Mechanical signals affect and regulate many aspects of the cell behaviour, including growth, differentiation, gene expression and cell death. This thesis investigates the manner by which mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, a combined fluorescence-atomic force microscope (AFM) was used to produce well defined nanomechanical perturbations while simultaneously tracking the real-time motion of fluorescently labelled intracellular organelles in live cells.
By tracking instantaneous displacements of mitochondria far from the point of indentation, insights can be gained into the long-distance propagation of forces and the role of the cytoskeleton in force transmission. Quantitative analysis and tracking of mitochondria, using several image registration and tracking techniques, revealed an increase of approximately 40% in the mean mitochondrial displacement following AFM perturbation. Furthermore, when either the actin cytoskeleton or microtubules were disrupted using anti-cytoskeletal drugs, no significant change in mitochondrial displacement was observed following indentation, revealing the crucial role of both cytoskeletal networks in the long-distance transmission of forces through the cell.
In addition, the effect of retinol and conjugated linoleic acid (CLA), compounds that have diverse effects on various cellular processes, on the mechanical behaviour of the cell was examined: both compounds were found to have a significant detrimental effect on the formation of focal adhesions, which was directly correlated to the measured cell elasticity (Young’s modulus) of the cell. Furthermore, quantification of mitochondrial displacements in response to applied AFM perturbations showed force propagation through the cytoskeleton to be blunted. Treatment of the two compounds in combination showed an additive effect. These results may broaden our understanding of the interplay between cell mechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease
