Elasticity, referred to as Young's modulus (given in Pa), is a parameter that reflects the actual condition of the cytoskeleton of a living cell and continuous elasticity measurement allows monitoring the dynamical behavior of this highly regulated system in real time. The principle of elasticity measurement is to physically indent a cell with a probe, to measure the applied force and to process this force-indentation data using an appropriate model.

Methods: 

We used a Multimode-AFM to indent living and fully polarized epithelial cells (16HBE14o^-) at 37°C with a colloidal probe to monitor the cell elasticity and the dynamic behavior of the cytoskeleton. Cells were indented continuously (0.25 Hz, one loading/unloading cycle every 4 seconds) with a tip velocity of 1mm/s and a loading force of 2 nN. This method creates 15 force-indentation curves per minute and therefore we developed semi-automatic analysis software allowing to process several hundred curves in less than an hour.

Results: 

During measurement of untreated cells we observed an oscillation of cell elasticity with a period of 200 seconds and a variation of the amplitude. It is known that the non-muscular motor-protein myosin II is crucial for cell motility, cell shape and tension. Blocking myosin II with blebbistatin resulted in (a) disappearance of the elasticity oscillations and (b) a decrease of the Young`s moduli. Myosin II is phosphorylated by myosin light chain kinase (MLCK) which in turn is activated by Ca²⁺ and calmodulin. Application of the MLCK specific inhibitor ML-7 decreased the cell elasticity as well as the reduction of intracellular Ca²⁺ with BAPTA-AM. Raising the intracellular Ca²⁺ by ionomycin or thapsigarin increases the elasticity. Application of ionomycin and blebbistatin simultaneously resulted in a decrease of elasticity showing that myosin II activity determines the elasticity of a living cell. We further tested this hypothesis by blocking Rac1, a small GTPase which reduces myosin II activity via p21-activated kinase (PAK). Inhibition of Rac1 therefore disinhibits myosin II activity and we observed an increase of cell elasticity.

We conclude that (i) continuous elasticity measurements enables to investigate cytoskeletal dynamics of living cells under physiological conditions and (ii) that the elasticity of a living cell is determined by myosin II activity.