Kanazawa University

KANAZAWA, Japan, Oct. 3, 2024 /PRNewswire/ -- Researchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa Universit y, demonstrate how morphogens combined with cell adhesion can generate tissue domains with a sharp boundar y in an in vitro model system. Figure 1 Caption Figure 1: How does the morphogen gradient regulate multicellular patterning? Recent advances that have enabled the growth of tissue cultures into organoids and embryoids have heightened interest as to how tissue growth is controlled during the natural processes of embryo development. It is known that the diffusion of signaling molecules called morphogens directs patterned tissue growth but what has been harder to understand is how the gradient of morphogens from this diffusion can lead to sharply defined domains in the resulting tissue (Fig. 1). Now Satoshi Toda at Kanazawa University NanoLSI (currently Osaka University, Institute for Protein Research), alongside Kosuke Mizuno at NanoLSI and Tsuyoshi Hirashima at the National University of Singapore, demonstrate a simple model system – SYnthetic Morphogen system for Pattern Logic Exploration using 3D spheroids (SYMPLE3D) – that sheds light on the process. Various previous studies have looked at the role of morphogens and cell adhesion during tissue growth separately. However, the researchers noted a couple of recent studies indicating how a morphogen involved in neural tube patterning controls expression of a family of adhesion proteins called cadherins to form sharply defined structures. Prompted by these insights, they devised their model system to investigate the interplay between morphogens and cadherins. They highlight how in vivo morphogens induce numerous changes in cellular properties simultaneously, making it hard to disentangle what is going on. For this reason, as they highlight in the discussion of their report, "SYMPLE3D provides a new synthetic biology approach for mechanistically studying tissue patterning and engineering organoid structures." SYMPLE3D uses two types of cells – one, the GFP secretors, which secrete GFP and express P-cadherin forming what they describe as "GFP-secreting organizer spheroids". The other is a GFP receiver cell, initially engineered to express a synthetic receptor called "synNotch" that recognizes GFP and induces mCherry reporter – "imC cells" (Fig. 2A). The first stage looked at the result of co-culturing the GFP secretors and receiver cells. They found that although the imC cells did capture the secreted GFP resulting in a GFP gradient, the resulting gradient contained ectopically active cells – expression of the high-level mCherry reporter in an inappropriate position of the gradient (Fig. 2B, upper panel). To deal with the issue of ectopically active cells, Mizuno and Toda engineered GFP receiver cells to induce mCherry-fused E-cadherin, a cell adhesion molecule. To their surprise, a uniformly activated tissue domain with sharp boundary emerged instead of a gradient between the secretor and receiver cells (Fig. 2B, lower panel). Figure 2: Caption Figure 2. Tissue domain formation through the coupling of morphogen signals and cell adhesion. (A) SYnthetic Morphogen system for Pattern Logic Exploration using 3D spheroids (SYMPLE3D). (B) mCherry (upper panel) or Ecad-mCherry (lower panel) was induced in response to the GFP gradient. The sharp boundary was also robust to changes in growth conditions. Since the addition of a single factor, E-cadherin, caused a significant change in the pattern, they then focused on the mechanism of the pattern formation process with a combination of molecular gradient and E-cadherin in their model system. By monitoring the real time process of tissue growth, they were able to identify activated GFP receiver cells engineered to induce mCherry-fused E-cadherin were initially scattered but aggregated over the course of time. Ectopically active cells were then gradually absorbed into this active domain resulting in a sharp cut off between the mCherry positive and negative domains. They also note "an intriguing aspect" of their synthetic tissue domain, in that across the active domain the distribution of induced E-cadherin-mCherry was uniformly high, whereas GFP was distributed with a gradient. Here, they revealed a key feature of E-cadherin for the synthetic tissue domain formation. They analyzed the behavior of cells that express various levels of E-cadherin in response to different amounts of GFP and found that the behavior was the same whether the cells induced low or high levels of E-cadherin. Furthermore, they showed that cells that induced more than a certain amount of E-cadherin were able to mix with each other and form a single cell population, regardless of the expression level. Therefore, the mixing of cells that induced different levels of E-cadherin within the GFP gradient allowed the cells to receive GFP uniformly and thus the expression level of E-cadherin became evenly high in the synthetic tissue domain (Fig. 3). A simple mathematical model, developed by Hirashima, based on cell movement governed by differential adhesion energy supported their experimental observations. "Our findings suggest the possibility of programming a new tissue domain with sharp boundaries in organoids by combining synthetic morphogens with cell adhesion control," they conclude in their report. Figure 3: Caption Figure 3. The pattern formation mechanism where morphogen signals and cadherin expression cooperate to generate tissue domains with a sharp boundary. Note about the contributions of Kosuke Mizuno to research at the NanoLSI Kosuke Mizuno, a second-year doctoral student in the Nano Life Science Program, has made notable contributions to NanoLSI research. He was awarded the NanoLSI Transdisciplinary Research Grant for FY2023 and FY2024. Glossary Morphogen A signaling molecule that controls cell fate decision dependently on its local concentration to regulate morphogenesis. Morphogens are secreted from source cells, diffuse within tissues and form a concentration gradient that works as positional information for cell differentiation. Representative morphogens include Wnt, BMP, Shh, and retinoic acid. Cadherins Cadherins are a family of transmembrane proteins that facilitate cell to cell adhesion. Mizuno, Hirashima and Toda use E-cadherin and P-cadherin in the current study, which belong to the classical cadherin family. Cadherins primarily bind to each other via their homophilic extracellular domains. However, cadherins also have an intracellular domain, which binds to adaptor proteins connected to actin cytoskeletons and contributes to tissue compaction and cell sorting. Synthetic Notch receptor (synNotch) Notch is a signal transduction receptor. When the extracellular domain of Notch receptor binds to its ligand Delta, Notch transmembrane region is cleaved, leading to the translocation of Notch intracellular domain into the nucleus to regulate the expression of target genes. The synNotch receptor is a modified version of Notch receptor, in which the extracellular domain of Notch is replaced by an antibody or nanobody and the intracellular domain by an artificial transcription factor. Using synNotch receptors, researchers can design what ligand molecule cells recognize and what target genes cells express in response, which enables manipulation of cell-cell communications. Reference Kosuke Mizuno, Tsuyoshi Hirashima, Satoshi Toda. Robust tissue pattern formation by coupling morphogen signal and cell adhesion EMBO Reports. 2024. DOI：10.1038/s44319-024-00261-z URL： Funding This research was supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan, Japan Science and Technology Agency (JST) PRESTO (JPMJPR2147), Grant-in-Aid for Scientific Research (20K15828, 21H05291, 21H05290), Japan Agency for Medical Research and Development (AMED) (22bm0704048h0003), Senri Life Science Foundation, Kato Memorial Bioscience Foundation, Kao Foundation, Yoshida Scholarship Foundation, and the Mechanobiology Institute, National University of Singapore. Contact Fujiko Imanaga (Ms) Project Planning and Outreach NanoLSI Administration Office, Nano Life Science Institute (WPI-NanoLSI) Kanazawa University Kakuma-machi, Kanazawa 920-1192, Japan Email: [email protected] Tel: +81 (76) 234-4555 About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University Understanding nanoscale mechanisms of life phenomena by exploring "uncharted nano-realms". Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale. About the World Premier International Research Center Initiative (WPI) The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS). See the latest research news from the centers at the WPI News Portal: Main WPI program site: About Kanazawa University As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities. The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas. SOURCE Kanazawa University WANT YOUR COMPANY'S NEWS FEATURED ON PRNEWSWIRE.COM? 440k+ Newsrooms & Influencers 9k+ Digital Media Outlets 270k+ Journalists Opted In GET STARTED

KANAZAWA, Japan, Sept. 6, 2024 /PRNewswire/ -- Researchers at the Nano Life Science Institute (WPI-NanoLSI) , Kanazawa University , develop a biosensor that improves sensitivity to 1-methylnicotinamide (1-MNA) in urine by orders of magnitude without the need for sample purification. Metabolites are a common indicator of disease. In particular 1-MNA levels are increased by cancer, liver disease, obesity and metabolic disease. At present, mass spectrometry and nuclear magnetic resonance measurements are commonly used to measure quantities of metabolites in samples but they are expensive and complicated limiting their widespread use. Instead Masaya Ueno, Tomoki Ogoshi and Atsushi Hirao at Kanazawa WPI-NanoLSI use a type of pillararene molecule (Figs. 1 and 2) as a biosensor, getting around the need for high purification of other 1-MNA biosensing molecules while improving sensitivity and specificity. 1-MNA is produced in the body through the methylation of nicotinamide (Nam) by nicotinamide N-methyltransferase (NNMT) during the metabolism of the vitamin B3 vitamer niacin. As such it provides a measure of NNMT activity, which is elevated in some cancers. There is evidence that 1-MNA levels correlate with the how aggressive the tumour is and suppressing the gene for NNMT diminished certain behavioral symptoms of disease. As such the researchers explain in their report "monitoring the NNMT expression and activity in patients by quantification of 1-MNA is important for elucidating and diagnosing their pathology." Previously they had demonstrated some potential for the molecule pillar[6]arene functionalized with 12 carboxylate anions (P6AC) as a 1-MNA sensor, since it binds to 1-MNA and inhibits fluorescence due to photo-induced electron transfer. However extensive sample purification was needed and even then, although the millimolar concentrations in murine urine could be detected, the P6AC biosensor lacked the sensitivity to detect the micromolar concentrations found in culture supernatants of human cancer cells. To find a biosensor with better sensitivity, the researchers investigated the binding between 1-MNA and pillar[6]arene functionalized with sulfonate groups (P6AS). They found that the binding affinity was 700 times greater than for P6AC, leading to a biosensor with sub micromolar sensitivity, even in unpurified human urine, although detection of the higher concentrations in mouse murine was better. However the researchers noted that detection in human serum was not possible due to the higher levels of autofluorescence. By comparison the detection sensitivity possible with mass spectrometry is nanomolar but the throughput is much lower. The researchers suggest that the high throughput of the P6AS biosensor could make it suitable for screening 1000s of potential NNMT inhibitors, which may help towards a treatment for diseases like liver disease and cancer. The researchers explain the higher sensitivity thanks to the stronger acidity of sulfonate groups compared to carboxylate groups. They conclude their report of the work, "Further improvement of our strategy will contribute to high-throughput screening of NNMT inhibitors, diagnosis of liver diseases, and imaging of human cancer cells in vivo." Related figures Fig. 1 Fig. 1 Caption: Chemical structure of Pillar[n]arene and 1-methylnicotinamide (1-MNA). Copyright 2024 Masaya Ueno Fig. 2 Fig. 2 Caption: Pillar[6]arene with 12 sulfonate groups (P6AS) specifically forms complexes with 1-methylnicotinamide (1-MNA)-a niacin metabolite-and works as a biosensor even in unpurified biological samples. Copyright 2024 Masaya Ueno Glossary Pillar[n]arenes The pillar[n]arenes take their names from their pillar shapes and macrocyclic structures. The researchers were prompted to investigate the potential of P[6]AS as a 1-MNA detector following previous work by another group demonstrating the high binding affinity of P[n]AS for ammonium ions. Binding affinity The binding affinity refers to the strength of bond between the ligands on the protein and the candidate molecule, typically through intermolecular interactions such as ionic or hydrogen bonding or van der waals forces. The is a higher binding affinity when the attractive forces are higher. Mass spectrometry Mass spectrometers identify molecules based on their mass. The molecule is ionized and then accelerated through a field. The trajectory the molecule takes gives an indication of the charge mass ratio of the ion. Reference Masaya Ueno, Hiroki Sugiyama, Feng Li, Tatsuya Nishimura, Hiroshi Arakawa, Xi Chen, Xiaoxiao Cheng, Shinji Takeuchi, Yumie Takeshita, Toshinari Takamura, Sakae Miyagi, Tadashi Toyama, Tomoyoshi Soga, Yusuke Masuo, Yukio Kato, Hiroyuki Nakamura, Hiromasa Tsujiguchi, Akinori Hara, Atsushi Tajima, Moeko Noguchi-Shinohara, Kenjiro Ono, Kenta Kurayoshi, Masahiko Kobayashi, Yuko Tadokoro, Atsuko Kasahara, Mahmoud I. Shoulkamy, Katsuhiro Maeda, Tomoki Ogoshi, Atsushi Hirao A supramolecular biosensor for rapid and high-throughput quantification of a disease-associated niacin metabolite Analytical Chemistry 2024. DOI：10.1021/acs.analchem.4c02653 URL: Funding acknowledgments M.U. was supported by a Grant-in-Aid for Scientific Research (C) (23K06672) from the Ministry of Education, Culture, Sports, Science, and Technology, (MEXT), Japan. A.H. was supported by a Grant-in-Aid for Scientific Research (A) (19H01033) from MEXT and a Project for Cancer Research and Therapeutic Evolution (P-CREATE) (19cm0106104h0004) from the Japan Agency for Medical Research and Development (AMED). T.O. was supported by the CREST program of the Japan Science and Technology (JST) agency (JPMJCR18R3) and a Grant-in-Aid for Scientific Research (A) (22H00334) from MEXT. This work was supported by a WPI-NanoLSI Transdisciplinary Research Grant from Kanazawa University. NanoLSI is supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan. This work was supported by WISE Program for Nano-Precision Medicine, Science, and Technology of Kanazawa University by MEXT. This work was also supported by MEXT Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence (CURE) Program (JPMXP1323015484). Contact: Kimie Nishimura (Ms) Project Planning and Outreach NanoLSI Administration Office, Nano Life Science Institute (WPI-NanoLSI) Kanazawa University Kakuma-machi, Kanazawa 920-1192, Japan Email: [email protected] Tel: +81 (76) 234-4555 About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University Understanding nanoscale mechanisms of life phenomena by exploring "uncharted nano-realms". Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale. About the World Premier International Research Center Initiative (WPI) The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS). See the latest research news from the centers at the WPI News Portal: Main WPI program site: About Kanazawa University As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities. The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas. SOURCE Kanazawa University

KANAZAWA, Japan, Aug. 26, 2024 /PRNewswire/ -- Researchers at the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, used high-speed atomic force microscopy to observe dynamic changes in AMPA receptors, which are vital for brain communication. Their findings, published in ACS Nano, reveal how these receptors adapt during signal transmission and suggest potential targets for neurological therapies. This study, led by Mikihiro Shibata, delves into the complex behavior of AMPA receptors (AMPARs), which are vital for communication between nerve cells in the brain. AMPARs are responsible for fast excitatory neurotransmission, a process crucial for learning, memory, and overall cognitive function. The research particularly focuses on the GluA2 subunit of AMPARs, a key component in transmitting signals at synapses, the junctions where neurons connect. The team employed an advanced imaging technique known as high-speed atomic force microscopy (HS-AFM) to observe the real-time behavior of the N-terminal domain (NTD) in the GluA2 subunit. The NTD is the starting segment of the protein, playing a critical role in how AMPARs function and cluster at synapses. The study also examined how the GluA2 subunit interacts with TARP γ2, a regulatory protein that fine-tunes the receptor's response to signals. One of the key findings is the behavior of the NTD in different states: resting, activated, and desensitized. The researchers discovered that in the activated state, the NTD dimers—pairs of NTDs—can split into single units or monomers. This process, known as subunit exchange, allows parts of one receptor to s another, potentially altering the receptor's function. This novel observation was supported by molecular dynamics simulations, which showed that these monomeric states are stable in their lipid environment, providing a potential mechanism for receptor adaptability and diversity. In the desensitized state, where the receptor becomes less responsive to signals, the NTD dimers separate, but their movement is more restricted compared to the activated state. This desensitization helps protect nerve cells from overstimulation, which could lead to cellular damage. The study's insights into the structural changes of the NTDs in different functional states highlight the dynamic nature of AMPARs and their ability to adapt to various conditions within the synaptic environment. The research also sheds light on the role of neuronal pentraxin 1 (NP1), a protein that aids in the clustering of AMPARs at synapses. NP1 forms a ring-shaped structure that binds to the tips of the NTDs, potentially facilitating the gathering of multiple AMPARs into clusters. This clustering is essential for efficient synaptic transmission, as it brings receptors closer together, allowing for more effective signaling between neurons. By linking multiple receptors, NP1 enhances the strength and reliability of the synaptic connection, contributing to the overall efficiency of neural communication. The study's findings contribute significantly to our understanding of how AMPARs function and adapt during neurotransmission. By revealing the dynamic structural changes in the NTDs and highlighting the role of NP1 in receptor clustering, the research offers new insights into the molecular processes that underlie synaptic plasticity—the ability of synapses to strengthen or weaken over time, which is essential for learning and memory. These discoveries could have important implications for developing treatments for neurological disorders where AMPAR function is disrupted, such as in epilepsy, Alzheimer's disease, and other cognitive impairments. As the authors conclude, "Our research reveals the dynamic structural changes that occur within AMPA receptors, underscoring their remarkable adaptability. Understanding these mechanisms not only deepens our knowledge of brain function but also opens new avenues for therapeutic interventions targeting synaptic transmission and plasticity." Glossary AMPA Receptors (AMPARs) Proteins on the surface of nerve cells essential for communication between cells in the brain. They respond to signals by allowing charged particles to enter the cell, triggering various processes like learning and memory formation. N-terminal domain (NTD) The beginning part of a protein that influences how the protein behaves and interacts with other molecules. For AMPARs, the NTD helps them cluster together at nerve cell connections. Synapse The point where two nerve cells connect and communicate, allowing signals to pass from one cell to another. TARP γ2 A regulatory protein that interacts with AMPARs to control their response to signals. High-speed atomic force microscopy (HS-AFM) This imaging technique uses a nanosized tip at the end of a cantilever that is scanned over a sample. It can be used to determine the topography of a sample surface from the change in the strength of forces between the tip and the sample with distance, and the resulting deflection of the cantilever. It was first developed in the 1980s but a number of modifications have augmented the functionality of the technique since. It is better suited to imaging biological samples than the scanning tunnelling microscope developed that had been developed because it does not require a conducting sample. In the 2000s Toshio Ando at Kanazawa University was able to improve the scanning speed to such an extent that moving images could be captured. This allowed people to use the technique to visualize molecular processes for the first time. Desensitized state A condition where the receptor becomes less responsive to signals, often to prevent overstimulation. Neuronal pentraxin 1 (NP1) A protein that clusters AMPARs at synapses, improving their signaling ability. Subunit exchange The process where parts of a protein (subunits) s receptors, potentially altering their function. Related figure and caption High-speed AFM directly visualized the real-space and real-time structural dynamics of AMPARs in the presence of ligands in a liquid environment. © 2024 Sumino, et al., ACS Nano Reference Ayumi Sumino, Takashi Sumikama, Yimeng Zhao, Holger Flechsig, Kenichi Umeda, Noriyuki Kodera, Hiroki Konno, Motoyuki Hattori, and Mikihiro Shibata, High-speed atomic force microscopy reveals dynamic dimer splitting of the N-terminal domain of GluA2 ionotropic glutamate receptor-auxiliary subunit complex, Published online 23 August 2024, ACS Nano 2024. DOI: 10.1021/acsnano.4c06295 URL: Funding acknowledgements This research was supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan (to M.S.), JSPS KAKENHI grant numbers JP20K21122 (to M.S.) and JP21H01771 (to M.S.), JSPS Bilateral Program Number JP120247404 (to M.S.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to M.S.), the Uehara Memorial Foundation (to M.S.), the National Natural Science Foundation of China (32071234, 32271244, 32250610205, and 32411540020 to M.H.), an innovative research team of high-level local universities in Shanghai and a key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005 to M.H.), and JST/CREST (JPMJCR1762 to N.K. and H.F.). Contact Kimie Nishimura (Ms) Project Planning and Outreach NanoLSI Administration Office, Nano Life Science Institute (WPI-NanoLSI) Kanazawa University Kakuma-machi, Kanazawa 920-1192, Japan Email: [email protected] Tel: +81 (76) 234-4555 About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University Understanding nanoscale mechanisms of life phenomena by exploring "uncharted nano-realms". Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale. About the World Premier International Research Center Initiative (WPI) The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS). See the latest research news from the centers at the WPI News Portal: Main WPI program site: About Kanazawa University As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities. The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas. SOURCE Kanazawa University