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206 Using Magnetic Tweezers to probe Mechanically-Induced Signalling in Endothelial Cells
  1. Matthew Bryan,
  2. Ioannis Xanthis,
  3. Sarah Hsiao,
  4. Paul Evans
  1. University of Sheffield

Abstract

Introduction Atherosclerosis is an inflammatory arterial disease that develops in regions exposed to disturbed flow, such as at bends. Blood flow affects endothelial cells through drag forces (shear stress) and by modifying biomolecular motion around the cell (mass transport). Flow systems commonly used in vitro to induce cellular signalling cannot distinguish between the shear stress and mass transport mechanisms and trigger multiple stress-sensitive receptors (mechanoreceptors) simultaneously. Magnetic tweezers are an alternative method, applying force directly to specific mechanoreceptors. Here, we describe the development of magnetic tweezers that can apply two-dimensional (2D) forces and demonstrate their potential for studying live-cell signalling processes.

Methods and results A four-poled electromagnet was built as part of a magnetic tweezers platform. Each pole was independently powered, enabling the 2D magnetic field profile between the poles to be controlled. ANSYS software was used to computationally model the magnetic field profile produced in the region between the pole pieces and the maximum force calculated as 16 pN per bead. The electromagnet was embedded within a fluorescence microscope fitted with an incubation chamber heated to 37ºC, enabling live-cell imaging during force application. Computer control of the magnetic field profile enabled the generation of forces of arbitrary magnitude, direction and oscillation frequency.

Human umbilical vein endothelial cells (HUVEC) were cultured in fibronectin-coated dishes until they reached confluency and were serum-starved prior to experimentation. Cells were loaded with a calcium-sensitive fluorescent dye prior to the application of superparamagnetic beads coated with antibodies that recognise integrin-β1. Cells were imaged for 3 min without force to define a signalling baseline, followed by 3 min of applied force. Force applied to the mechanoreceptors initiated calcium influx around the beads. Using single-cell analysis, the amplitudes of the calcium influx peaks were quantified. It was observed that peak amplitude significantly increased under constant 16 pN force and that the response under force was similar to that induced by 15 dyne/cm2 laminar flow (Figure 1).

Abstract 206 Figure 1

Ca peak amplitudes under (a) 16 pN force and (b) 15 dyne/cm2 flow. Bars show mean ± SEM; unpaired two-tailed t-test (n = 4–13)

Conclusions Magnetic tweezers were built around a fluorescence microscope to enable live-cell imaging of cell signalling during force application. Using calcium signalling to validate the approach, the feasibility of using the magnetic tweezers to induce a mechanoresponse was demonstrated. The flexible design of the magnetic tweezers means it has the potential to uncover the mechanisms through which shear stress is converted into a biological signal, since it can be adapted to study a variety of mechanoreceptors, signalling pathways and force profiles.

  • Shear stress
  • Mechanosensing
  • Magnetic tweezers

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