Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts

Abstract

The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells

“…On the basis of these and earlier (29,35) studies, we suggest that the initial Ca 2ϩ "flash" or "bump" is associated with the endoplasmic reticulum or a component of the endoplasmic reticulum closely associated with the plasma membrane. Alternatively, the TRP channel could be responding to cytoskeletal interactions or changes in membrane stress (63,64). In either case, we speculate that TRP or TRP-like channels communicate this signal in a Gd 3ϩ -sensitive fashion to the plasma membrane.…”

Smooth Muscle Actin Determines Mechanical Force-induced p38 Activation

“…For all experiments, the pole face was parallel with and 2 cm from the surface of the cell culture dish. As the surface area of the magnet was larger than the culture dishes, and as the bead covering was relatively uniform for all cells, the forces applied to cells across the width of the culture dish were relatively uniform (23). Constant forces (0.65 pN/m 2 projected cell area) but of varying duration were used for all experiments.…”

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule–actin crosstalk

“…Moreover, studies of the local viscoelastic moduli of the cell envelope or the cytoplasm can yield valuable insights into the structure of the membrane associated actin cortex and structural changes induced by cell stimulating or damaging agents [7,10], deceases [21] or by application of external forces [22]. A variety of colloidal probe microrheometry techniques have been developed over the last few years based on the analysis of Brownian motion [10,23,24] or of the viscoelastic responses evoked by local forces applied through optical traps [10,23] and magnetic tweezers.…”