Mechanical Cell Membrane stimulation using Magnetic Tweezers increases cell membrane stiffness through integrin dependent and independent mechanisms

Abstract

Several cellular processes, such as proliferation, migration tissue morphogenesis and tissue regeneration, are closely regulated by the cells ability to sense their microenvironment and translate mechanical stimuli to actionable intracellular biochemical signals. Integrins play a crucial role in these mechanotransduction mediated pathways, as they play a critical role linking a cells actin cytoskeleton to the extracellular matrix (ECM) and thus operating as an intracellular and extracellular hotspot of biochemical signals and mechanosensing. The cell’s ability to sense ECM characteristics such as stiffness is primarily mediated through Integrins, that relay this information through Focal adhesions and the connection to the actin cytoskeleton. ECM ligands like fibronectin (FN) showcase high affinity binding to integrins receptors through their RGD binding sequence and enable the adhesion of cells to the extracellular matrix. The aim of this study was to address the ability of the cell to sense external mechanical stimuli through integrin dependent and independent mechanotransduction, upon force application to the cell membrane. To address this and be able to exert sufficient forces to deform the cell membrane we constructed a custom-made magnetic tweezer that was able to generate up to 50 nN of force from 5 μm distance on 4.5 μm superparamagnetic beads. We confirmed the magnitude of the applied magnetic field of the magnetic tweezer using previous reported methods and Force-Distance calibration curves. Additionally using a high initial magnetic permeability and low hysteresis soft-alloy core we were able to apply rapid magnetization and demagnetization regimes to the area of interest, thus creating a regime of high magnetic force-pulses with high temporal resolution. Furthermore, we showcased the ability of the magnetic tweezer to apply sufficient forces to displace 4.5 μm superparamagnetic beads embedded in elastic polyacrylamide gels using magnetic pulses followed by relaxation displacements upon rapid demagnetization of the electromagnet. We then applied the same methodology on HeLa cells via Fibronectin and Concanavalin A(CONA) conjugated superparamagnetic beads. We functionalized the superparamagnetic beads using p-toluene-sulfonyl (Tosyl) chemistry resulting in the covalent binding of CONA and FN through primary amine groups on the bead surface. We went on to use live cell imaging to study the displacements of these microspheres upon applied magnetic field with and without treatment with the actin polymerization inhibitor, Cytochalasin D(CYTO), and the cell contractility inhibitor Rho Kinase, Y-27632 (ROCK). Decreasing displacements of FN coated superparamagnetic beads of a magnitude of -61%(p<0.05) over time where observed, an effect that was not observed upon treatment of HeLa cells with the CYTO and ROCK. CONA conjugated beads also displayed decreases of a magnitude of -29% (p<0.05), but treatment with CYTO and ROCK didn’t have an effect, as same magnitude reductions where observed. The above results suggest that PM mechanical stimulation results in the stiffening of the cell through integrin dependent and independent mechanisms. It also suggests that integrin-based stiffening is entirely dependent on the actin cytoskeleton and actomyosin contractility while integrin independent changes in cortical stiffness may reflect changes in PM lipid composition or changes in the polymerization of other cytoskeletal elements such as intermediate filaments.