Multi-Scale Force Transmission to and Within the Nucleus.

PhD Theses.The mechanical state of cells, controlled primarily by cytoskeletal (CSK) networks (actin, microtubules and intermediate filaments) is a critical component of maintaining healthy function. Forces transmitted through the cytoskeleton influence the organisation and state of nuclear material, leading to changes in gene expression. This thesis aims to increase our understanding of the role of the CSK networks, specifically the intermediate filament keratin, and their interplay in integrating mechanical forces. We primarily use immunofluorescence imaging of the CSK networks and the nucleus, supported by Atomic Force Microscopy. We work in human epidermal keratinocytes (HEKs), as they are rich in keratin, whose role in cytoskeletal force transmission is under-studied. Since drugs to disrupt keratin are scarce, we first established that Withaferin-A, a compound previously used to disrupt vimentin intermediate filaments, can disrupt keratin at non cyto-toxic doses; impacting cell mechanics and migration. Following from this, Withaferin-A was used alongside established cyto-modulatory drugs to disrupt CSK networks, quantifying a range of properties describing their organisation. These data were fitted to nuclear parameters that described opposing functions on the nuclear state of HEKs for keratin and tubulin, with keratin protecting the nucleus from mechanical force. Finally, machine and deep learning techniques were used to expand the mathematical modelling of data. By training networks to predict nuclear location from only CSK images, a causative relationship between CSK organisation and nuclear location can be derived. In addition, we develop new models to rapidly analyse Atomic Force Microscopy curves and generate synthetic cell images. These results demonstrate the important role of keratin in protecting the nucleus from mechanical force and that deep learning techniques can be used in the study of cell mechanics to gain new insights