ORCID Profile
0000-0002-4575-6501
Current Organisation
Tokyo Daigaku
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Publisher: Springer Science and Business Media LLC
Date: 16-02-2022
DOI: 10.1007/S00249-021-01587-5
Abstract: Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand–receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability.
Publisher: Springer Science and Business Media LLC
Date: 03-2022
DOI: 10.1007/S00249-022-01595-Z
Abstract: Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca 2+ ) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca 2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca 2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (− 2.94 to 0 mmH 2 O) and a pneumatic high-speed pressure cl (− 25 to 0 mmHg). To visualize Ca 2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca 2+ levels immediately after aspirating in idual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic cl showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic cl as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.
Publisher: MyJove Corporation
Date: 20-11-2021
DOI: 10.3791/62490
Publisher: American Society of Hematology
Date: 11-05-2023
DOI: 10.1182/BLOODADVANCES.2022008650
Abstract: von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds. From differential cysteine alkylation and mass spectrometry experiments, 13 VWF disulfide bonds at the FVIII binding site were found to exist in formed and unformed states, and binding of FVIII results in partial formation of 12 of the VWF bonds. Force spectroscopy studies indicate that the VWF-FVIII bond stiffens in response to force and this feature of the interaction is ablated when VWF disulfide bonds are prevented from forming, resulting in slip-only bond behavior. Exposure of VWF to pathological fluid shear forces ex vivo and in vivo causes partial cleavage of all 13 disulfide bonds, further supporting their malleable nature. These findings demonstrate that FVIII binding to VWF involves dynamic changes in the covalent states of several VWF disulfides that are required for productive interaction in physiological shear forces.
Publisher: Research Square Platform LLC
Date: 07-08-2023
DOI: 10.21203/RS.3.RS-3218836/V1
Abstract: Microgeometry profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains enigmatic. We introduce a fluorescent micropipette aspiration assay to concurrently observe intracellular calcium mobilization and cytoskeletal restructuring in real-time under distinct microscale geometric alterations. Utilizing finite element analyses, coupled with PIEZO1-specific transgenic models, we discern that steeper micropipette tip angles markedly enhance PIEZO1-mediated calcium influx. This mechanical stress on the aspirated cell induces a significant F-actin reorganization, resulting in a "mechanical buckle" that lifies PIEZO1 activity within the aspirated region. Remarkably, when this F-actin network is disrupted, PIEZO1 gating is significantly inhibited, signifying its indispensable role in mechanosensing under geometrical changes. This mechanobiology study illuminates the profound relationship between biomechanical microenvironment, cytoskeletal adaptation, and PIEZO1 activation, inspiring future bioengineering applications.
Publisher: Frontiers Media SA
Date: 15-03-2022
Abstract: Cells sense and respond to mechanical stimuli for activation, proliferation, migration, and differentiation. The associated mechanosensing and biomechanical properties of cells and tissues are significantly implicated in the context of cancer, fibrosis, dementia, and cardiovascular diseases. To gain more mechanobiology insights, dynamic force spectroscopies (DFSs), particularly optical tweezers (OT), have been further advanced to enable in situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside of a cell. In this review, we first explain the classic OT-DFS rationales and discuss their applications to protein biophysics, extracellular biomechanics, and receptor-mediated cell mechanosensing. As a non-invasive technique, optical tweezers’s unique advantages in probing cytoplasmic protein behaviors and manipulating organelles inside living cells have been increasingly explored in recent years. Hereby, we then introduce and highlight the emerging OT rationales for intracellular force measurement including refractive index matching, active–passive calibration, and change of light momentum. These new approaches enable intracellular OT-DFS and mechanical measurements with respect to intracellular motor stepping, cytosolic micro-rheology, and biomechanics of irregularly shaped nuclei and vesicles. Last but not least, we foresee future OT upgrades with respect to overcoming phototoxicity and system drifting for longer duration live-cell measurements multimodal integration with advanced imaging and nanotechnology to obtain higher spatiotemporal resolution and developing simultaneous, automated, and artificial intelligence–inspired multi-trap systems to achieve high throughput. These further developments will grant unprecedented accessibility of OT-DFS and force measurement nanotools to a wider biomedical research community, ultimately opening the floodgates for exciting live-cell mechanobiology and novel therapeutic discoveries.
Publisher: Institute of Electrical and Electronics Engineers (IEEE)
Date: 2023
Publisher: Wiley
Date: 11-07-2023
DOI: 10.1002/EXP.20230004
Abstract: Mechanical forces play a vital role in biological processes at molecular and cellular levels, significantly impacting various diseases such as cancer, cardiovascular disease, and COVID‐19. Recent advancements in dynamic force spectroscopy (DFS) techniques have enabled the application and measurement of forces and displacements with high resolutions, providing crucial insights into the mechanical pathways underlying these diseases. Among DFS techniques, the biomembrane force probe (BFP) stands out for its ability to measure bond kinetics and cellular mechanosensing with pico‐newton and nano‐meter resolutions. Here, a comprehensive overview of the classical BFP‐DFS setup is presented and key advancements are emphasized, including the development of dual biomembrane force probe (dBFP) and fluorescence biomembrane force probe (fBFP). BFP‐DFS allows us to investigate dynamic bond behaviors on living cells and significantly enhances the understanding of specific ligand‐receptor axes mediated cell mechanosensing. The contributions of BFP‐DFS to the fields of cancer biology, thrombosis, and inflammation are delved into, exploring its potential to elucidate novel therapeutic discoveries. Furthermore, future BFP upgrades aimed at improving output and feasibility are anticipated, emphasizing its growing importance in the field of cell mechanobiology. Although BFP‐DFS remains a niche research modality, its impact on the expanding field of cell mechanobiology is immense.
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2CB00010E
Abstract: The N-AIM of VWF-A1 forms a Rotini-like structure, therefore partially autoinhibit VWF-A1–GPIbα interaction. The N-AIM acts as a defending sword to protect and stabilize the VWF-A1 structure under harsh environments.
Publisher: MDPI AG
Date: 06-01-2023
DOI: 10.3390/BIOS13010095
Abstract: Cancer cells interacting with the extracellular matrix (ECM) in the tumor microenvironment is pivotal for tumorigenesis, invasion, and metastasis. Cell–ECM adhesion has been intensively studied in cancer biology in the past decades to understand the molecular mechanisms underlying the adhesion events and extracellular mechanosensing, as well as develop therapeutic strategies targeting the cell adhesion molecules. Many methods have been established to measure the cell–ECM adhesion strength and correlate it with the metastatic potential of certain cancer types. However, those approaches are either low throughput, not quantitative, or with poor sensitivity and reproducibility. Herein, we developed a novel acoustic force spectroscopy based method to quantify the cell–ECM adhesion strength during adhesion maturation process using the emerging z-Movi® technology. This can be served as a fast, simple, and high-throughput platform for functional assessment of cell adhesion molecules in a highly predictive and reproducible manner.
No related grants have been discovered for Haoqing Wang.