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Article

Enhancement of Cytoskeletal Tension Promotes Amyloid-β Aggregation on the Neuronal Cell Surface

by
Juri Nakayama
,
Yuna Fujiya
,
Kiyotaka Tokuraku
* and
Masahiro Kuragano
*
Graduate School of Engineering, Muroran Institute of Technology, Mizumoto 27-1, Muroran 050-8585, Hokkaido, Japan
*
Authors to whom correspondence should be addressed.
Molecules 2026, 31(4), 718; https://doi.org/10.3390/molecules31040718
Submission received: 27 January 2026 / Revised: 13 February 2026 / Accepted: 18 February 2026 / Published: 19 February 2026
(This article belongs to the Special Issue 30th Anniversary of Molecules—Recent Advances in Chemical Biology)
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Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that accounts for the majority of dementia cases. The accumulation of amyloid-β (Aβ) aggregates on neuronal surfaces is a known important event that typifies AD. Although cell membrane architecture and cytoskeletal tension are thought to be involved in the process of Aβ aggregation, it remains unclear how cytoskeleton-derived tension alters the function of cell membranes, which serve as a scaffold for Aβ aggregation. In this study, we investigated whether cytoskeletal tension promotes Aβ aggregation on neuroblastoma, SH-SY5Y cells. Cytoskeletal tension was enhanced by jasplakinolide, an actin depolymerization inhibitor, and calyculin A, a serine/threonine phosphatase inhibitor that promotes myosin II activation. Real-time imaging with quantum-dot-labeled Aβ nanoprobes revealed that both pharmacological treatments significantly increased Aβ deposition on the surface of living cells. Our findings suggest that cytoskeletal tension promotes Aβ aggregation over the membrane barrier, providing new insights into the biophysical mechanisms underlying Aβ accumulation in AD pathogenesis.

1. Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that accounts for the majority of dementia cases [1]. According to the amyloid-β (Aβ) hypothesis, the accumulation of Aβ in the brain is considered a major factor in the development of AD [2]. Aβ peptides are generated during the cleavage of amyloid precursor protein by β-secretase and γ-secretase, and subsequently aggregate and deposit on the surface of neurons in the brain [3].
Previously, we succeeded in the development of real-time imaging methods to observe the Aβ aggregation process [4]. Fluorescent semiconductor nanocrystals, known as quantum dots (QDs), have been utilized to visualize Aβ aggregation. By chemically conjugating QDs to Aβ40, a fluorescent nanoprobe (QDAβ) was developed. After incubating QDAβ with unlabeled Aβ42, the two species co-polymerized, allowing QDAβ to be periodically incorporated into Aβ fibrils. This incorporation enabled real-time observation of the aggregation process under a fluorescence microscope. Furthermore, we also reported the quantitative estimation of aggregate amounts based on measuring the standard deviation (SD) value of fluorescence images [5]. Using QD-based imaging methods, we demonstrated successful real-time, three-dimensional visualization of Aβ aggregation on the surfaces of PC12 cells [6]. Additionally, another study demonstrated that Aβ aggregation on the surfaces of normal human brain microvascular endothelial cells induced abnormal cell adhesion to gelatin substrates, resulting in disruption of actin cytoskeletal organization and causing apoptosis [7]. Further, we reported that Aβ inhibits endothelial cell motility and inhibits wound healing through abnormal actin filament reorganization, and in that case, Aβ aggregates preferentially formed at the leading edge of motility [8]. Thus, it has become clear that various cell motility phenomena are impaired through abnormalities in the cytoskeleton, although the mechanism by which cell motility promotes Aβ aggregation on the cell membrane remains unclear.
The cytoskeleton is responsible for maintaining the movement and morphology of various cellular structures [9,10]. Furthermore, the interaction of F-actin and non-muscle myosin II (NMII) plays a crucial role in the formation and maintenance of the morphology of axons and dendrites in neurons [11,12]. The dynamics of F-actin are associated with the contractile forces generated by NMII. The activation of NMII is regulated by the phosphorylation of its regulatory light chain (RLC) at Ser19 and Thr18 [11]. NMII filaments organize stress fibers by pulling and aligning F-actin, thereby maintaining cell morphology [13]. During this process, the contractile force of NMII exerts substantial tension on the actomyosin system and stress fibers [14], and this tension is transmitted to the cell membrane via membrane linkers [15,16]. When the tension is applied to the membrane, lipid molecules spread out, increasing the total surface area per lipid molecule. This loosening of lipid packing leads to enhanced membrane fluidity [17]. Previously, we suggested that actin polymerization inhibition showed Aβ aggregation at the cell periphery was significantly suppressed [6], lending credence to the prediction that cell-generated “force” controls Aβ aggregation via the cell membrane. However, it remains unclear how cytoskeleton-derived tension alters the function of the cell membrane as a scaffold for Aβ aggregation. Therefore, in this study, we attempt to investigate the mechanism by which changes in tension associated with neuronal morphological changes and movement promote Aβ aggregation. To analyze the effect of tension on Aβ aggregation, we adopt two pharmacological inhibitors, jasplakinolide and calyculin A, to enhance cellular tension. Jasplakinolide, a cyclic peptide derived from the marine sponge Jaspis johnstoni, induces actin polymerization by inhibiting actin filament depolymerization [18,19]. This stabilizes F-actin and increases the stiffness of the cytoskeletal structure [20]. Meanwhile, calyculin A, which was initially isolated from a marine sponge Discodermia calyx, is a serine/threonine phosphatase inhibitor that promotes the phosphorylation of RLC [21]. In addition, calyculin A has been shown to increase cellular contractility [22].
This study confirms that enhancing cytoskeletal tension using both inhibitors could promote Aβ aggregation on cell membranes, and provides new insights into the contribution of Aβ aggregation, aiding in understanding of the pathogenesis of AD.

2. Results

2.1. The Deposition of Aβ Aggregation on the Cell Surface

First, we observed Aβ aggregates deposited on the cell surfaces. To visualize cytoskeletons of SH-SY5Y cells, we established SH-SY5Y-EGFP-Lifeact stable cells. Lifeact is a 17-amino-acid peptide derived from the Abp140 protein of budding yeast [23]. By this method, we had already confirmed that only QD and QDAβ could not be aggregated and deposited on living cells using conventional microscopy [6,8]. Here, SH-SY5Y cells were incubated with 20 µM Aβ42 and 25 nM QDAβ for 24 h at 37 degrees. Our previous works reported that Aβ exhibited saturation of the aggregation process in the condition with or without cell culture [5,6]. As shown in Figure 1A, Aβ aggregates emerged around the cell edge. Interestingly, Aβ aggregates were localized along the cell outline (Figure 1A, boxed region). Then, to obtain the high-resolution images of deposited Aβ aggregates near the cells, we utilized super-resolution fluorescence microscopy, Nikon Structured Illumination Microscopy (N-SIM) (Nikon, Tokyo, Japan) [24]. As seen in Figure 1B, we succeeded in super-resolution observation of Aβ aggregates using QD imaging methods. Aβ aggregates exhibited a cluster of red dots depositing each cell part (Figure 1C; box a, base of cell process; box b, cell body; box c, lateral side of cell process). Previously, we reported that QDAβ was incorporated in fibril formation of Aβ42 and that single QDs in amyloid fibril were periodicity distributed [4]. Further, we reported the sedimentation process of Aβ particles with several µm in the solution of physiological conditions such a PBS [4]. We speculate that the single red dot was a rolled-up aggregate of amyloid fibrils incorporating QDAβ, and that the cluster of red dots might be observed as one particle under a conventional microscope. It might be that a certain number of red dots aggregate to grow to a size where they begin to sediment in solution. Thus, we confirmed Aβ aggregation around cell edges with high-resolution images.
Next, we analyzed three-dimensional (3D) distribution of Aβ aggregates around cells. Using confocal laser microscopy, we obtained Z-stack images and reconstructed 3D images (Figure 2A). 3D images provided layer information of Aβ aggregates and SH-SY5Y cells, indicating that Aβ aggregates covered the cell top surface. Figure 2B shows slice views of XY, XZ and YZ planes. At the bottom side (left panel), F-actin was localized in ventral surfaces. Meanwhile, the top of the cell was covered by Aβ aggregates (Right panel). Enlarged XZ plane displayed cells embedded in Aβ aggregates (Figure 2C). These results correspond with our past findings of differentiated PC12 cells and endothelial cells [6,7,8]. Remarkably, almost-aggregates were not localized within the cell body, indicating that Aβ aggregates were seldom incorporated into inner cells during aggregation. Although we reported that microglia phagocytose carry small amounts of Aβ and accumulate it in lysosomes, no large amounts of Aβ aggregates appeared inside the cells [4]. Thus, these results indicate that Aβ aggregates were deposited on the cell membrane.

2.2. Appropriate Concentrations of Jasplakinolide and Calyculin A Do Not Impact Aβ Aggregation

Previously, we confirmed decrease in cytoskeletal contraction force dramatically inhibited the Aβ aggregate on the surface of differentiated PC12 cells through the depolymerization of F-actin using cytochalasin D [6]. In this study, we attempt to elucidate the relationship between cytoskeletal tension and Aβ aggregation on the cell membrane. To increase cytoskeletal tension, two cytoskeletal inhibitors, jasplakinolide and calyculin A, were adopted. First, we confirmed whether the inhibitors themselves affect Aβ aggregation under conditions without cells using the Microliter-Scale High-throughput Screening (MSHTS) system [5]. We report that the variability of fluorescence intensity in fluorescence micrograph images is correlated with the amount of Aβ aggregates [5]. Using the MSHTS system, we obtained images of Aβ aggregates after treatment with each inhibitor (Figure 3A). Then, we measured the SD values of QDAβ fluorescence images at each concentration of both inhibitors (Figure 3B). The addition of jasplakinolide and calyculin A did not affect Aβ aggregation at concentrations below 20 nM and 0.625 nM, respectively.

2.3. Enforcement of the Actin Cytoskeleton Through the Inhibition of Actin Depolymerization Promotes Aβ Aggregation on Cell Surfaces

To clarify the contribution of cortical cellular tension on Aβ aggregation, SH-SY5Y-EGFP-Lifeact stable cells were treated with 0 to 40 nM jasplakinolide and were co-incubated with 20 µM Aβ42 and 25 nM QDAβ for 24 h (Figure 4A). Generally, 50–1000 nM jasplakinolide was used to induce the change in cytoskeletal structure [25,26]. Here, to avoid cell toxicity and morphological cell changes, we adopted processing at a lower concentration range than in past reports. At 24 h, Aβ aggregates visualized by QDAβ emerged in all samples (Figure 4A, bottom). Cells treated with 40 nM jasplakinolide lost most of their cytoskeletal structure and had a round appearance, but in ≤20 nM concentrations, cells maintained their proper morphology and cytoskeletal structures. We then quantified the amount of Aβ aggregates deposited on cell surfaces. The integrated density (IntDen) was calculated from the entire image of Aβ (Figure 4A, bottom) and normalized using the cell area calculated from the F-actin fluorescence image (Figure 4A, top). Interestingly, the amount of Aβ aggregates significantly increased at 1.25 nM jasplakinolide (Figure 4B). We evaluated the increase in F-actin intensity due to 1.25 nM jasplakinolide. The mean gray value per pixel, representing the fluorescence intensity of F-actin, was measured by the profile plot, the function of Image J software (version 1.54r, NIH, Bethesda, MD, USA) (Figure 4C,D). After treatment with 1.25 nM jasplakinolide, F-actin intensity along a line on cell images increased. These results suggest that jasplakinolide-induced promotion of actin polymerization stabilizes actin filaments and consequently enhances Aβ aggregation at the cell cortical region.

2.4. Activation of Myosin Through Phosphorylation of the Myosin Light Chain Promotes Aβ Aggregation on Cell Surfaces

The activity of NMII is responsible for the proper organization of F-actin, thereby maintaining cell morphology. Next, we focused on cellular tension promoted by the NMII-dependent contraction force. Here, we treated SH-SY5Y cells using calyculin A that enhances RLC phosphorylation [21] and analyzed the effects of NMII activation on Aβ aggregation. In general, 5–10 nM calyculin A was used to induce the change in cytoskeletal structure [22,27]. Here, to capture changes in Aβ aggregation due to subtle changes in the cytoskeleton, we adopted processing at a lower concentration range than in past reports. After 24 h of time-lapse initiation, cells treated with 1.25 nM calyculin A or higher concentrations had detached from the substrate (Figure 5A). In the presence of 0.156 to 0.625 nM calyculin A, cells maintained their proper morphology and cytoskeletal structure. We then quantified the amount of Aβ aggregates deposited on cell surfaces (Figure 5B). IntDen was calculated from the entire image of Aβ (Figure 5A, bottom) and normalized using the cell area calculated from the F-actin fluorescence image (Figure 5A, top). The amount of Aβ aggregates, which was calculated by IntDen/total area, significantly increased in the presence of 0.625 nM calyculin A (Figure 5B). Fluorescence intensity profiles of F-actin along a line drawn on cell images 1 h after the start of time-lapse observation showed a slight increase when calyculin A concentration was 0.625 nM (Figure 5C,D). This suggests that the activation of NMII through the inhibition of dephosphorylation of RLC using calyculin A caused an increase in Aβ aggregation on cell surfaces.

2.5. Co-Treatment Promoted Aβ Aggregation, and the Phosphorylated State of RLC

We added two types of inhibitors to cells, jasplakinolide and calyculin A, each acting on actin or NMII, respectively. To examine the effect of simultaneous treatment, we carried out co-treatment experiments of both inhibitors at concentrations that promoted Aβ aggregation in each individual inhibitor treatment. Co-treatment with 1.25 nM jasplakinolide and 0.625 nM calyculin A significantly increased Aβ aggregation compared to untreated samples, but the amount of Aβ aggregates did not increase compared to treatment with the individual inhibitors (Figure 6A,B).
To analyze whether β-sheet structure and deposition of Aβ fibrils increased or not, we performed the Thioflavin T (ThT) assay to study the effect of jasplakinolide and calyculin A. ThT is a fluorescent dye used to detect and quantify the formation of β-sheet-rich Aβ fibrils [28]. Fluorescence images of ThT were also captured using an inverted fluorescence microscope (Figure 6C). Additionally, we measured fluorescence intensity of ThT using a fluorescence microplate reader (Figure 6D). The increase in fluorescence intensity of ThT at 24 h demonstrated the promotion effect of 1.25 nM jasplakinolide and 0.625 nM calyculin A on Aβ deposition. Under co-treatment conditions, ThT fluorescence intensity tended to increase. These results indicate that increased Aβ aggregates on the cell surface are due to the promotion of β-sheet structure and Aβ fibrillization.
Furthermore, to confirm whether these treatments activated NMII and consequently generated cellular tension, immunofluorescence staining of 2P-RLC was performed in SH-SY5Y cells to visualize intracellular tension. The level of 2P-RLC phosphorylation was determined by IntDen. Compared to cells not treated with inhibitors, the IntDen value increased significantly both when using either the inhibitor alone or when using both inhibitors simultaneously (Figure 6E,F). These results confirm that the treatment with either or both inhibitors increased tension in cells. This suggests that Aβ aggregation is associated with cellular tension.

3. Discussion

This study demonstrates that the improvement of cytoskeletal structure induced by jasplakinolide and calyculin A promotes Aβ aggregation on the surface of living cells. It is suggested that two distinct pathways of cytoskeletal changes, namely, stabilization of stress fibers by inhibiting actin depolymerization and enhancement of motor activity by inhibiting dephosphorylation of the NMII light chain, may both affect the dynamics of Aβ aggregates present across the cell membrane (Figure 7).
First, we argued the relation between drug-induced cytoskeletal force enhancement and the Aβ aggregation process. Jasplakinolide, an actin depolymerization inhibitor, has previously been shown to stabilize SFs [29] and inhibit actin turnover rate in various cell types [30]. It has been reported to inhibit F-actin turnover within neuronal growth cones [31]. Thus, jasplakinolide alters the physical strength of cells and the tension. Through stabilizing the structure of SFs, cells exhibited the increase in surface stiffness [20]. Faure et al. reported that jasplakinolide treatment could mimic the increase in cell surface tension caused by cell spreading [32]. Here, we found that treatment with 1.25 nM jasplakinolide increased the fluorescence intensity of F-actin in SH-SY5Y cells, consistent with previous reports (Figure 4C). Furthermore, jasplakinolide treatment significantly increased phosphorylated RLC in SH-SY5Y cells, which indicates upregulation of NMII (Figure 6E,F), consistent with the previous report. Zhao et al. demonstrated that jasplakinolide upregulated F-actin levels and the phosphorylated RLC/RLC ratio in glucose–oxygen-deprived brain microvascular endothelial cells [33]. Possibly, jasplakinolide treatment increases the amount of F-actin directly beneath the plasma membrane in SH-SY5Y cells. We hypothesize that by reinforcing the scaffolding on which NMII exerts stable contractile force, the tension exerted on the cytoskeleton structure would increase.
Meanwhile, the protein phosphatases PP1 and PP2A of inhibitor calyculin A are also widely used for cytoskeletal reorganization. It was reported that calyculin A promotes SF formation in NIH-3T3 fibroblasts and gerbil fibroma cells through upregulation of RLC phosphorylation, thereby enhancing contractile force [22]. Asano et al. reported that calyculin A increases cortical contractile force in sea urchin eggs [34]. Consistent with these studies, we showed that treatment with calyculin A enhanced RLC phosphorylation (Figure 6E,F) and slightly increased F-actin fluorescence intensity. Indeed, while it has been reported that SF was enhanced, nM-range concentrations of calyculin A induced the movement of F-actin to the plasma membrane, but did not significantly change the amount of actin incorporated into cytoskeletal components [35]. In migrating newt lung epithelial cells, the addition of calyculin A increases the actin turnover rate [36]. Thus, calyculin A treatment might increase the activity of NMII directly beneath the plasma membrane, thereby increasing contractile force and therefore the tension exerted on the cytoskeletal structure. The increase in 2P-RLC following co-treatment of jasplakinolide and calyculin A remained at the same level as with treatment with each inhibitor alone (Figure 6E,F). This suggests that with treatment of each inhibitor, the total number of NMII filaments that can be incorporated into the cytoskeleton and exert contractile force is already saturated. There was no difference in the amount of Aβ aggregates between co-treatment and treatment with each inhibitor alone which suggests that the tension exerted on the cell surface was already saturated.
The transmission of cytoskeletal tension to the cell surface requires a connection between the plasma membrane and the cytoskeleton [15,37]. Membrane tension in migrating cells is determined by the function of the cytoskeleton [38]. Interaction between the plasma membrane and cortical actomyosin is essential, and this is regulated by a protein capable of simultaneously interacting with both the plasma membrane and F-actin [39]. ERM (ezrin/radixin/moesin) proteins are well-known cross-linkers between the cytoskeleton and the plasma membrane [40,41,42]. Ezrin depletion resulted in reorganization of the actin cytoskeleton and a decrease in membrane tension [43]. In vitro studies have shown that ezrin slides along F-actin [44]. It was reported that radixin-specific depletion induced defects in cell motility [45]. The specific suppression of moesin expression using short hairpin RNA weakened and destabilized actin bundles in epithelial cells, reducing their invasive ability [46]. Moreover, increased cortical stiffness provided by moesin is important for spindle formation and normal chromosome segregation [47]. We predict that the increase in actomyosin-derived contractile force by jasplakinolide and calyculin A, via ERM proteins, leads to a modification of tension on the cell membrane surface, thereby affecting the interaction between Aβ and the cell membrane.
Next, we will focus on the mechanism by which increased tension increases Aβ aggregation. The interaction between Aβ and the cell membrane is an important factor in the development of AD [48,49]. Various studies have shown that the state of the cell membrane is also involved in Aβ aggregation. Changes in membrane architecture alter lipid composition [3] and affect the distribution and stability of lipid rafts enriched in cholesterol and sphingolipids [50]. Since lipid rafts serve as platforms that promote the conformational transition of Aβ from an α-helix to a β-sheet and accelerate Aβ aggregation [51], it is considered that stabilization of these rafts may further enhance Aβ aggregation. Aβ fibrillation occurs in the presence of phospholipid membrane bilayers [52]. Aβ oligomers/protofibrils interpenetrate the lipid bilayer, as observed by cryo-electron tomography [53]. Furthermore, membrane curvature affects Aβ aggregation [54,55]. The state of the cell membrane changes in response to mechanical stimuli [56]. Molecular dynamics simulations revealed the effect of membrane tension on the properties of the lipid bilayer [17]. Increased tension leads to a decrease in membrane thickness and an increase in fluidity. Modification in the behavior of the lipid bilayer is also controlled by temperature: low temperatures increase lipid order and membrane rigidity whereas high temperatures decrease lipid order and membrane fluidity [57,58,59]. Actually, tension is applied to neurites and the actin cytoskeleton contributes to maintaining this tension [60]. The increased fluidity of phospholipids is dependent on tension derived from the cytoskeleton, and may create spaces between the phospholipids that make up the membrane, making it easier for Aβ to enter the hydrophobic regions, potentially promoting the aggregation of hydrophobic Aβ.
Finally, we discuss the limitations of our study. A major challenge is our inability to quantify the tension applied to SH-SY5Y cells. The degree to which tension is altered by jasplakinolide and calyculin A needs to be assessed using atomic force microscopy-based force spectroscopy or optical tweezers [61,62]. Detailed analysis of the distribution of intracellular tension and Aβ aggregation using molecular-based tension sensors is also important [63,64]. Further analysis of the effects of factors in the cell membrane (e.g., lipid rafts) on promoting Aβ aggregation is also needed [51]. Analysis of the chemical composition of disease-related amyloid has shown that lipids are involved in the tissue deposition of amyloid fibrils [65]. It was reported that Aβ aggregation was promoted by GM1-ganglioside using nerve-growth factor-treated PC12 cells [66,67]. Given that Aβ aggregation on the cell membrane is a complex event regulated by numerous parameters, future analyses that closely integrate cell biology and biophysics are desirable.

4. Materials and Methods

4.1. Cell Culture

SH-SY5Y cells were purchased from K.A.C. Co., Ltd. (Kyoto, Japan). Cells were cultured in D-MEM medium (043-30085, Wako, Osaka, Japan) at 37 °C in humidified air containing 5% CO2. The medium was supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, France) and 1% penicillin-streptomycin solution (Wako). pEGFP-Lifeact was a kind gift from Dr. Keiju Kamijo (Tohoku Medical and Pharmaceutical University, Sendai, Japan). To establish a stable SH-SY5Y-EGFP-Lifeact clone, cells were transfected with plasmids using Superfect (Qiagen, Hilden, Germany) in OPTI-MEM (Gibco, Grand Island, NY, USA), according to the manufacturers’ protocols. Transfected cells were selected by treatment with 200 µg/mL G-418 (Wako) for 2 weeks. Resistant colonies were cloned by a limiting dilution method and then screened by fluorescence microscopy (TE-2000, Nikon). SH-SY5Y-EGFP-Lifeact stable cells were cultured in medium supplemented with 200 µg/mL G-418.

4.2. Immunofluorescence

Indirect immunofluorescence was performed as described by Kuragano et al. [68]. The following primary antibodies were used: anti-diphosphorylated RLC at Thr18 and Ser19 (2P-RLC) pAb (Cell Signaling Technology, Beverly, MA, USA) (kindly gifted from Dr. Masayuki Takahashi, Hokkaido University, Sapporo, Japan). Cy3-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as the secondary antibody. The IntDen and cell area were measured by Image J software (version 1.54r).

4.3. Preparation of QDAβ Nanoprobes

The QDAβ nanoprobe was prepared using QD-PEG-NH2 (Qdot 605 ITK Amino (PEG) Quantum-dot; Q21501MP, Thermo Fisher Scientific, Waltham, MA, USA) according to our previous report [4]. The QDAβ nanoprobe was prepared as follows: 10 µM QD-PEG-NH2 was first reacted with 1 mM sulfo-EMCS (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) in PBS for 1 h at room temperature. After quenching and eliminating unreacted sulfo-EMCS, the QD-PEG-NH2-bound sulfo-EMCS was reacted with 74 μM Cys-conjugated Aβ40 for 1 h at room temperature. The concentration of QDAβ was determined by comparing absorbance at 350 nm relative to that of unlabeled QD-PEG-NH2.

4.4. Microliter-Scale High-Throughput Screening System

SD values in existence of jasplakinolide (J210700, HPLC; 97% purity, Toronto Research Chemicals, Vaughan, ON, Canada) and calyculin A (038-14453, HPLC; 95% purity, Wako) were determined by a modified MSHTS system, as was described in our previous report [5]. More specifically, various concentrations of jasplakinolide and calyculin A, 20 μM Aβ42 and 25 nM QDAβ in PBS were incubated in a 1536-well plate (782096, Greiner Bio-One, Kremsmünster, Austria) at 37 °C for 24 h. The QDAβ-Aβ42 aggregates that formed in each well were observed by an inverted fluorescence microscope (TE2000) equipped with a color CCD camera (DP72, Olympus, Hachioji, Japan) and an objective lens (Plan Fluor 4×/0.13 PhL DL, Nikon). SD values of fluorescence intensities of 40,000 pixels (200 × 200 pixels) around the central region of each well were measured by Image J software (version 1.54r).

4.5. Time-Lapse Observation

To observe live cells, time-lapse images were captured with an inverted microscope (Ti-E; Nikon) equipped with a color CMOS camera (DS-Ri2; Nikon) and an objective lens (Plan Apo 60×/0.95 NA, Nikon) and FITC (FITC-A-Basic-NTE, ex: 457–492 nm, em: 508–551 nm, Semrock Rochester, NY, USA) and TRITC (TRITC-A-Basic-NTE, ex: 532–552 nm, em: 594–646 nm, Semrock) filter sets. During observation, the cells were replated onto a glass-bottom 96 well-plate (IWAKI, AGC Techno Glass Co., Ltd., Shizuoka, Japan) precoated with 10 mg/mL fibronectin (Gibco), and were maintained in DMEM/F12 (Gibco) supplemented with 10% FBS and warmed in a chamber (INUBTF-WSKM; Tokai Hit, Fujinomiya, Japan) set at 37 °C. Images were captured every 10 min and analyzed using NIS-Elements AR software 4.51.00 (Nikon).

4.6. Confocal Laser Scanning Microscopy

Z-stacks images were captured using an inverted microscope (Ti-E) and a confocal laser microscope system (C2 Plus; Nikon) equipped with an objective lens Plan Apo λ 100×/1.45 NA Oil; Nikon). Images were captured and analyzed using NIS-Elements AR software 4.51.00. F-actin and QD were excited by a laser at 488 and 561 nm, respectively.

4.7. Super-Resolution Microscopy

For super-resolution microscopy, the images were captured with an inverted microscope (Ti-E; Nikon) equipped with a CMOS camera (ORCA-Flash v.4.30; Hamamatsu Photonics, Hamamatsu, Japan) and N-SIM unit (Nikon) and objective lens (Apo TIRF 100× Oil 1.49 NA, Nikon). F-actin and QD were excited by a laser at 488 and 561 nm, respectively. Images were analyzed using NIS-Elements AR software 4.51.00 (Nikon).

4.8. ThT Assay

SH-SY5Y cells were seeded and incubated in a 10 µg/mL fibronectin (Gibco, USA)-coated 96-wellplate for 24 h at 37 °C and 5% CO2. The cells were treated with 20 µM Aβ alone and with 20 µM ThT and incubated for 24 h at 37 °C, 5% CO2. The solutions were removed and fresh DMEM/F12 was dispensed. Fluorescence intensity of ThT was measured using a fluorescence microplate reader (SH-9000, Yamato, Tokyo, Japan) at 490 nm. Images were captured using a Ti-E inverted fluorescence microscope combined with NIS-Elements AR software 4.51.00 (Nikon) and analyzed using Image J software (version 1.54r).

5. Conclusions

In summary, we confirmed that Aβ aggregation and deposition on living cells are promoted by the inhibition of actin depolymerization and protein phosphatases using pharmacological treatment. These results indicate that cellular cortical tension might promote Aβ assembly over the cell membrane. These findings will contribute greatly to elucidating the mechanism of Aβ accumulation in the development of AD.

Author Contributions

J.N.: writing—original draft, formal analysis, data curation. Y.F.: writing—review and editing, data curation. K.T.: writing—review and editing, supervision, funding acquisition. M.K.: writing—review and editing, supervision, project administration, funding acquisition, conceptualization. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by a SUNBOR GRANT from the Suntory Foundation for Life Sciences (to M. Kuragano), JST JPMJPF2213 (to K. Tokuraku) and JSPS JP24K08627 (to K. Tokuraku).

Data Availability Statement

The data presented in this study are available in article.

Acknowledgments

We are grateful to the Nikon Imaging Center (Hokkaido University) for assistance with super-resolution microscopy (N-SIM), image acquisition, and analysis.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

The following abbreviations are used in this manuscript:
ADAlzheimer’s disease
IntDenIntegrated density
MSHTSMicroliter-scale high-throughput screening
NMIINonmuscle myosin II
N-SIMNikon structured illumination microscopy
QDQuantum dot
RLCRegulatory light chain
SDStandard deviation
ThTThioflavin T

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Figure 1. Aβ aggregates around cell edges. (A) Representative image of SH-SY5Y-EGFP-Lifeact stable cells and Aβ aggregates. Cells were incubated with 20 µM Aβ42 and 25 nM QDAβ at 37 °C for 24 h. Images were captured using a conventional fluorescence microscope. White arrowhead in top middle image indicates the localization of Aβ aggregates. White box indicates enlarged region displayed on the bottom panel. Note that localization of Aβ aggregates corresponds with outline of EGFP-Lifeact. (B) Representative super-resolution fluorescence microscopy image of SH-SY5Y-EGFP-Lifeact stable cells and Aβ aggregates. (C) Enlarged images of boxed region of panel B (box a, base of cell process; box b, cell body; box c, lateral side of cell process).
Figure 1. Aβ aggregates around cell edges. (A) Representative image of SH-SY5Y-EGFP-Lifeact stable cells and Aβ aggregates. Cells were incubated with 20 µM Aβ42 and 25 nM QDAβ at 37 °C for 24 h. Images were captured using a conventional fluorescence microscope. White arrowhead in top middle image indicates the localization of Aβ aggregates. White box indicates enlarged region displayed on the bottom panel. Note that localization of Aβ aggregates corresponds with outline of EGFP-Lifeact. (B) Representative super-resolution fluorescence microscopy image of SH-SY5Y-EGFP-Lifeact stable cells and Aβ aggregates. (C) Enlarged images of boxed region of panel B (box a, base of cell process; box b, cell body; box c, lateral side of cell process).
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Figure 2. 3D localization of Aβ aggregation with SH-SY5Y cells. (A) Cells were incubated with 20 µM Aβ42 and 25 nM QDAβ at 37 °C for 24 h. Images were captured using a confocal laser microscope. White arrowhead indicates Aβ aggregates covering the top of cell surface. (B) Enlarged slice image of panel A. Bottom side and top side indicate lower and higher position of Z-stack in 3D observation. White arrowhead indicates Aβ aggregates. Note that Aβ aggregation was localized over the cell. Orange lines indicate the location of each slice view. (C) Enlarged and inverted image of slice view of XZ plane in panel B. White arrowheads indicate Aβ aggregates.
Figure 2. 3D localization of Aβ aggregation with SH-SY5Y cells. (A) Cells were incubated with 20 µM Aβ42 and 25 nM QDAβ at 37 °C for 24 h. Images were captured using a confocal laser microscope. White arrowhead indicates Aβ aggregates covering the top of cell surface. (B) Enlarged slice image of panel A. Bottom side and top side indicate lower and higher position of Z-stack in 3D observation. White arrowhead indicates Aβ aggregates. Note that Aβ aggregation was localized over the cell. Orange lines indicate the location of each slice view. (C) Enlarged and inverted image of slice view of XZ plane in panel B. White arrowheads indicate Aβ aggregates.
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Figure 3. Appropriate concentrations of jasplakinolide and calyculin A did not affect Aβ aggregation. (A) 25 µM Aβ42 and 25 nM QDAβ were incubated with 0 to 40 nM jasplakinolide (top) or 0 to 2.50 nM calyculin A (bottom) at 37 °C for 24 h. Images were captured using a conventional fluorescence microscope. Bar, 100 µm. (B) SD values of fluorescence intensity of each pixel, which were correlated with the amount of Aβ aggregates, in the presence of various concentrations of each inhibitor (left, jasplakinolide; right, calyculin A). Data represent the mean ± SD (n ≥ three independent experiments). NS indicates no significant differences among the groups (Student’s t-test, * p < 0.05, ** p < 0.01).
Figure 3. Appropriate concentrations of jasplakinolide and calyculin A did not affect Aβ aggregation. (A) 25 µM Aβ42 and 25 nM QDAβ were incubated with 0 to 40 nM jasplakinolide (top) or 0 to 2.50 nM calyculin A (bottom) at 37 °C for 24 h. Images were captured using a conventional fluorescence microscope. Bar, 100 µm. (B) SD values of fluorescence intensity of each pixel, which were correlated with the amount of Aβ aggregates, in the presence of various concentrations of each inhibitor (left, jasplakinolide; right, calyculin A). Data represent the mean ± SD (n ≥ three independent experiments). NS indicates no significant differences among the groups (Student’s t-test, * p < 0.05, ** p < 0.01).
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Figure 4. Effects of jasplakinolide on F-actin fluorescence intensity and Aβ aggregation. (A) Fluorescent images of SH-SY5Y EGFP-Lifeact-stable cells and Aβ aggregates observed by a 20× objective lens after cells were fixed 24 h after starting time-lapse observation. The top image shows F-actin fluorescence, the bottom image shows QDAβ fluorescence. The brightness and contrast of the Aβ images were adjusted to improve visibility. Bar, 100 µm. (B) Differences in the amount of Aβ aggregates at each jasplakinolide concentration. IntDen/total area indicates the amount of Aβ aggregates on the cell surface. Error bars represent ± SD of the mean values from n ≥ 32 different images in two independent experiments. Statistically significant differences observed by one-way ANOVA, Tukey’s test (**** p < 0.001). Values are represented by dots on graph. (C) Fluorescence images of SH-SY5Y EGFP-Lifeact-stable cells were treated with the indicated condition. The images were captured 1 h after time-lapse observation. A white line (distance in 25 µm) was drawn on the cell in each image. Bar, 20 µm. (D) Actin fluorescence intensity profiles along the lines in the images of panel C at each concentration. Data represents the mean ± SD (0 µM; 9 lines, 1.25 µM; 20 lines).
Figure 4. Effects of jasplakinolide on F-actin fluorescence intensity and Aβ aggregation. (A) Fluorescent images of SH-SY5Y EGFP-Lifeact-stable cells and Aβ aggregates observed by a 20× objective lens after cells were fixed 24 h after starting time-lapse observation. The top image shows F-actin fluorescence, the bottom image shows QDAβ fluorescence. The brightness and contrast of the Aβ images were adjusted to improve visibility. Bar, 100 µm. (B) Differences in the amount of Aβ aggregates at each jasplakinolide concentration. IntDen/total area indicates the amount of Aβ aggregates on the cell surface. Error bars represent ± SD of the mean values from n ≥ 32 different images in two independent experiments. Statistically significant differences observed by one-way ANOVA, Tukey’s test (**** p < 0.001). Values are represented by dots on graph. (C) Fluorescence images of SH-SY5Y EGFP-Lifeact-stable cells were treated with the indicated condition. The images were captured 1 h after time-lapse observation. A white line (distance in 25 µm) was drawn on the cell in each image. Bar, 20 µm. (D) Actin fluorescence intensity profiles along the lines in the images of panel C at each concentration. Data represents the mean ± SD (0 µM; 9 lines, 1.25 µM; 20 lines).
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Figure 5. Effects of calyculin A on F-actin fluorescence intensity and Aβ aggregation. (A) Fluorescent images of SH-SY5Y EGFP-Lifeact-stable cells and Aβ aggregates observed by a 20× objective lens after cells were fixed 24 h after starting time-lapse observation. Top image shows F-actin fluorescence, bottom image shows QDAβ fluorescence. Brightness and contrast of Aβ images were adjusted to improve visibility. Bar, 100 µm. (B) Differences in the amount of Aβ aggregates at each calyculin A concentration. IntDen/total area indicates the amount of Aβ aggregates on the cell surface. Error bars represent ± SD of the mean values from n ≥ 16 different images in two independent experiments. Statistically significant differences observed by one-way ANOVA, Tukey’s test (*** p < 0.005). Values are represented by dots on graph. (C) Fluorescence images of SH-SY5Y EGFP-Lifeact-stable cells treated with the indicated condition. The images were captured 1 h after time-lapse observation. A white line (distance in 25 µm) was drawn on the cell in each image. Bar, 20 µm. (D) Actin fluorescence intensity profiles along the lines in the images of panel C at each concentration. Data represents the mean ± SD (0 nM, 4 lines; 0.625 nM, 4 lines).
Figure 5. Effects of calyculin A on F-actin fluorescence intensity and Aβ aggregation. (A) Fluorescent images of SH-SY5Y EGFP-Lifeact-stable cells and Aβ aggregates observed by a 20× objective lens after cells were fixed 24 h after starting time-lapse observation. Top image shows F-actin fluorescence, bottom image shows QDAβ fluorescence. Brightness and contrast of Aβ images were adjusted to improve visibility. Bar, 100 µm. (B) Differences in the amount of Aβ aggregates at each calyculin A concentration. IntDen/total area indicates the amount of Aβ aggregates on the cell surface. Error bars represent ± SD of the mean values from n ≥ 16 different images in two independent experiments. Statistically significant differences observed by one-way ANOVA, Tukey’s test (*** p < 0.005). Values are represented by dots on graph. (C) Fluorescence images of SH-SY5Y EGFP-Lifeact-stable cells treated with the indicated condition. The images were captured 1 h after time-lapse observation. A white line (distance in 25 µm) was drawn on the cell in each image. Bar, 20 µm. (D) Actin fluorescence intensity profiles along the lines in the images of panel C at each concentration. Data represents the mean ± SD (0 nM, 4 lines; 0.625 nM, 4 lines).
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Figure 6. Effects of co-treatment of jasplakinolide and calyculin A on F-actin fluorescence intensity and Aβ aggregation. (A) Fluorescent images of SH-SY5Y EGFP-Lifeact-stable cells treated simultaneously with two inhibitors and Aβ aggregates observed by a 20× objective lens after cells were fixed 24 h after starting time-lapse observation. The brightness and contrast of Aβ images were adjusted to improve visibility. Bar, 100 µm. (B) Effects of co-treatment with two inhibitors on Aβ aggregation. Error bars represent ± SD of the mean values from n ≥ 45 different images in two independent experiments. Statistically significant differences observed by nonparametric Kruskal–Wallis test (*** p < 0.005, **** p < 0.001). (C) SH-SY5Y cells were incubated with 20 µM Aβ42, 20 µM ThT. Images were captured by an inverted fluorescence microscope. (D) Fluorescence intensity of ThT at 24 h. The data represent the mean ± SD from three independent experiments. Statistically significant differences observed by one-way ANOVA, Dunnett’s test (* p < 0.05). (E) Effects of treatment with each inhibitor on the activation state of NMII of SH-ST5Y cells. Immunofluorescence images of F-actin and 2P-RLC in SH-SY5Y cells. Cells were treated with the indicated conditions. F-actin was stained with Alexa Fluor488-phalloidin. Bar, 20 µm. (F) Integrated density of 2P-RLC in immunofluorescence-stained cells. Error bars represent ± SD of the mean values from n ≥ 41 cells. Statistically significant differences observed by one-way ANOVA, Tukey’s test (*** p < 0.005, **** p < 0.001). Values are represented by dots on graph.
Figure 6. Effects of co-treatment of jasplakinolide and calyculin A on F-actin fluorescence intensity and Aβ aggregation. (A) Fluorescent images of SH-SY5Y EGFP-Lifeact-stable cells treated simultaneously with two inhibitors and Aβ aggregates observed by a 20× objective lens after cells were fixed 24 h after starting time-lapse observation. The brightness and contrast of Aβ images were adjusted to improve visibility. Bar, 100 µm. (B) Effects of co-treatment with two inhibitors on Aβ aggregation. Error bars represent ± SD of the mean values from n ≥ 45 different images in two independent experiments. Statistically significant differences observed by nonparametric Kruskal–Wallis test (*** p < 0.005, **** p < 0.001). (C) SH-SY5Y cells were incubated with 20 µM Aβ42, 20 µM ThT. Images were captured by an inverted fluorescence microscope. (D) Fluorescence intensity of ThT at 24 h. The data represent the mean ± SD from three independent experiments. Statistically significant differences observed by one-way ANOVA, Dunnett’s test (* p < 0.05). (E) Effects of treatment with each inhibitor on the activation state of NMII of SH-ST5Y cells. Immunofluorescence images of F-actin and 2P-RLC in SH-SY5Y cells. Cells were treated with the indicated conditions. F-actin was stained with Alexa Fluor488-phalloidin. Bar, 20 µm. (F) Integrated density of 2P-RLC in immunofluorescence-stained cells. Error bars represent ± SD of the mean values from n ≥ 41 cells. Statistically significant differences observed by one-way ANOVA, Tukey’s test (*** p < 0.005, **** p < 0.001). Values are represented by dots on graph.
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Figure 7. Schematic model of promotion of Aβ aggregation on the cell surface by enhancement of cytoskeletal tension. Jasplakinolide and calyculin A promote Aβ aggregation on SH-SY5Y cells through the activation of NMII contraction with enhancement of tension of cellular surfaces.
Figure 7. Schematic model of promotion of Aβ aggregation on the cell surface by enhancement of cytoskeletal tension. Jasplakinolide and calyculin A promote Aβ aggregation on SH-SY5Y cells through the activation of NMII contraction with enhancement of tension of cellular surfaces.
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Nakayama, J.; Fujiya, Y.; Tokuraku, K.; Kuragano, M. Enhancement of Cytoskeletal Tension Promotes Amyloid-β Aggregation on the Neuronal Cell Surface. Molecules 2026, 31, 718. https://doi.org/10.3390/molecules31040718

AMA Style

Nakayama J, Fujiya Y, Tokuraku K, Kuragano M. Enhancement of Cytoskeletal Tension Promotes Amyloid-β Aggregation on the Neuronal Cell Surface. Molecules. 2026; 31(4):718. https://doi.org/10.3390/molecules31040718

Chicago/Turabian Style

Nakayama, Juri, Yuna Fujiya, Kiyotaka Tokuraku, and Masahiro Kuragano. 2026. "Enhancement of Cytoskeletal Tension Promotes Amyloid-β Aggregation on the Neuronal Cell Surface" Molecules 31, no. 4: 718. https://doi.org/10.3390/molecules31040718

APA Style

Nakayama, J., Fujiya, Y., Tokuraku, K., & Kuragano, M. (2026). Enhancement of Cytoskeletal Tension Promotes Amyloid-β Aggregation on the Neuronal Cell Surface. Molecules, 31(4), 718. https://doi.org/10.3390/molecules31040718

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