Transport Regulation of Two-Dimensional Receptor-Ligand Association

AbstractThe impact of flow disturbances on platelet adhesion is complex and incompletely understood. At the molecular scale, platelet glycoprotein Ibα (GPIbα) must associate with the von Willebrand factor A1 domain (VWF-A1) with a rapid on-rate under high hemodynamic forces, as occurs in arterial thrombosis, where various transport mechanisms are at work. Here, we theoretically modeled the coupled transport-reaction process of the two-dimensional (2D) receptor-ligand association kinetics in a biomembrane force probe to explicitly account for the effects of molecular length, confinement stiffness, medium viscosity, surface curvature, and separation distance. We experimentally verified the theoretical approach by visualizing association and dissociation of individual VWF-A1-GPIbα bonds in a real-time thermal fluctuation assay. The apparent on-rate, reciprocal of the average time intervals between sequential bonds, decreased with the increasing gap distance between A1- and GPIbα-bearing surfaces with an 80-nm threshold (beyond which bond formation became prohibitive) identified as the combined contour length of the receptor and ligand molecules. The biomembrane force probe spring constant and diffusivity of the protein-bearing beads also significantly influenced the apparent on-rate, in accordance with the proposed transport mechanisms. The global agreement between the experimental data and the model predictions supports the hypothesis that receptor-ligand association behaves distinctly in the transport- and reaction-limited scenarios. To our knowledge, our results represent the first detailed quantification of physical regulation of the 2D on-rate that allows platelets to sense and respond to local changes in their hemodynamic environment. In addition, they provide an approach for determining the intrinsic kinetic parameters that employs simultaneous experimental measurements and theoretical modeling of bond association in a single assay. The 2D intrinsic forward rate for VWF-A1-GPIbα association was determined from the measurements to be (3.5 ± 0.67) × 10−4 μm2 s−1

Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin‐related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F‐actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3‐knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon‐like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three‐dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3‐dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities

Autoregulation of von Willebrand factor function by a disulfide bond switch

Force-dependent binding of platelet glycoprotein Ib (GPIb) receptors to plasma von Willebrand factor (VWF) plays a key role in hemostasis and thrombosis. Previous studies have suggested that VWF activation requires force-induced exposure of the GPIb binding site in the A1 domain that is autoinhibited by the neighboring A2 domain. However, the biochemical basis of this “mechanopresentation” remains elusive. From a combination of protein chemical, biophysical, and functional studies, we find that the autoinhibition is controlled by the redox state of an unusual disulfide bond near the carboxyl terminus of the A2 domain that links adjacent cysteine residues to form an eight-membered ring. Only when the bond is cleaved does the A2 domain bind to the A1 domain and block platelet GPIb binding. Molecular dynamics simulations indicate that cleavage of the disulfide bond modifies the structure and molecular stresses of the A2 domain in a long-range allosteric manner, which provides a structural explanation for redox control of the autoinhibition. Significantly, the A2 disulfide bond is cleaved in ~75% of VWF subunits in healthy human donor plasma but in just ~25% of plasma VWF subunits from heart failure patients who have received extracorporeal membrane oxygenation support. This suggests that the majority of plasma VWF binding sites for platelet GPIb are autoinhibited in healthy donors but are mostly available in heart failure patients. These findings demonstrate that a disulfide bond switch regulates mechanopresentation of VWF.: This study was supported by grants from the National Health and Medical Research Council of Australia (P.J.H.), Royal College of Pathologists Foundation Kanematsu/Novo Nordisk Research Award (F.P. and L.J.), Diabetes Australia Research Trust grant G179720 and Sydney Medical School Early-Career Researcher Kickstart Grant (L.J.), National Heart Foundation of Australia Postdoctoral Fellowship (101285) (L.J.) and British Heart Foundation Intermediate Basic Science Research Fellowship (FS/11/51/28920) (B.M.L.), Deutsche Forschungsgemeinschaft (research unit FOR 1543 to C.A.-S., C.B., and F.G.), the Center for Modelling and Simulation in the Biosciences postdoctoral program of the Heidelberg University (A.B.), and the Klaus Tschira Foundation (F.G.). B.L. was supported by the Dutch Thrombosis Foundation through grant number 2016-03.

Single-molecue study on GPIb-alpha and von Willebrand factor mediated platelet adhesion and signal triggering

The binding between the 45 kDa N-terminal domain of the a subunit of the GPIb-IX-V complex (GPIbαN) on the platelet membrane and the A1 domain of von Willebrand factor (VWF-A1), a multimeric protein circulating in the plasma, plays a key role in platelet adhesion and thrombus initiation at sites of cut-injury and atherosclerotic plaque rupture where blood vessels are subjected to high haemodynamic shear. A fundamental yet unresolved issue is how haemodynamic force upregulates this interaction (binding kinetics) and how a mechanical stimulus is translated into a biochemical signal (mechanotransduction). In order to address above issues, we setup a new biomembrane force probe (BFP) with the drifting reduction, temperature control and concurrent fluorescence imaging. My research findings are summarized into three aims: 1. VWF regions surrounding A1 hinder A1-GPIbα interaction at zero force, which is relieved by increasing force that stabilizes the interaction, giving rise to a VWF-GPIbα catch bond. 2. Three transport-related physical factors: receptor-ligand separation distance, Brownian motion and diffusivity govern the VWF-GPIbα association. 3. Mechanical force and structural variation regulate platelet signaling via the engagement duration of GPIbα mechanosensor. My thesis study advances our understanding of the biophysical and structural basis of how the VWF activation, its interaction with GPIbα and signal transduction are regulated by force when platelets' haemostatic functions are most needed.Ph.D

Tensile and compressive force regulation on cell mechanosensing

Receptor-mediated cell mechanosensing plays critical roles in cell spreading, migration, growth, and survival. Dynamic force spectroscopy (DFS) techniques have recently been advanced to visualize such processes, which allow the concurrent examination of molecular binding dynamics and cellular response to mechanical stimuli on single living cells. Notably, the live-cell DFS is able to manipulate the force “waveforms” such as tensile versus compressive, ramped versus clamped, static versus dynamic, and short versus long lasting forces, thereby deriving correlations of cellular responses with ligand binding kinetics and mechanical stimulation profiles. Here, by differentiating extracellular mechanical stimulations into two major categories, tensile force and compressive force, we review the latest findings on receptor-mediated mechanosensing mechanisms that are discovered by the state-of-the-art live-cell DFS technologies

Platelet Mechanobiology Inspired Microdevices: From Hematological Function Tests to Disease and Drug Screening

Platelet function tests are essential to profile platelet dysfunction and dysregulation in hemostasis and thrombosis. Clinically they provide critical guidance to the patient management and therapeutic evaluation. Recently, the biomechanical effects induced by hemodynamic and contractile forces on platelet functions attracted increasing attention. Unfortunately, the existing platelet function tests on the market do not sufficiently incorporate the topical platelet mechanobiology at play. Besides, they are often expensive and bulky systems that require large sample volumes and long processing time. To this end, numerous novel microfluidic technologies emerge to mimic vascular anatomies, incorporate hemodynamic parameters and recapitulate platelet mechanobiology. These miniaturized and cost-efficient microfluidic devices shed light on high-throughput, rapid and scalable platelet function testing, hematological disorder profiling and antiplatelet drug screening. Moreover, the existing antiplatelet drugs often have suboptimal efficacy while incurring several adverse bleeding side effects on certain individuals. Encouraged by a few microfluidic systems that are successfully commercialized and applied to clinical practices, the microfluidics that incorporate platelet mechanobiology hold great potential as handy, efficient, and inexpensive point-of-care tools for patient monitoring and therapeutic evaluation. Hereby, we first summarize the conventional and commercially available platelet function tests. Then we highlight the recent advances of platelet mechanobiology inspired microfluidic technologies. Last but not least, we discuss their future potential of microfluidics as point-of-care tools for platelet function test and antiplatelet drug screening

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

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

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

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