fluorescence-micropipette-aspiration-assay-to-investigate-red-blood

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to ...

The University of Sydney

Heart Research Institute

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.

Mechano-covalent protection of coagulation factor VIII by von Willebrand factor.

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.

Acoustic Force-Based Cell-Matrix Avidity Measurement in High Throughput.

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.

Biomembrane force probe (BFP): Design, advancements, and recent applications to live-cell mechanobiology.

Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (ER-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMβ2) on neutrophils remains elusive.

Article	Year
Mechano-covalent protection of coagulation factor VIII by von Willebrand factor. Blood advances\| PubMed ID: 36240294	2023
Acoustic Force-Based Cell-Matrix Avidity Measurement in High Throughput. Biosensors\| PubMed ID: 36671930	2023
Biomembrane force probe (BFP): Design, advancements, and recent applications to live-cell mechanobiology. Exploration (Beijing, China)\| PubMed ID: 37933233	2023
Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 38205640	2024

Haoqing Jerry Wang

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