and that the transition between the dwell and growth phases did not correspond to the incorporation or dissociation of peptides (peptide concentration independence of the dwell phase needs to be examined) (Figure 4d).

**Figure 4.** Stepwise growth of Aβ42 fibrils. (**a**) Time courses of the fast (red) and the slow (green) ends of the spiral and straight fibrils in Figure 2. The insets are the enlarged time courses of the fast ends. The open and closed triangles indicate the start and end of pause states (dwell time). (**b**–**d**) Distribution of the dwell time (**b**), time for step (**c**), and step size (**d**) with single exponential fits giving the mean life times of the pause and growing states in (**e**), and mean step sizes, for the spiral (open circles with solid lines) and straight (closed circles with dashed lines) type fibrils from the LMW Aβ42 incubation. (**e**) Mechanistic model of the stepwise growth of the spiral and straight Aβ42 fibrils with the kinetic parameters from (**b**) and (**c**). Reproduced from [93].

The similar stepwise growth and its kinetics were characterized in Aβ25–35 fibril growth by AFM [126]. The transitions between the pause and growing phases were also identified as first-order kinetic processes with the structure conversion at the fibril ends [126]. The fibril ends can neither incorporate nor release peptides at the pause phase (the blocked state), while they consecutively grow with 7 nm or its integer multiples during the growing phase [126]. The differences in the kinetic parameters between the spiral and the straight growth modes of Aβ42 indicate that the energy landscape for the transition between the dwell and growth modes was different between the two types of fibrils. The fibril end structure at the dwell phase may also be different between the spiral and straight fibrils (Figure 4e).

#### **3. Mechanical Force Modulates the Transition from Oligomeric to Filamentous States of A**β

HS-AFM can be used to investigate the force-dependent molecular processes. The physical environment is also one of essential factors that determine biochemical responses [127]. In the amyloid research field, the application of external mechanical forces, such as shaking, agitation, and sheer stress, has been used to accelerate aggregation and fibrillation [128–132]. A recent study by Wang et al. showed the effects of mechanical sample rotation in the commonly used magic angle spinning NMR experiments to study amyloid-β aggregation and the effect of (−)-epigallocatechin gallate (EGCG), which is a small molecule polyphenolic compound found in green tea extract [133]. However, the underlying mechanism of the physical effects has remained unclear. Although the exerted force to the sample by the AFM tip is usually kept as small as possible (see Section 8.4), methods for controlling and actively utilizing the applied force to characterize the molecular processes have been developed [134,135].

Using the external force applied by HS-AFM, Tashiro et al. found the linearly organized globular Aβ42 oligomers that showed the force-dependent thin filament extrusion [112]. They first observed the aggregate species in 1-day Aβ42 incubation bound to the stage surface, and then temporarily increased the tapping force of the cantilever tip, which is immediately followed by the observation of the same area with the original tapping force [112]. As shown in Figure 5, the breakage of the connected oligomers induced the extension of the thin straight filaments [112], which suggests that the stability of the oligomers may suppress their structural conversion into the filaments and that its

removal may be promoted by the applied force. This result provides insights into the importance of the mechanistic stability of amyloid oligomers.

**Figure 5.** HS-AFM imaging of tip-induced Aβ42 filament growth. The HS-AFM tapping amplitude was transiently increased at the time indicated by the red arrows. Reprint with permission [112]; Copyright (2019), John Wiley and Sons.
