Visualising the role of tropomyosins in cell adhesion and mechanosensing using light and electron microscopy.

Mammalian cells have complex cytoskeletons that form structures for cells to sense, interact with, attach, and move through the dynamic extracellular environment. Primary components of the cytoskeleton are co-filaments of actin and tropomyosin. These filaments are rapidly assembled to fulfill cellular needs in response to environmental cues (nutrient concentration, suitability of the matrix, etc.), and they undergo constant remodelling. The actin component of the filament has relatively minor molecular variability when compared to the tropomyosin component. In mammals, there are over 20 different tropomyosin isoforms, and many of them participate in unique cellular functions. Studying tropomyosin isoforms is important because their expression is substantially altered in cancer cells. Tropomyosins 1.6 and 2.1 are classified as tumour suppressors because they inhibit transformed phenotypes. Conversely, tropomyosin 3.1 promotes tumour cell survival and is a potential target for chemotherapeutic drugs. Notably, two major isoforms of tropomyosin, 1.8 and 1.9, are downregulated in cancer but their role(s) has not been investigated in depth. The studies described in this thesis address the knowledge gap by demonstrating an essential role for tropomyosins 1.8/1.9 in cell-substrate adhesion at the single filament level using cryo-electron tomography. Expressing these isoforms in transformed and cancer cells that naturally downregulate them restored the ability of the cells to form adhesions. This is of significance as adhesion loss is one of the first steps in cancer metastasis. Furthermore, the mechanism by which these isoforms moderate adhesion assembly and adhesion maturation was elucidated: actin/tropomyosin 1.8/1.9 filaments directly anchor to nascent adhesion sites and support paxillin activation (phosphorylation in residue Y118) by focal adhesion kinases. This increases the proportion of nascent adhesion sites that mature into focal adhesions. In the absence of tropomyosins 1.8/1.9, cells dramatically increase the ...