Tropomyosins are coiled-coil proteins that bind actin filaments and regulate multiple cytoskeletal functions, including actin network dynamics near the leading edge of motile cells. Previous work demonstrated that tropomyosins inhibit actin nucleation by the Arp2/3 complex and prevent filament disassembly by cofilin. We find that the Arp2/3 complex and cofilin, in turn, regulate the binding of tropomyosin to actin filaments. Using fluorescence microscopy we show that tropomyosin (non-muscle Drosophila Tm1A) polymerizes along actin filaments, starting from “nuclei” that appear preferentially on ADP-bound regions of the filament, near the pointed end. Tropomyosin fails to bind dendritic actin networks created in vitro by the Arp2/3 complex, in part because the Arp2/3 complex blocks pointed ends. Cofilin promotes phosphate dissociation and severs filaments, generating new pointed ends and rendering Arp2/3-generated networks competent to bind tropomyosin. Tropomyosin’s attraction to pointed ends reflects a strong preference for conformations localized to that region of the filament and reveals a basic molecular mechanism by which lamellipodial actin networks are insulated from the effects of tropomyosin.

ABSTRACT:Dynamically unstable polymers capture and move cellular cargos in both bacteria and eukaryotes, but the regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial Actin-Like Protein (ALP) that segregates the low copynumber plasmid pLS20 in Bacillus subtilis. Purified Alp7A forms dynamically unstable polymers with two critical points: an intrinsic critical concentration (0.6 µM), observed when ATP hydrolysis is blocked, and a dynamic critical concentration (10.3 µM), observed when ATP hydrolysis occurs. From biochemical and kinetic analysis, the intrinsic critical concentration reflects a balance between filament elongation and shortening, while the dynamic critical concentration reflects a balance between filament nucleation and catastrophic disassembly. Although Alp7A does not form stable polymers at physiological concentrations, rapid nucleation by an accessory factor, Alp7R, decreases the dynamic critical concentration into the physiological range. Intrinsic and dynamic critical concentrations are fundamental parameters that can be used to describe the behavior of all dynamically unstable polymers.

The sequence, structure, and assembly dynamics of eukaryotic actins are conserved across phyla. In contrast, actin-like proteins (ALPs) from eubacteria share little sequence homology, form polymers with different architectures, and assemble with different kinetics. The structural and functional diversity of the bacterial ALPs appears to arise from their (i) high degree of functional specialization and (ii) small number of regulatory factors. To understand the molecular mechanism by which a given ALP carries out its biological function, we must, therefore, understand its unique architecture and assembly dynamics. In this chapter, we provide a basic toolbox for studying the self-assembly of uncharacterized ALPs, including methods for characterizing the architecture and stability of polymers, specifying the mechanism of their nucleation, and measuring their rate of growth. Determining these basic properties provides a stable base for more complex reconstitutions of biological function.

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