Unravelling the molecular interactions of tropomyosin on actin at the single molecule and single filament level

Tropomyosin is a well-known regulator of the dynamics of the actin cytoskeleton, yet its molecular interactions with actin are not understood. Single filament techniques have proved to be a powerful tool for understanding mechanisms for various actin binding proteins. The main aim of this thesis was to dissect the steps in the assembly pathway of tropomyosin on preformed actin filaments using fluorescence microscopy and microfluidics. Actin filaments were grown from seeds attached to the cover glass such that they were anchored at one end and could be aligned parallel to the surface using fluid flow. Labelling of tropomyosins for visualisation in such assays has been challenging, and after screening various labelling techniques it was found that fusion of the mNeonGreen at the N terminus of tropomyosin was a generic method for labelling different tropomyosin isoforms. Tpm1.8, a human cytosolic tropomyosin isoform found in lamellipodia and stress fibres was then used in this assay to dissect the nucleation, assembly and disassembly processes of this isoform on preformed actin filaments. An asymmetry in the assembly and disassembly process was seen, with the kinetics for Tpm1.8 being consistently greater at the barbed end of the actin filament as compared to the pointed end. Further, it was observed that these processes occur independently for the two strands of the actin filament. At the single molecule level, it was observed that nucleation, elongation as well as dissociation occur as single tropomyosins, indicating that tropomyosin does not need a ‘nucleus’ of a minimum size. This assay was then extended to Tpm3.1, another human cytosolic isoform widely studied due to its role in cancer. Asymmetry in the assembly and disassembly kinetics was confirmed for this isoform as well. Anti-Tpm3.1 compounds previously shown to have anti-cancer activity in cells were then added to the TIRF assay to understand their effect on the kinetics of this isoform