Actin Tropomyosin Assembly Intermediates

Actin filaments play a critical role as part of the cytoskeleton, where they are involved in most cellular processes including determination of cell shape, cell migration, cell division, membrane function and intracellular transport. In muscle cells actin filaments are a key component of the contractile apparatus. This enormous functional specialisation is associated with differences in filament organisation, dynamics and interaction with actin-binding proteins. Recent evidence reveals that tropomyosins are key players in this dynamic regulation of the functions of actin filaments. However, the molecular mechanisms underlying the assembly of tropomyosin strands on actin filaments, the competition between tropomyosin isoforms and the differences in stability are not known. The main roadblock to addressing these questions is a lack of techniques to follow the dynamics of the process at the molecular level.Here we have developed a single-molecule fluorescence imaging approach to visualise and quantify snapshots of the assembly process by reconstituting actin filaments in the presence of labelled tropomyosin isoforms. Cytoskeletal and skeletal tropomyosin isoforms were labelled using maleimide chemistry. Biochemical assays showed that labelled tropomyosins bind cooperatively to actin filaments. We then competitively bound different tropomyosin isoforms to actin filaments in a microfluidic flow channel and visualised early assembly intermediates utilising TIRF microscopy. Our observations reveal nucleation of short stretches of tropomyosin polymers at multiple locations along the actin filaments. Our results have implications for the assembly pathways of specialised actin filaments in cells.