A correlative bioimaging force and multiomics platform for skin cell mechanosensing identification for new responsive proteins and the effects of agin

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External mechanical forces and ECM stiffness are important in regulating cell behaviour, from single cell migration to the biology of tissue development and disease. Studies of animal cells under external dynamic directional stretch have revealed novel mechanobiological responses including cytoskeletal reorganization. Although some molecular players have been identified these cellular rearrangements have not been studied at high dynamic and special resolution during stretch and, to our knowledge, high resolution imaging has not be directly correlated with other omics approaches or utilised to understand cellular ageing. We have developed an on-microscope stretch system which enables high resolution fluorescent microscopy of skin cells undergoing unidirectional stretch whilst incorporating live force measurements and accurate control of strain. Using this system we will image cells undergoing stretch and then isolate protein and RNA from these cell populations for downstream proteomic and transcriptomic analysis, allowing us to not only observe novel fine dynamic responses but coordinate this with the changes in protein expression to identify the proteins mediating the responses and how these responses depend on the force applied, the bulk properties of the cells, and the age of the cells. Thus, the overall objective of this project is to link individual high resolution cellular features (such as the cytoskeleton, cell junctions, and ECM) to proteomic and transcriptomic analysis and the mechanical response / properties of cells during stretch. This unique resource of integrated datasets can be mined for markers and master regulators of cellular mechanical stress in both young and old cells. These same correlated datasets can also be produced +/- bioactives to identify which responsive components are attenuated by particular interventions, linking cell biology, proteomics and transcriptomics to product efficacy and mode of action. Overall the project will contribute to our understanding of how skin cells respond to mechanical stimuli at the molecular and cellular level. The key objectives are: 1) Bioimaging of cellular responses of skin cells (fibroblasts and keratinocytes, including primary cells, to mechanical stress including stretch at different rates or cyclical stretching. 2) Generation of correlated proteomic and transcriptomic datasets using quantitative proteomics (SWATH) and RNA sequencing 3) Analysing compounds known to affect mechanical properties of cells and selected compounds of interest to our industrial partner, as well as compounds known to impact skin structural properties in vivo. This will highlight potential modes of action and identify structural changes which can be linked to modification of cell properties 4) Understanding the mechanical responses to ageing. Deploy the techniques described above to generate a matched dataset for ageing (Imaging/force/proteome/transcriptomics) which we can interrogate to identify potential links between protein or transcript and differing cellular responses seen in aged cells.