Investigating the dynamics and organisation of the fibroblast cytoskeleton in aged vs young cells.

The cytoskeleton, comprised of microtubules, actin filaments and intermediate filaments, plays a vital role in the normal function of the cell. Both the individual networks and the interactions between them are responsible for functions such as cell motility, cargo trafficking, cell division and maintenance of cell structure, while they are also integral to the communication of the cell with its environment, responding to chemical and mechanical cues. Research has shown that cytoskeletal proteins undergo changes in aged cells and tissues. These include higher critical concentration, longer lag phase and lower turnover rates of actin filaments as a result of oxidation from reactive oxygen species (ROS), a reduction in γ-tubulin which is necessary for microtubule nucleation, and advanced glycation end-product (AGE) modification of vimentin, an intermediate filament, which leads to aggregation around the nucleus. The dynamics and organisation of these three cytoskeletal components have not been investigated in conjunction with the observed changes in ageing cells. Using previously and newly validated fluorescently tagged expression constructs, live imaging stains and antibodies, we aim to elucidate changes in cytoskeletal dynamics, using bioimage analysis methods to segment and extract quantitative information from high resolution confocal and super-resolution images. Low passage and aged fibroblasts will be fixed and stained or live-imaged along with etoposide-treated senescence control cells. Cells will also be subjected to chemical and mechanical stressors for observation of cytoskeletal dynamics under stress, while additional targets for investigation will be identified using bioinformatics approaches in collaboration with Procter & Gamble.