Episódios
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D. Carlero et al, ACS Nano 2024, 18, 30, 19518–19527
Researchers from Kanazawa University's NanoLSI, IMDEA Nanoscience, and CNB-CSIC studied influenza A replication using high-speed atomic force microscopy. They observed that recombinant ribonucleoprotein complexes (rRNPs) can undergo multiple transcription cycles, with RNA structure stability influencing synthesis rates. Their findings offer new insights into viral replication mechanisms and RNA synthesis regulation, opening doors for further research on gene expression control.
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M.S. Alam et al, Small Methods 2024, 2400287
Atomic force microscopy (AFM) was initially developed to visualize surfaces at nanoscale resolution. Researchers at WPI NanoLSI, Kanazawa University, have now extended AFM for 3D imaging, particularly for flexible nanostructures like carbon nanotubes. They demonstrated that dynamic mode AFM, which uses a vibrating tip, causes less friction and damage than static mode, making it ideal for imaging delicate biological systems like cells, organelles, and vesicles.
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Madhu Biyani and colleagues at NanoLSI, Kanazawa University, have developed a sensitive and selective electrochemical biosensor for detecting the cancer biomarker ADAR1, using new aptamers. This cost-effective tool enables rapid ADAR1 detection in diluted samples, promising improved cancer prognosis and monitoring.
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Yanjun Zhang, Yuri Korchev, and colleagues used hopping probe scanning ion conductance microscopy to study hydrogen peroxide eustress on colorectal cancer cells, revealing varying cell stiffness and gradients. Their findings could lead to new cancer and inflammatory disease therapies.
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Mikihiro Shibata and collaborators used high-speed atomic force microscopy to study nucleosome dynamics. They found that nucleosomes without histone tails, particularly H2B and H3, showed increased sliding and DNA unwrapping. These findings highlight the importance of histone tails in chromatin stability and structure.
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Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Japan, collaborating with Professor Sarikaya, Seattle, USA.
The research described in this podcast was published in Small in February 2024
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
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Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Holger Flechsig and Clemens Franz from WPI-NanoLSI, at Kanazawa University, in collaboration with Vincent Torre from the International School of Advanced Studies in Italy and former WPI-NanoLSI members Leonardo Puppulin and Arin Marchesi.
The research described in this podcast was published in Nature Communications in January 2024
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/Researchers observe the structural heterogeneity of a lipid scramblase
Researchers from Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University report in Nature Communications that TMEM16F, a transmembrane protein that facilitates the passive movement of phospholipids and ions across membranes, explores a larger conformational landscape than previously thought to perform its unique functions. The finding refines our molecular understanding of crucial physiological processes such as blood coagulation and COVID-19 pathogenesis, and highlights the importance of probing membrane proteins in native-like environments.
Lipid scramblases are proteins embedded in cell membranes that play a crucial role in shuffling phospholipids between the two lipid layers that form such cellular boundaries. TMEM16F, a member of the TMEM16 protein family, acts as both a calcium-activated ion channel and a lipid scramblase, meaning that it can facilitate the transfer of both, lipids and ions across the chemical environment outside and inside of the cell. These movements regulate several biological functions such as blood clotting, bone development, and viral entry and are therefore of great physiological and clinical interest. At the molecular level, the TMEM16F architecture has a double-barrelled shape in which two identical polypeptide chains (called subunits), each formed by ten transmembrane (TM) helices, stick together (a process known as dimerization) to form two separate and presumably independent ion and lipid pathways.
Previously, it was thought that TMEM16F might work like a simple gate, with calcium ions serving as keys to unlock the two permeation pathways. Opening and closing the gate to different extents would let lipids and ions cross the plasma membrane alternately. However, structural investigations using cryo-electron microscopy (cryo-EM) -an in vitro technique that can reveal the 3D architecture of purified and frozen proteins at near-atomic resolution – have mostly captured TMEM16F snapshots in inactive conformations, with the ion and lipid gates presumably trapped in a closed state, raising questions about the validity of existing models.
So how did the researchers set about shedding light on how TMEM16F works?
To gain a better understanding of TMEM16F’s structure and function relationship, Holger Flechsig and Clemens Franz from WPI-NanoLSI, Kanazawa University, in collaboration with Vincent Torre from the International School of Advanced Studies (Italy) and former WPI-NanoLSI members Leonardo Puppulin and Arin Marchesi, used advanced techniques such as single-molecule force spectroscopy (SMFS) and high-speed atomic force microscopy (HS-AFM) imaging. These methods allowed them to observe TMEM16F behaviour at the molecular level in physiological environments, providing insights into its structure, dynamics, and mechanical properties.
The study uncovered that TMEM16F exhibits a wide range of structural conformations that have been overlooked so far. The research revealed unexpected changes in the dimerization interface and TMEM16F subunit arrangements, suggesting that TMEM16F operates in a more dynamic and versatile manner than previously thought. The authors propose tha
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Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz at the Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University.
The research described in this podcast was published in the Journal of Cell Science in January 2024.
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
A novel role for S100A11 in focal adhesion regulation
Researchers at Kanazawa University report in the Journal of Cell Science on a novel role of the small Ca2+ion-binding protein S100A11 [S one hundred A eleven] in focal adhesion disassembly.S100A11 is a small Ca2+ion-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays F-actin binding activity and localizes to actin stress fibers, but its precise role in regulating these structures remained unclear.
In their study, Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz together with colleagues from the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, in Japan, and Karlsruhe Institute of Technology, in Germany, report a novel localization of S100A11 to disassembling focal adhesions at the end of contractile stress fibers in HeLa and U2OS cells. Specifically, S100A11 transiently appears at the onset of focal adhesion disassembly, reliably marking the targeted adhesion sites for subsequent disassembly. Interestingly, S100A11 leaves focal adhesion sites before the completion of disassembly, indicating that S100A11 plays a specific role in the initiation of adhesion site disassembly, rather than the disassembly process itself.
So what are focal adhesions anyway and what can we learn from them?
Focal adhesions are integrin-containing cell/matrix adhesion sites enabling cells to adhere to the cellular environment and to apply cellular contraction forces during extracellular matrix remodeling. Directed cell migration requires the coordinated assembly of new adhesion sites at the front, and disassembly at the rear of the cell, and better understanding mechanisms regulating focal adhesion turnover is, therefore, an important goal in cell migration and invasion research. The newly discovered role of S100A11 in focal adhesion disassembly extends insights into the molecular mechanisms underlying focal adhesion site disassembly.
The authors furthermore delineate a force-dependent recruitment mechanism for S100A11 to adhesion sites involving non-muscle myosin II-driven stress fiber contraction, activation of mechanosensitive, Ca2+ ion-permeable Piezo1 channels, and intracellular Ca2+ ion influx at mechanically stressed focal adhesions. In turn, locally elevated Ca2+ ion levels activates and recruits S100A11 to the adhesion sites targeted for disassembly.
So how did they work this out?
The force-dependent recruitment of S100A11 to stressed focal adhesions was confirmed using a micropipette pulling assay able to apply pulling forces onto individual focal adhesion sites. Even when myosin II-dependent intracellular contractility was inhibited, external pulling forces still recruited S100A11 to stretched focal adhesion sites, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ ion and Piezo1 function was still indispensable, indicating that myosin II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ ion influx, in turn leading to S100A11 activation and focal adhesion recruitment.
Lastly, the authors show impaired focal adhesion translocation and disassembly rat
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Researchers observe what ubiquitination hinges on
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Hiroki Konno and Holger Flechsig at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University.
The research described in this podcast was published in Nano Letters in December 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers observe what ubiquitination hinges on
Researchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University report in Nano Letters how the flexibility of a protein hinge plays a crucial role in the transfer of proteins in key cell processes.
Ubiquitination – the addition of the protein ubiquitin – is a key stage in many cell processes, such as protein degradation, DNA repairs, and signal transduction. Using high-speed atomic force microscopy (AFM) and molecular modelling, researchers led by Hiroki Konno and Holger Flechsig at WPI-NanoLSI, Kanazawa University have identified how the mobility of a ubiquitination related enzyme hinge allows ubiquitination to take place.
So what was known already about ubiquitination?
Previous studies have identified a number of enzymes that facilitate ubiquitination, including an enzyme that activates ubiquitin (E1), an enzyme that conjugates it (E2), and an enzyme that catalyzes ubiquitin protein joining (that is, a ligase, E3) to a target protein. The HECT-type E3 ligase is characterized by a HECT domain that comprises an N lobe with the E2-binding site and a C lobe with a catalytic Cys residue, A flexible hinge connects the two lobes, leading to the hypothesis that ubiquitination is facilitated by the rearrangement of the protein around this hinge. Konno and their collaborators deployed their high-speed atomic force microscope to hunt for evidence that this was the case.
So what did they find out?
The researchers noted that when the HECT domain was crystallized with a type of E2 enzyme, it formed an L shape such that the distance between the catalytic Cys residue of the HECT domain and the catalytic Cys of the E2 enzyme was 41 Å – too far for the transfer of ubiquitin. However, in its catalytic conformation the HECT domain has a different shape where the distance between the two catalytic Cys residues is much closer – just 8 Å – so this is thought to be a “catalytic conformation”.
Analysis of high-speed-AFM images of a wild-type HECT domain of E6AP revealed two conformations – one of which looked spherical and the other oval. Using AFM simulations they attributed the oval shapes to the L conformation and spherical shapes are either the catalytic conformation or the so called inverted T conformation, which had been observed in the another type of HECT domain where the distance between the Cys residues is 16 Å. To overcome the spatio-temporal resolution limitations of imaging, the experiments were complemented by molecular modelling to visualize HECT domain conformational motions at the atomistic level. Simulation AFM was used to generate a corresponding pseudo AFM movie, which clearly showed the change from spherical to the oval shaped topography.
“Although experimental limitations do not allow us to resolve the intermediate conformations,” explain the researchers in their report of the work. “The performed modeling provides evidence that the transitions between spherical and oval HECT domain shapes observed under high-speed-AFM correspond to functional conformational motions under which the C-lobe rotates relative to the N-lobe, thereby allowing the change between catalytic and L-shape HECT conformations.”
Further experiments with mutant HECT domains with less flexibility in the h
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Chromatin Accessibility: A new avenue for gene editing
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, led by Yusuke Miyanari.
The research described in this podcast was published in Nature Genetics in February 2024
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Chromatin Accessibility: A new avenue for gene editing
In a study recently published in Nature Genetics, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University explore chromatin accessibility, that is, endogenous access pathways to the genomic DNA, and its use as a tool for gene editing.
Our DNA is protected from unwanted external modifications by forming structures called nucleosomes that consist of threads of DNA wound around chunks of special proteins known as histones. This unique coiled shape prevents the access of undesirable molecules to a cell’s DNA. However, for vital genetic functions—such as DNA repair—the right set of proteins require access to these DNA fragments. This phenomenon known as ‘chromatin accessibility’ involves a privileged set of protein molecules, many of which are still unknown.
Now, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University, led by Yusuke Miyanari, have used advanced genetic screening methods to unravel chromatin accessibility and its pathways.
So how did they go about it?
For the investigation the team used a combination of two technologies—CRISPR screening and ATAC-see. While the former is a method to suppress the function of a desired set of genes, the latter is a means to identify which ones are essential for chromatin accessibility. Thus, using this method all genes playing a crucial role in chromatin accessibility could be pinned down.
With the help of these assays, novel pathways and individual players involved in chromatin accessibility were uncovered—some playing a positive role and some negative. Of these, one particular protein, TFDP1, showed a negative effect on chromatin accessibility. When it was suppressed, a significant increase in chromatin accessibility was observed, accompanied by nucleosome reduction. A deeper dive into the mechanism of TFDP1 revealed that it functions by regulating the genes responsible for production of certain histone proteins.
The team then focused their study towards exploring biotechnological applications of their findings. After suppressing TFDP1, two different approaches were tried. The first approach involved gene editing using the CRISPR/Cas9 tool. This revealed that deletion of TFDP1 made the gene editing process easier. Now, most chromatin accessibility occurs in nucleosome-depleted regions or NDRs. However, by suppressing TFDP1 chromatin accessibility occurred not only in NDRs but across other regions as well. Secondly, the depletion of TFDP1 aided the process of converting regular cells into stem cells, a massive step forward in cellular transformation.
This study identified new chromatin accessibility pathways and channels for exploring its potential even further. “Our study shows the significant potential to manipulate chromatin accessibility as a novel strategy to enhance DNA-templated biological applications, including genome editing and cellular reprogramming,” conclude the researchers.
Reference
Satoko Ishii, Taishi Kakizuka, Sung-Joon Park, Ayako Tagawa, Chiaki Sanbo, Hideyuki Tanabe, Yasuyuki Ohkawa, Mahito Nakanishi, Kenta Nakai, Yusuke Miyanari. Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility. Nature Genetics, Feb
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Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Romain Amyot, Noriyuki Kodera, and Holger Flechsig at the Kanazawa University NanoLSI.
The research described in this podcast was published in Frontiers in Molecular Biosciences in November 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers predict protein placement on AFM substrates
Researchers at Kanazawa University report in Frontiers in Molecular Biosciences a computational method to predict the placement of proteins on AFM substrates based on electrostatic interactions.
The observation of biomolecular structures using atomic force microscopy (AFM) and the direct visualization of functional conformational dynamics in high-speed AFM (HS-AFM) experiments have significantly advanced the understanding of biological processes at the nanoscale. In experiments, a biological sample is deposited on a supporting surface (AFM substrate) and is scanned by a probing tip to detect the molecular shape and its dynamical changes. The observation of protein dynamics under HS-AFM is a delicate balance between immobilizing the structure on the supporting surface while at the same time preventing too strong perturbations by immobilization.
The process of placing a biomolecular sample on the supporting surface and controlling its proper attachment is a challenge at the very start of every AFM observation. By the chemical composition of the buffer, interactions between the sample and substrate can be modified. Such surface modifications are often critical for successful AFM observations of protein structures and their functional motions. However, the molecular orientation of the sample is a priori unknown, and due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis.
Romain Amyot, Noriyuki Kodera, and Holger Flechsig from Kanazawa University have now developed a physical model to predict the placement of biomolecular structures on AFM substrates based on electrostatic interactions. The method considers the substrates commonly used in AFM experiments (mica, APTES-mica, lipid bilayers) and takes into account buffer conditions. In computer simulations, a large number of possible molecular arrangements on the AFM substrate are sampled, and from evaluating the corresponding interaction energies, the most favorable placement is determined. Furthermore, the analysis allows predictions of the imaging stability under tip scanning.
The authors provide several applications of the new method and obtain remarkable agreement of model predictions with previous experimental HS-AFM imaging of proteins. The findings can explain, for example, why HS-AFM observations of the Cas9 endonuclease, a protein playing a key role in genetic engineering applications, can reliably visualize functional relative motions of target DNA and Cas9 and capture DNA cleavage events at the single molecule level (see Fig. 1). Furthermore, as demonstrated for the ATP-powered chaperone machine ClpB, the model can explain how buffer conditions affect the stability of the sample-substrate complex and validate observations of previous HS-AFM experiments.
In summary, the new method allows to employ the enormous amount of available structural data for biomolecules to make predictions of the sample placement on AFM substrates even prior to an actual experiment, and it can also be applied for post-experimental analysis of AFM imaging data. The developed method is implemented within the freely available BioAFMviewer software package, providing a convenient platform for applications by the broad BioAFM community.
Reference
R. Amyot, K. Nakamoto, N. Kodera, H. Flechsi
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Sodium channel investigation
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Ayumi Sumino and Takashi Sumikama at the Kanazawa University NanoLSI.
The research described in this podcast was published in Nature Communications in December 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Sodium channel investigation
Researchers at Kanazawa University report in Nature Communications a high-speed atomic force microscopy study of the structural dynamics of sodium ion channels in cell membranes. The findings provide insights into the mechanism behind the generation of cell-membrane action potentials.
The transport of ions to and from a cell is controlled by pore-forming proteins embedded in the cell membrane. In particular, so-called voltage-gated sodium channels (VGSCs) govern the transfer of sodium (Na+) ions, and play an important role in the regulation of the membrane potential — the voltage difference between the cell’s exterior and interior. In electrically excitable cells such as neurons and muscle cells, VGSCs participate in the generation of action potentials; these are rapid changes in the membrane potential enabling the transmission of e.g. neural signals. The precise structural changes occurring in VGSCs are not completely understood, however. Now, Ayumi Sumino and Takashi Sumikama from Kanazawa University in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues have succeeded in observing the structural dynamics of VGSC by means of high-speed atomic force microscopy (high speed-AFM), a method capable of imaging the nanostructure and subsecond dynamics of biomolecules.
VGSCs can be in three different states: resting, inactive and active. In the latter state, Na+ ions can pass through the channel; in the resting and inactive states, which are structurally different, ions cannot pass. The basic structure of a VGSC consists of two modules: voltage sensor domains and pore domains. These domains form a square arrangement, with the ion pore at its center. An important open question is whether the voltage sensor domains dissociate from the pore domains when the channel closes.
So how did they go about determining this?
Sumino and colleagues performed experiments on three VGSCs. One is the sodium channel of a particular bacterium (Arcobacter butzleri), the other two are mutants of it. These three VGSCs have different voltage dependencies, with activation voltages starting at -120 mV, -50 mV and 0 mV, so that at the experimental conditions (0 mV), the VGSCs are in different states.
In order to provide insights into the structural dynamics of these three VGSCs, the researchers applied high speed-AFM, a powerful technique for producing image sequences of biochemical compounds. A single AFM image is generated by laterally moving a tip just above the sample’s surface; during this xy-scanning motion, the tip’s position in the direction perpendicular to the xy-plane (the z-coordinate) will follow the sample’s height profile. The variation of the z-coordinate of the tip then produces a height map — the image of the sample. The generation of such AFM images in rapid succession then produces a video recording of the sample.
The HS-AFM results revealed that for the mutant VGSC in the resting state, the voltage sensor domains are indeed dissociated from the pore domains. Furthermore, the researchers found that the dissociated voltage sensor domains of neighboring channels connect to form pairs — this is referred to as dimerization.
The observation of the dissociation of voltage sensor domains, as well as the dimerization between pore channels,
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Researchers fix the chirality of helical proteins
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Naoki Ousaka, Mark J. MacLachlan and Shigehisa Akine at the Kanazawa University NanoLSI.
The research described in this podcast was published in Nature Communications in October 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers fix the chirality of helical proteins
Researchers at Kanazawa University report in Nature Communications how they can control chirality inversion in α helical peptides.
The function of a protein is determined by its structure – prompting great interest in how to manipulate these structures. The structure is defined not just by the sequence of amino acids that make it, but the shape these acids make – the secondary structure – as well as how that shape is then folded. The most common secondary protein structure is the α-helix, which can coil to the right or left. This coiling direction in turn determines how it engages with other chiral structures, which may be the form of a light beam or another molecule. Although molecular components and environmental factors can favor a particular coiling direction over the other, helical molecules tend to flip between the two coil directions. Now Naoki Ousaka, Mark J. MacLachlan and Shigehisa Akine at Kanazawa University in Japan have shown how they can control and fix the coil direction.
Helical proteins are chiral molecules, which means that the molecule’s shape cannot be fitted into its mirror image. In nature helical proteins often have other chiral components, such as sugars or amino acids, and these will determine which way the protein coils. However, there is a lot of interest in synthesizing artificial helical proteins that have different chemical components and hence functions not found in nature, and these may not have other chiral components. Nonetheless having both types or “enantiomers” of the chiral molecule can be hazardous because of the significant differences in behavior between the two chiral forms, one of which may be benign or even therapeutic while the other is toxic. Hence, there is demand for other ways of selecting and fixing the chirality.
So how did they go about this?
Ousaka, MacLachlan and Akine synthesized α helical molecules solely from achiral components. They included bulky segments so that the molecule tended towards the larger rings of the α helical structure, as well as side chains of piperidine – molecular components that are common in pharmaceuticals. These side chains can be cross linked to “staple” the molecule into either the righthanded or lefthanded coil, inhibiting flipping between the two – chiral inversion. Finally they added another molecular component, known as an ester – the L-Val-OH residue. This would switch the direction of the coil in response to acidic or basic environments due to preferences in the interaction between oxygen atoms in the ester and the amino acid backbone.
The researchers used a range of chiral characterization methods including circular dichroism, nuclear magnetic resonance and liquid chromatography. They found that with the molecule stapled just once, it would slow down the flipping between enantiomers by a factor of 106, although this still occurred over minutes. Changing the solution to acid or alkali also successfully determined which enantiomer was favoured. However, stapling the molecule twice slowed down the chirality inversion by a factor of 1012, so that the molecular chirality was stable for years. This increased energy barrier to chirality inversion could then be overcome by heating the sample to very high temperatures to switch bet
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Genetic switches in tumor development
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Masanobu Oshima at the Kanazawa University NanoLSI.
The research described in this podcast was published in Cancer Research in November 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Genetic switches in tumor development
Researchers at Kanazawa University report in Cancer Research how Kras and p53 mutations influence the tumor suppressor and promoter functions of a TGF- ß pathway. The findings may lead to a new approach for colorectal cancer therapy.
Both the progression and the suppression of tumors are governed by biomolecular processes. Often, a particular process is involved in either cancer progression or suppression. Cancer treatment in the form of drugs then typically focuses on the respective deactivation or activation of the relevant biomolecular process. However, it has been established that a process known as transforming growth factor ß (TGF-ß) signaling*1 plays a role in both tumor suppression and progression. Now, Masanobu Oshima from Kanazawa University and colleagues have studied the precise genetic conditions underlying the outcome of TGF-ß signaling. Their findings may help the development of new therapeutic strategies for particular cancers.
The suppressive effect of TGF-ß signaling happens through the stimulation of cell differentiation — the process through which dividing cells acquire their type or function. The malignant progression of cancers, on the other hand, comes from a process called epithelial-mesenchymal transition (EMT), in which an epithelial cell transforms into a mesenchymal cell type. The former is a ‘stationary’ type of cell, found in epithelial tissue, whereas the latter is a more ‘migratory’ type of cell found in development and cancer.
So how did they investigate these processes and what did they found out?
Oshima and colleagues performed experiments with tumor-derived organoids. They confirmed that TGF-ß family cytokine, activin plays a role in tumor suppression and progression dependent on the mutation types of driver genes. In certain cancer cells treated with activin, the researchers noted that the partial EMT is induced with tumor aggressiveness and development. On the other hand, certain mutated activin receptors were found to have cancer suppressor capabilities, which made the scientists conclude that genetic alterations underlie the dual function of activins.
One of the two relevant genes is Kras which relays signals that regulate cell growth, division and differentiation. Oshima and colleagues found that a mutation of Kras blocks TGF-ß/activin-induced growth suppression. The other gene is known as Trp53, which encodes tumor protein 53, playing an important role in cancer regulation. A combination of Kras and Trp53 mutations at hot spots, known as gain-of-function mutation, was found to not just block tumor suppression but promote partial EMT and tumor proliferation.
The experiments were done with mouse intestinal tumor-derived organoids with defined genetic backgrounds, which makes the results relevant for therapeutic strategies for human colorectal cancer. Quoting the scientists: “Based on these results, the control of TGF- ß/activin signaling appears to be an important preventive and therapeutic strategy against the malignant progression of colorectal cancer carrying […] mutations”.
Reference
Dong Wang, Mizuho Nakayama, Chang Pyo Hong, Hiroko Oshima, and Masanobu Oshima. Gain-of-function p53 mutation acts as a genetic switch for T
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Researchers tune the speed of chirality switching
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Shigehisa Akine at the Kanazawa University NanoLSI.
The research described in this podcast was published in Science Advances in November 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers tune the speed of chirality switching
Researchers at Kanazawa University report in Science Advances how they can accelerate and decelerate chirality inversion in large cage molecules using alkali metal ion binding.
Chiral molecules can have dramatically different functional properties while sharing identical chemical formulae and almost identical structures. The molecular structure of two types of a chiral molecule – so-called enantiomers – are mirror images of each other where one cannot be superposed on the other any more than your right hand can fit front-to-back on the left. While a lot of chiral molecules are traditionally considered fixed as left- or right-handed, chiral molecules based on helices are known to be able to switch in response to changes in their environment. Now researchers led by Shigehisa Akine at Kanazawa University have demonstrated how environmental changes can also accelerate or decelerate this chiral inversion process, providing “a novel time-programmable switchable system”.
The researchers focused their study on “metallocryptand (R6)-LNi3”, an organic molecule featuring metal atoms in a cage-like molecular structure that can exist in one of two possible forms described as the P or M type (right- and left-handed, respectively). In its pure form (R6)-LNi3 has a preferred ratio of P type to M type of 12:88. Starting from a 50:50 ratio, the molecules will flip between one form and the other with a preference for flipping towards the M type to meet that ratio. The researchers measured this change in ratio using NMR and circular dichroic spectroscopy. However, add an alkali metal into the cage cavity and this preference can change.
By adding alkali metal ions to the solution of the (R6)-LNi3 the researchers could confirm that the metal ions readily bound to the metallocryptand from the changes in the spectroscopic signatures of the molecules. In addition, the bound ion also shifted the preferred ratio by a margin and with a speed that depended on which alkali metal was used.
So what is causing this effect?
The researchers attribute the different rates and ratios to differences in binding constants not just between the metal ion and the two forms of the molecule but also a virtual binding constant for the molecule transitioning between the two. The binding between a caesium ion and the P type molecule was more than 20 times greater than that with the M type so the solution eventually switched to a higher proportion of the P type with a P:M ratio of 75:25 over the course of 21 hours. The final ratio with a rubidium ion was similarly bias to the P type reaching a slightly lower ratio of 72:28 but in just 100 minutes. With potassium ion the equilibrium ratio was lower again at 68:32 but reached within just a minute, three orders of magnitude faster than for the caesium ion. The researchers attribute this speed to the large virtual bonding constant with the transitioning molecule.
With smaller ions – lithium and sodium ions – the preferred molecular type did not actually change but the final ratio was reached much faster. It is the first time researchers have demonstrated that such chiral inversion can be sped up and slowed down by tuning the molecules environment.
“This research can provide a new insight into the development of an on-de
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Researchers identify the dynamic behavior of a key SARS-CoV-2 accessory protein
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Richard Wong at Kanazawa University alongside Noritaka Nishida at Chiba University.
The research described in this podcast was published in the Journal of Physical Chemistry Letters in September 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers identify the dynamic behavior of a key SARS-CoV-2 accessory protein
Researchers at Kanazawa University report in the Journal of Physical Chemistry Letters high-speed atomic force microscopy studies that shed light on the possible role of the open reading frame 6 (or, ORF6) protein in COVID19 symptoms.
While many countries across the world are experiencing a reprieve from the intense spread of SARS-CoV-2 infections that led to tragic levels of sickness and multiple national lockdowns at the start of the decade, cases of infection persist. A better understanding of the mechanisms that sustain the virus in the body could help find more effective treatments against sickness caused by the disease, as well as arming against future outbreaks of similar infections. With this in mind there has been a lot of interest in the accessory proteins that the virus produces to help it thrive in the body.
“Similar to other viruses, SARS-CoV-2 expresses an array of accessory proteins to re-program the host environment to favor its replication and survival,” explain Richard Wong at Kanazawa University and Noritaka Nishida at Chiba University and their colleagues in this latest report. Among those accessory proteins is ORF6. Previous studies have suggested that ORF6 potently interferes with the function of interferon 1 (that is, IFN-I), a particular type of small protein used in the immune system, which may explain the instances of asymptomatic infection with SARS-CoV2. There is also evidence that ORF6 causes the retention of certain proteins in the cytoplasm while disrupting mRNA transport from the cell, which may be means for inhibiting IFN-I signalling. However, the mechanism for this protein retention and transport disruption was not clear.
So how did they figure it out?
Well, to shed light on these mechanisms the researchers first looked into what clues various software programs might give as to the structure of ORF6. These indicated the likely presence of several intrinsically disordered regions. Nuclear magnetic resonance measurements also confirmed the presence of a very flexible disordered segment. Although the machine learning algorithm AlphaFold2 has proved very useful for determining how proteins fold, the presence of these intrinsically disordered regions limits its use for establishing the structure of ORF6 so the researchers used high-speed atomic force microscopy (or AFM), which is able to identify structures by “feeling” the topography of samples like a record player needle feels the grooves in vinyl.
Using high speed AFM the researchers established that ORF 6 is primarily in the form of ellipsoidal filaments of oligomers – strings of repeating molecular units but shorter than polymers. The length and circumference of these filaments was greatest at 37 °C and least at 4 °C, so the presence of fever could be beneficial for producing larger filaments. Substrates made of lipids – fatty compounds – also encouraged the formation of larger oligomers. Because high speed AFM captures images so quickly it was possible to grasp not just the structures but also some of the dynamics of the ORF6 behavior, including circular motion, protein assembly and flipping. In addition, further computer anal
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Researchers define a nanopipette fabrication protocol for high resolution cell imaging
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Yasufumi Takahashi at the Kanazawa University NanoLSI.
The research described in this podcast was published in Analytical Chemistry in August 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Researchers define a nanopipette fabrication protocol for high resolution cell imaging
Researchers at Kanazawa University report in Analytical Chemistry how to produce nanopipettes that reliably provide nanoscale resolution scanning ion conductance microscopy images of living cells.
A nanoscale view of living cells can provide valuable insights into cell structure and function. Over the years, various microscopy techniques have been enrolled to obtain a window into biological specimens at the nanoscale but all with their limitations and challenges. Although scanning ion conductance microscopy has demonstrated the capability to image living biological samples in solution with nanoscale resolution, it has been hampered by challenges in reliably producing nanopipettes with the optimum geometry for the job. Now researchers led by Yasufumi Takahashi at Kanazawa University’s Nano LSI and Nagoya University have devised a protocol for reproducibly fabricating nanopipettes with the preferred geometry for high quality imaging.
So what is scanning ion conductance microscopy and what kind of nanopipette does it need?
Scanning ion conductance microscopy uses a nanopipette to control the distance between nanopipette and sample using an ion current as feedback signal. The shape of the nanopipette significantly influences the performance of the device. For instance, a wide aperture limits the possible resolution, a long shunt can lead to rectification effects that warp the ion current measurements, and if the glass of the nanopipette is too thick it can deform the sample before the proximity of the aperture has reached the point needed for constant ion current topographical mapping. As a result, the ideal nanopipette has a short shunt, small aperture and thin glass walls.
The standard procedure for fabricating the nanopipette is to pull a capillary tube with a laser puller that heats the capillary tube it is manipulating. The capillary then narrows where it lengthens until it is finally drawn into two separate pieces. Although quartz can allow a little more control in the process of drawing the capillary tube into shape it is hydrophobic, which raises complications in actually filling the nanopipette with the aqueous solution needed for the ion current. For this reason, the researchers developed a protocol by which they could draw nanopipettes from borosilicate glass capillaries with the required control and reproducibility.
Takahashi and his collaborators noted that ideally the starting capillary should have thick walls and a narrow inner diameter, however it is not easy to obtain capillary tubes to these requirements from commercial suppliers. Instead, they preheat the capillary for 5 s without pulling it, which causes the glass walls to the thicken and reduces the inner diameter. They also optimized the parameters for pulling the tube, such as the velocity.
So did it work? Apparently so
The researchers demonstrated the performance of the nanopipettes they produced by imaging a cell undergoing a type of endocytosis, where it engulfs and absorbs some external material. They were able to image the microvilli – that is, tiny cellular membrane protrusions – found on the cell surface, as well as the endocytic pits that form and the formation of a cap c
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Hydration matters: The interaction patterns of water and oxide crystals revealed
Transcript of this podcast
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Keisuke Miyazawa at the Kanazawa University NanoLSI.
The research described in this podcast was published in Nanoscale in July 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Hydration matters: The interaction patterns of water and oxide crystals revealed.
https://nanolsi.kanazawa-u.ac.jp/en/highlights/28428/
In a study recently published in the journal Nanoscale, researchers from Kanazawa University and AGC Inc. use three-dimensional atomic force microscopy to study the hydrated form and structure of commonly occurring oxide crystals.
While sapphire and quartz are oxide crystals used in a wide range of industrial applications, the atomic-scale structures of these materials are not well understood. The major chemical components of sapphire and quartz are aluminum oxide and silicon dioxide, respectively. These components have a high affinity for water, which affects the chemical reactivity of the crystals. Thus, a thorough knowledge of the water-binding properties of these oxides is important for further innovative applications.
To date, traditional microscopic methods have only provided insights into the two-dimensional topography of their surfaces. Now, a research team led by Keisuke Miyazawa from the NanoLSI at Kanazawa University has developed three-dimensional (3D) microscopy technique for a detailed study of the interaction of the surfaces of these materials with water.
So how did they do it?
The team started by looking at the surface structures and its hydration structures of sapphire and α-quartz in water. For this, they used an advanced form of microscopy known as 3D atomic force microscopy (3D-AFM). Oxide crystals usually have hydroxyl (OH) groups, which are the main “water-binding” molecules, closely linked with the oxides. Hence, the team studied the OH groups and its hydration structures on both crystals when immersed in water. They found that the hydration layer on sapphire was not uniform because of the nonuniform local distributions of the surface OH groups. On the other hand, the hydration layer on α-quartz was uniform because of the atomically flat distributions of the surface OH groups.
When the interaction force of these oxides with water was subsequently measured, it was found that a greater force was required to break the water-crystal bonds in sapphire than in α-quartz. Lastly, it was also discovered that this affinity was much higher in regions where the oxides were in close proximity to the OH groups.
This study showed that the hydration structures of oxides are dependent on the location and density of OH groups, in addition to the strength of the OH groups’ hydrogen bonding (the chemical bond used to bind to water). What’s more, it was successfully shown here that 3D-AFM can be used in unraveling the interaction of water with several surfaces, a potential avenue for understanding solid-liquid interactions better. “This study contributes to the application of 3D-AFM in exploring atomic scale hydration structures on various surfaces, and hence, to a wide range of solid–liquid interfacial research fields,” conclude the researchers.
Reference
Sho Nagai, Shingo Urata, Kent Suga, Takeshi Fukuma, Yasuo Hayashi and Keisuke Miyazawa. Three-dimensional ordering of water molecules reflecting hydroxyl groups on sapphire (001) and α-quartz (100) surfaces
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Ion channel block unraveled
Transcript of this podcast
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takashi Sumikama at the Kanazawa University NanoLSI in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues.
The research described in this podcast was published in Nature Communications in July 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Ion channel block unraveled
Researchers at Kanazawa University report in Nature Communications how calcium ions can block sodium ion channels located in cell membranes. Structural analysis and computer simulations made it possible to identify where and why calcium ions get stuck.
Ion channels are structures within cell membranes that enable specific ions to travel to and from the cell. Such transfer is essential for a variety of physiological processes like muscle cell contraction and nerve excitation. In so-called tetrameric cation channels, the ion selectivity results from the unique structural and chemical environment of the part referred to as the selectivity filter, which is located between two intertwined helical structures. Tetrameric ion channels are prone to ‘divalent cation block’, the blocking of the channel by ions like calcium (as in Ca2+). Such blocking regulates the ionic current, which is involved in various neural activities such as memory formation. How divalent cation block happens exactly is still unclear at the moment — in particular, a direct observation of the cation actually blocking the ion pathway has not been reported yet. Now, Takashi Sumikama from Kanazawa University in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues has discovered the mechanism behind divalent cation block in NavAb, a well-known tetrameric sodium (Na) channel. Through structural analysis and computer simulations, the researchers were able to reveal the relevant structural features and molecular processes at play.
So how did they go about this structural analysis?
NavAb is a sodium channel cloned from a bacterium (Arcobacter butzleri) and has a well-known structure. Sumikama and Irie’s colleagues performed experiments with NavAb and three mutants. The structures of the mutants were determined for environments with and without calcium. The scientists focused on the differences in electron densities for the different structures, as these provide insights into the locations of the calcium ions. They found that for the mutants displaying calcium blocking, one or two calcium ions are located at the bottom of the selectivity filter. They also discovered that two other divalent cations — magnesium (as in Mg2+) and strontium (Sr2+) ions — blocked the calcium-blocking mutant sodium channels.
The researchers then performed computer simulations to obtain a detailed understanding of the interaction between the calcium ions and the mutated NavAb channels. The simulations reproduce the dynamics of ions passing — or not passing — the channel. In the absence of calcium ions, sodium ions were observed to penetrate the channel. In the presence of calcium ions, penetration significantly decreased in the calcium-blocking mutants. The simulations also confirmed that the blocking calcium ions are ‘stuck’ at the bottom of the selectivity filter, and revealed that this ‘sticking’ is related to the increased hydrophilicity (affinity to water) of relevant structural parts of the mutated channels.
The results of Sumikama and Irie’s colleagues provide an important step forward towards a full understanding of the mechanism of divalent cation block in NavAb, an important and representa
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Brain cancer linked to nuclear pore alterations
Transcript of this podcast
Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Masaharu Hazawa and Richard Wong at the Kanazawa University NanoLSI, alongside Mitsutoshi Nakada and colleagues at Kanazawa University.
The research described in this podcast was published in Cell Reports in August 2023
Kanazawa University NanoLSI website
https://nanolsi.kanazawa-u.ac.jp/en/
Brain cancer linked to nuclear pore alterations
Researchers at Kanazawa University report in Cell Reports how alterations in nuclear pores lead to the degradation of anti-tumor proteins.
Several types of cancer are believed to be linked to alterations of macromolecular structures known as nuclear pore complexes. These structures are embedded in the nuclear envelope, a membrane barrier that separates the nucleus of a cell from the cytoplasm (the liquid filling the rest of the cell). They consist of proteins called nucleoporins, which regulate the transport of molecules across the nuclear envelope, including enzymes that enable the synthesis of DNA. Whether nuclear pore complex alterations play a role in glioblastoma, the most common type of cancer originating in the brain, is unclear at the moment. Now, Masaharu Hazawa, Mitsutoshi Nakada and Richard Wong from Kanazawa University and colleagues have found a link between the functioning of nuclear pore complexes and glioblastoma — specifically, they demonstrated the inactivation of a tumor-suppressing protein called p53.
The protein p53 is crucial in cancer prevention. The corresponding gene TP53 encodes proteins that prevent mutations of the genome and is the most frequently mutated gene in human cancers. Gaining insights into how p53 inactivation happens is crucial for understanding tumorigenesis in general and glioblastoma in particular.
So how did the researchers go about it?
Mitsutoshi Nakada and Richard Wong and colleagues first checked whether any nuclear pore complex proteins were amplified (that is ‘overexpressed’) in glioblastoma. They found that one such protein, called NUP107, showed overexpression. Further investigations revealed that NUP107 is a potential oncoprotein in glioblastoma; its overexpression degrades the function of the cancer-suppressing p53 protein. They also found that MDM2, another protein, is overexpressed simultaneously with NUP107. MDM2 is also known to mediate p53 protein degradation.
Further studies will be necessary to uncover the full molecular pathways at play, but the scientists speculate that the increased amount of NUP107 proteins in the nuclear pore complexes of glioblastoma cells results in nuclear pore complex structures that regulate the transport of molecules from the nucleus to the cytoplasm in a way that promotes p53 degradation. This scenario is referred to as nuclear transport surveillance. Experiments in which NUP107 proteins were depleted re-activated p53, consistent with NUP107 providing the structural stability of glioblastoma NPCs.
The findings of Mitsutoshi Nakada and Richard Wong and colleagues confirm that alterations of nuclear pore complexes contribute to the pathogenesis of glioblastoma. As Mitsutoshi Nakada and Richard Wong put it : “Together, our findings establish roles of nuclear pore complexes in transport surveillance and provide insights into p53 inactivation in glioblastoma.”
Reference
Dini Kurnia Ikliptikawati, Nozomi Hirai, Kei Makiyama, Hemragul Sabit, Masashi Kinoshita, Koki Matsumoto, Keesiang Lim, Makiko Meguro-Horike, Shin-ichi Horike, Masaharu Hazawa, Mitsutoshi Nakada, and Richard&
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