Tropomodulin and Leiomodin Differentially Nucleate Tropomyosin Assembly on Actin Filaments

Abstract

Tropomyosins play a central role in the regulation of the actin cytoskeleton by assembling along filaments into rope-like, head-to-tail polymers ("clusters") along filaments that regulate the binding and activity of numerous actin-binding proteins. Importantly, in cells, tropomyosins do not decorate actin filaments uniformly; some filaments are devoid of tropomyosins, whereas others recruit specific isoforms. To explore the diversity of tropomyosin-isoform binding kinetics and identify factors that control their assembly, we designed tropomyosin constructs, bearing a natural N-terminal acetylation and targeted cysteine mutations for fluorescence labelling. We analysed the binding of different tropomyosin isoforms to individual actin filaments reconstituted inside a microfluidic chamber. We show that, despite significant differences in spontaneous nucleation and elongation rates, all tested tropomyosin isoforms can coassemble within the same clusters. In addition, we identified tropomodulin and leiomodin, two pointed-end-binding proteins, to efficiently nucleate the formation of tropomyosin clusters on actin filaments, for all tested tropomyosin isoforms. Notably, while tropomodulin nucleates tropomyosin clusters from the pointed end, leiomodin does so along the entire filament.