A variety of biological roles of mechanical forces have been proposed in cell biology, such as cell signaling pathways for survival, development, growth, and differentiation. Mechanical forces alter the mechanical conditions within cells and their environment, which strongly influences the reorganization of the actin cytoskeleton. Single-molecule imaging studies of actin filaments have led to the hypothesis that the actin filament acts as a mechanosensor; e.g., increases in actin filament tension alter their conformation and affinity for regulatory proteins. However, our understanding of the molecular mechanisms underlying how tension modulates the mechanical behavior of a single actin filament is still incomplete. In this study, a direct measurement of the twisting and bending of a fluorescently labeled single actin filament under different tension levels by force application (0.8-3.4 pN) was performed using single-molecule fluorescence polarization (SMFP) microscopy. The results showed that the amplitude of twisting and bending fluctuations of a single actin filament decreased with increasing tension. Electron micrograph analysis of tensed filaments also revealed that the fluctuations in the crossover length of actin filaments decreased with increasing filament tension. Possible molecular mechanisms underlying these results involving the binding of actin-binding proteins, such as cofilin, to the filament are discussed.

Depolymerization and polymerization of the actin filament are indispensable in eukaryotes. The DNase I binding loop (D-loop), which forms part of the interface between the subunits in the actin filament, is an intrinsically disordered loop with a large degree of conformational freedom. Introduction of the double mutation G42A/G46A to the D-loop of the beta cytoskeletal mammalian actin restricted D-loop conformational freedom, whereas changes to the critical concentration were not large, and no major structural changes were observed. Polymerization and depolymerization rates at both ends of the filament were reduced, and cofilin binding was inhibited by the double mutation. These results indicate that the two glycines at the tip of the D-loop are important for actin dynamics, most likely by contributing to the large degree of conformational freedom.

Eukaryotic microtubules are typically 13-protofilament tubes assembled from α/β-tubulin heterodimers that combine mechanical rigidity with dynamic instability. Homologous tubulins have been identified in Asgard archaea, the closest prokaryotic relatives to eukaryotes. Here, we characterize a heterodimeric α/β-tubulin system from Heimdallarchaeales. Biochemical reconstitution shows that Heim-α/β-tubulin forms a heterodimer that undergoes guanosine 5'-triphosphate-dependent polymerization with coupled nucleotide hydrolysis. Cryo-electron microscopy reveals that the polymers are composed of four-protofilament tubules, with microtubule-like lattices formed by conserved longitudinal interfaces and ball-and-socket lateral contacts. Single-filament imaging demonstrates intrinsic kinetic polarity and dynamic instability, while liposome encapsulation shows that microtubule growth generates forces sufficient to deform membranes. Despite their reduced protofilament number, Heim-α/β-microtubules share key structural and dynamic features with eukaryotic microtubules but exhibit lower bending stiffness and polymerization force. Thus, microtubule-like polymers can form from a range of protofilament numbers, with reduced architectures potentially adapted to small cellular dimensions and lower mechanical loads. Together, our results indicate expansion in microtubule protofilament number during eukaryogenesis.

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