High-Speed Three-Dimensional Scanning Force Microscopy Visualization of Subnanoscale Hydration Structures on Dissolving Calcite Step Edges.

Hydration at solid-liquid interfaces plays an essential role in a wide range of phenomena in biology and in materials and Earth sciences. However, the atomic-scale dynamics of hydration have remained elusive because of difficulties associated with their direct visualization. In this work, a high-speed three-dimensional (3D) scanning force microscopy technique that produces 3D images of solid-liquid interfaces with subnanoscale resolution at a rate of 1.6 s per 3D image was developed. Using this technique, direct 3D images of moving step edges were acquired during calcite dissolution in water, and hydration structures on transition regions were visualized. A Ca(OH)2 monolayer was found to form along the step edge as an intermediate state during dissolution. This imaging process also showed that hydration layers extended from the upper terraces to the transition regions to stabilize adsorbed Ca(OH)2. This technique provides information that cannot be obtained via conventional 1D/2D measurement methods.

Vitamin K2 sensitizes the efficacy of venetoclax in acute myeloid leukemia by targeting the NOXA-MCL-1 pathway.

Promising outcomes have been reported in elder patients with acute myeloid leukemia (AML) using combined therapy of venetoclax (VEN) and azacytidine (AZA) in recent years. However, approximately one-third of patients appear to be refractory to this therapy. Vitamin K2 (VK2) shows apoptosis-inducing activity in AML cells, and daily oral VK2 (menaquinone-4, GlakayR) has been approved for patients with osteoporosis in Japan. We observed a high response rate to AZA plus VEN therapy, with no 8-week mortality in the newly diagnosed AML patients consuming daily VK2 in our hospital. The median age of the patients was 75.9 years (range 66-84) with high-risk features. Patients received AZA 75 mg/m2 on D1-7, VEN 400 mg on D1-28, and daily VK2 45 mg. The CR/CRi ratio was 94.7% (18/19), with a CR rate of 79%. Complete cytogenetic CR was achieved in 15 of 19 (79%) patients, and MRD negativity in 2 of 15 (13%) evaluable CR patients. Owing to the extremely high response rate in clinical settings, we further attempted to investigate the underlying mechanisms. The combination of VK2 and VEN synergistically induced apoptosis in all five AML cell lines tested. VK2, but not VEN, induced mitochondrial reactive oxygen species (ROS), leading to the transcriptional upregulation of NOXA, followed by MCL-1 repression. ROS scavengers repressed VK2 induced-NOXA expression and led to the cancellation of pronounced apoptosis and the downregulation of MCL-1 by VK2 plus VEN. Additionally, knockdown and knockout of NOXA resulted in abrogation of the MCL-1 repression as well as enhanced cytotoxicity by the two-drug combination, indicating that VK2 suppresses MCL-1 via ROS-mediated NOXA induction. These data suggest that the dual inhibition of BCL-2 by VEN and MCL-1 by VK2 is responsible for the remarkable clinical outcomes in our patients. Therefore, large-scale clinical trials are required.

Revealing the Mechanism Underlying 3D-AFM Imaging of Suspended Structures by Experiments and Simulations.

The invention of 3D atomic force microscopy (3D-AFM) has enabled visualizing subnanoscale 3D hydration structures. Meanwhile, its applications to imaging flexible molecular chains have started to be experimentally explored. However, the validity and principle of such imaging have yet to be clarified by comparing experiments and simulations or cross-observations with an alternative technique. Such studies are impeded by the lack of an appropriate model. Here, this difficulty is overcome by fabricating 3D carbon nanotube (CNT) structures flexible enough for 3D-AFM, large enough for scanning electron microscopy (SEM), and simple enough for simulations. SEM and 3D-AFM observations of the same model provide unambiguous evidence to support the possibility of imaging overlapped nanostructures, such as suspended CNT and underlying platinum (Pt) nanodots. Langevin dynamics simulations of such 3D-AFM imaging clarify the imaging mechanism, where the flexible CNT is laterally displaced to allow the AFM probe access to the underlying structures. These results consistently show that 3D-AFM images are affected by the friction between the CNT and AFM nanoprobe, yet it can be significantly suppressed by oscillating the cantilever. This study reinforces the theoretical basis of 3D-AFM for imaging various 3D self-organizing systems in diverse fields, from life sciences to interface sciences.

Dynamics of Molecular Self-Assembly of Short Peptides at Liquid-Solid Interfaces - Effect of Charged Amino Acid Point Mutations.

Self-organizing solid-binding peptides on atomically flat solid surfaces offer a unique bio/nano hybrid platform, useful for understanding the basic nature of biology/solid coupling and their practical applications. The surface behavior of peptides is determined by their molecular folding, which is influenced by various factors and is challenging to study. Here, the effect of charged amino acids is studied on the self-assembly behavior of a directed evolution selected graphite-binding dodecapeptide on graphite surface. Two mutations, M6 and M8, are designed to introduce negatively and positively charged moieties, respectively, at the anchoring domain of the wild-type (WT) peptide, affecting both binding and assembly. The questions addressed here are whether mutant peptides exhibit molecular crystal formation and demonstrate molecular recognition on the solid surface based on the specific mutations. Frequency-modulated atomic force microscopy is used for observations of the surface processes dynamically in water at molecular resolution over several hours at the ambient. The results indicate that while the mutants display distinct folding and surface behavior, each homogeneously nucleates and forms 2D self-organized patterns, akin to the WT peptide. However, their growth dynamics, domain formation, and crystalline lattice structures differ significantly. The results represent a significant step toward the rational design of bio/solid interfaces, potent facilitators of a variety of future implementations.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Three-dimensional ordering of water molecules reflecting hydroxyl groups on sapphire (001) and α-quartz (100) surfaces.

Water molecules on oxide surfaces influence the chemical reactivity and molecular adsorption behavior of oxides. Herein, three-dimensional atomic force microscopy (3D-AFM) and molecular dynamics simulations are used to visualize the surface hydroxyl (OH) groups and their hydration structures on sapphire (001) and α-quartz (100) surfaces at the atomic-scale. The obtained results revealed that the spatial density distributions and hydrogen-bonding strengths of surface OH groups affect their local hydration structures. In particular, the force curves obtained by 3D-AFM suggest that the hydration forces of water molecules intensify at sites where water molecules strongly interact with the surface OH groups. The insights obtained in this study deepen our understanding of the affinities of Al2O3 and SiO2 for water molecules and contribute to the use of 3D-AFM in the investigation of atomic-scale hydration structures on various surfaces, thereby benefiting a wide range of research fields dealing with solid-liquid interfaces.

Protocol for live imaging of intracellular nanoscale structures using atomic force microscopy with nanoneedle probes.

Atomic force microscopy (AFM) is capable of nanoscale imaging but has so far only been used on cell surfaces when applied to a living cell. Here, we describe a step-by-step protocol for nanoendoscopy-AFM, which enables the imaging of nanoscale structures inside living cells. The protocol consists of cell staining, fabrication of the nanoneedle probes, observation inside living cells using 2D and 3D nanoendoscopy-AFM, and visualization of the 3D data. For complete details on the use and execution of this protocol, please refer to Penedo et al. (2021)1 and Penedo et al. (2021).2.

Identification of a novel type of focal adhesion remodelling via FAK/FRNK replacement, and its contribution to cancer progression.

Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.

Highly Stable Triangular Single-Layer 2D Assemblies: Synthesis and Their Stimuli-Responsive Elastic and Anisotropic Curling.

Synthesis of highly stable two-dimensional single-layer assemblies (SLAs) is a key challenge in supramolecular science, especially those with long-range molecular order and well-defined morphology. Here, thin (thickness <2 nm) triangular AuI -thiolate SLAs with high thermo-, solvato- and mechano- stability have been synthesized via a double-ligand co-assembly strategy. Furthermore, the SLAs show assembly-level elastic and anisotropic deformation responses to external stimuli as a result of the long-range anisotropic molecular packing, which provides SLAs with new application potentials in bio-mimic nanomechanics.

Azithromycin, a potent autophagy inhibitor for cancer therapy, perturbs cytoskeletal protein dynamics.

BACKGROUND: Autophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy. METHODS: We identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model. RESULTS: We elucidated that keratin-18 (KRT18) and α/ß-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue. CONCLUSIONS: As drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.

p53 regulates lysosomal membrane permeabilization as well as cytoprotective autophagy in response to DNA-damaging drugs.

Lysosomes are single-membraned organelles that mediate the intracellular degradation of macromolecules. Various stress can induce lysosomal membrane permeabilization (LMP), translocating intralysosomal components, such as cathepsins, to the cytoplasm, which induces lysosomal-dependent cell death (LDCD). This study reports that p53 regulates LMP in response to DNA-damaging drugs. Treating wild-type TP53 A549 cells with DNA-damaging drugs (namely, doxorubicin, carboplatin, and etoposide) induced LMP and accelerated cell death more rapidly than treating TP53-knockout (KO) A549 cells. This suggested p53-dependent LMP and LDCD induction in response to DNA damage. LMP was induced by p53-dependent BID upregulation and activation, followed by translocation of truncated BID to lysosomes. Simultaneously, autophagy for damaged lysosome elimination (lysophagy) was activated via the p53-mTOR-TEFB/TFE3 pathways in response to DNA damage. These data suggested the dichotomous nature of p53 for LMP regulation; LMP induction and repression via the p53-BID axis and p53-mTOR-TFEB/TFE3 pathway, respectively. Blocking autophagy with hydroxychloroquine or azithromycin as well as ATG5 KO enhanced LMP and LDCD induction after exposure to DNA-damaging drugs. Furthermore, lysosomal membrane stabilization using U18666A, a cholesterol transporter Niemann-Pick disease C1 (NPC1) inhibitor, suppressed LMP as well as LDCD in wild-type TP53, but not in TP53-KO, A549 cells. Thus, LMP is finely regulated by TP53 after exposure to DNA-damaging drugs.

Molecular insights on the crystalline cellulose-water interfaces via three-dimensional atomic force microscopy.

Cellulose, a renewable structural biopolymer, is ubiquitous in nature and is the basic reinforcement component of the natural hierarchical structures of living plants, bacteria, and tunicates. However, a detailed picture of the crystalline cellulose surface at the molecular level is still unavailable. Here, using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we revealed the molecular details of the cellulose chain arrangements on the surfaces of individual cellulose nanocrystals (CNCs) in water. Furthermore, we visualized the three-dimensional (3D) local arrangement of water molecules near the CNC surface using 3D AFM. AFM experiments and MD simulations showed anisotropic water structuring, as determined by the surface topologies and exposed chemical moieties. These findings provide important insights into our understanding of the interfacial interactions between CNCs and water at the molecular level. This may allow the establishment of the structure-property relationship of CNCs extracted from various biomass sources.

Visualizing Molecular-Scale Adsorption Structures of Anti-freezing Surfactants on Sapphire (0001) Surfaces at Different Concentrations by 3D Scanning Force Microscopy.

Anti-freezing surfactants form an adsorption layer at the solid-water interface to inhibit the nucleation and growth of ice. However, this mechanism has not been elucidated at the molecular scale because of the difficulties in visualizing such adsorption structures. In this study, we overcome this limitation by directly visualizing the three-dimensional (3D) adsorption structures of anti-freezing surfactants, hexadecyltrimethylammonium bromide (C16TABs), on sapphire (0001) surfaces through 3D scanning force microscopy. We present molecularly resolved two-dimensional/3D images of the adsorption structures in solutions of 1, 10, and 100 ppm. At 1 ppm, the molecules form a monolayer with a flat-lying configuration. At 10 ppm, the molecular orientation is closer to the upright configuration, with a relatively large tilt angle. At 100 ppm, the molecules form a bilayer with almost upright configurations, providing excellent screening of the sapphire surface from water. Owing to the steric and electrostatic repulsion between adjacent molecular head groups, the surface of the bilayer exhibits relatively large fluctuations, inhibiting the formation of stable ice-like structures. The understanding of molecular-level mechanisms provides important guidelines for improving the design of anti-freezing surfactants for practical applications such as car coolants.

Computed Three-Dimensional Atomic Force Microscopy Images of Biopolymers Using the Jarzynski Equality.

Three-dimensional atomic force microscopy (3D-AFM) has resolved three-dimensional distributions of solvent molecules at solid-liquid interfaces at the subnanometer scale. This method is now being extended to the imaging of biopolymer assemblies such as chromosomes or proteins in cells, with the expectation of being able to resolve their three-dimensional structures. Here, we have developed a computational method to simulate 3D-AFM images of biopolymers by using the Jarzynski equality. It is found that some parts of the fiber structure of biopolymers are indeed resolved in the 3D-AFM image. The dependency of 3D-AFM images on the vertical scanning velocity is investigated, and optimum scanning velocities are found. It is also clarified that forces in nonequilibrium processes are measured in 3D-AFM measurements when the dynamics of polymers are slower than the scanning of the probe.

Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins.

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.

Ricolinostat enhances adavosertib­induced mitotic catastrophe in TP53­mutated head and neck squamous cell carcinoma cells.

TP53 mutation is one of the most frequent gene mutations in head and neck squamous cell carcinoma (HNSCC) and could be a potential therapeutic target. Recently, the WEE1 G2 checkpoint kinase (WEE1) inhibitor adavosertib (Adv) has attracted attention because of its selective cytotoxicity against TP53­mutated cells and has shown promising activity in early phase clinical trials. In the present study, it was demonstrated that combined treatment with Adv and a selective histone deacetylase 6 (HDAC6) inhibitor, ricolinostat (RCS), synergistically enhanced cell death induction in four out of five HNSCC cell lines with TP53 mutation (CAL27, SAS, HSC­3, and OSC­19), one HNSCC cell line with impaired TP53 function by HPV­infection (UPCI­SCC154), and TP53­knockout human lung cancer cell line (A549 TP53­KO), but not in TP53 wild­type A549 cells. Time­lapse imaging showed that RCS enhanced the Adv­induced mitotic catastrophe. Consistent with this, RCS treatment suppressed checkpoint kinase 1 (Chk1) (Ser345) phosphorylation and co­administration of RCS with Adv suppressed cyclin­dependent kinase 1 (Tyr15) phosphorylation along with increased expression of γ­H2A.X, a marker of DNA double­strand breaks in CAL27 cells. These data showed that RCS enhanced Adv­induced premature mitotic entry and cell death induction in the mitotic phase. However, although HDAC6 knockdown enhanced Adv­induced cell death with γ­H2A.X elevation, HDAC6 knockdown did not repress Chk1 phosphorylation in CAL27 cells. Our data demonstrated that the co­administration of RCS with Adv in HNSCC cells resulted in the suppression of Chk1 activity, leading to synergistically enhanced apoptosis via mitotic catastrophe in a p53­dependent manner. This enhanced cell death appeared to be partially mediated by the inhibition of HDAC6 activity by RCS.

Lysosome­targeted drug combination induces multiple organelle dysfunctions and non­canonical death in pancreatic cancer cells.

Pancreatic cancer is one of the leading causes of cancer­related mortality and has the lowest 5­year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC­3, KP­4, PANC­1 and MIA PaCa­2, were examined. It was observed that FTY720 enhanced the lapatinib­induced cytotoxic effect and caused non­canonical and lysosome­dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome­targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

Visualizing intracellular nanostructures of living cells by nanoendoscopy-AFM.

Atomic force microscopy (AFM) is the only technique that allows label-free imaging of nanoscale biomolecular dynamics, playing a crucial role in solving biological questions that cannot be addressed by other major bioimaging tools (fluorescence or electron microscopy). However, such imaging is possible only for systems either extracted from cells or reconstructed on solid substrates. Thus, nanodynamics inside living cells largely remain inaccessible with the current nanoimaging techniques. Here, we overcome this limitation by nanoendoscopy-AFM, where a needle-like nanoprobe is inserted into a living cell, presenting actin fiber three-dimensional (3D) maps, and 2D nanodynamics of the membrane inner scaffold, resulting in undetectable changes in cell viability. Unlike previous AFM methods, the nanoprobe directly accesses the target intracellular components, exploiting all the AFM capabilities, such as high-resolution imaging, nanomechanical mapping, and molecular recognition. These features should greatly expand the range of intracellular structures observable in living cells.

Targeted disruption of GAK stagnates autophagic flux by disturbing lysosomal dynamics.

The autophagy­lysosome system allows cells to adapt to environmental changes by regulating the degradation and recycling of cellular components, and to maintain homeostasis by removing aggregated proteins and defective organelles. Cyclin G­associated kinase (GAK) is involved in the regulation of clathrin­dependent endocytosis and cell cycle progression. In addition, a single nucleotide polymorphism at the GAK locus has been reported as a risk factor for Parkinson's disease. However, the roles of GAK in the autophagy­lysosome system are not completely understood, thus the present study aimed to clarify this. In the present study, under genetic disruption or chemical inhibition of GAK, analyzing autophagic flux and observing morphological changes of autophagosomes and autolysosomes revealed that GAK controlled lysosomal dynamics via actomyosin regulation, resulting in a steady progression of autophagy. GAK knockout (KO) in A549 cells impaired autophagosome­lysosome fusion and autophagic lysosome reformation, which resulted in the accumulation of enlarged autophagosomes and autolysosomes during prolonged starvation. The stagnation of autophagic flux accompanied by these phenomena was also observed with the addition of a GAK inhibitor. Furthermore, the addition of Rho­associated protein kinase (ROCK) inhibitor or ROCK1 knockdown mitigated GAK KO­mediated effects. The results suggested a vital role of GAK in controlling lysosomal dynamics via maintaining lysosomal homeostasis during autophagy.