Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently-tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1 C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1 C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Eukaryotic actin filaments play a central role in numerous cellular functions, with each function relying on the interaction of actin filaments with specific actin-binding proteins. Understanding the mechanisms that regulate these interactions is key to uncovering how actin filaments perform diverse roles at different cellular locations. Several distinct classes of actin regulatory mechanisms have been proposed and experimentally supported. However, these mechanisms vary in their nature and hierarchy. For instance, some operate under the control of others, highlighting hierarchical relationships. Additionally, while certain mechanisms are fundamental and ubiquitous across eukaryotes, others are lineage-specific. Here, we emphasize the fundamental importance and functional significance of the following actin regulatory mechanisms: the biochemical regulation of actin nucleators, the ATP hydrolysis-dependent aging of actin filaments, thermal fluctuation- and mechanical strain-dependent conformational changes of actin filaments, and cooperative conformational changes induced by actin-binding proteins.

Recent advances in protein design have ushered in an era of constructing intricate higher-order structures. Nonetheless, orchestrating the assembly of diverse protein units into cohesive artificial structures akin to biological assembly systems, especially in tubular forms, remains elusive. To this end, we develop a methodology inspired by nature, which utilises two distinct protein units to create unique tubular structures under carefully designed conditions. These structures demonstrate dynamic flexibility similar to that of actin filaments, with cryo electron microscopy revealing diverse morphologies, like microtubules. By mimicking actin filaments, helical conformations are incorporated into tubular assemblies, thereby enriching their structural diversity. Notably, these assemblies can be reversibly disassembled and reassembled in response to environmental stimuli, including changes in salt concentration and temperature, mirroring the dynamic behaviour of natural systems. This methodology combines rational protein design with biophysical insights, leading to the creation of biomimetic, adaptable, and reversible higher-order assemblies. This approach deepens our understanding of protein assembly design and complex biological structures. Concurrently, it broadens the horizons of synthetic biology and material science, holding significant implications for unravelling life’s fundamental processes and enabling future applications.

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.

Phalloidin (Ph) is widely used for fluorescent labeling of actin filaments. We observed ADP-actin filaments labeled with rhodamine phalloidin (RhPh) or Alexa488-Ph in vitro and discovered that the fluorescence intensities along the filaments showed a mottled pattern of bright and dark regions. Filaments labeled with substoichiometric RhPh exhibited more significant fluorescence inhomogeneities than those labeled with excess RhPh. Because the quantum yield of Alexa488 fluorescence is hardly affected by the environment, we concluded that the inhomogeneities arise from nonuniform Ph binding density rather than locally inhomogeneous quantum yield of the fluorophores. Simulations assuming random RhPh binding alone partially produced fluorescence inhomogeneities, but the degree of inhomogeneities was significantly smaller than the experimental results. Furthermore, filaments colabeled with RhPh and Alexa488-Ph showed a positive correlation in fluorescence intensities of Rh and Alexa488. Moreover, addition of inorganic phosphate suppressed the fluorescence inhomogeneities and the correlation between the Rh and Alexa488 fluorescence intensities. These results indicated that two mechanisms contribute to the nonuniform binding density of Ph: (i) stochastic binding and (ii) local differences in Ph binding affinity caused by inorganic phosphate-sensitive structural polymorphism of actin filaments. This structural polymorphism may also affect the binding of various actin-binding proteins, contributing to the functional differentiation of actin filaments in vivo. Moreover, those mottled fluorescence patterns dynamically fluctuated over time. These temporal fluorescence fluctuations required glucose and glucose oxidase but were suppressed by Trolox, likely reflecting photophysical properties of fluorophores influenced by oxygen scavengers and triplet-state quenchers. Taken together, we provide new insights into the structural polymorphism of actin filaments.

Recent advances in protein design have ushered in an era of constructing intricate higher-order structures ¹ . Despite this progress, orchestrating the assembly of diverse protein units into cohesive artificial structures akin to biological assembly systems, especially in tubular forms, remains an elusive goal. To address this, we introduce the Nature-Inspired Protein Assembly Design (NIPAD), a novel methodology that utilises two distinct protein units to create unique tubular structures under carefully designed conditions. These structures demonstrate dynamic flexibility similar to that of actin filaments, with cryo-electron microscopy revealing diverse morphologies, like microtubules. By mimicking actin filaments, helical conformations were incorporated into tubular assemblies, thereby enriching their structural diversity. Remarkably, these assemblies can be reversibly disassembled and reassembled in response to environmental stimuli, such as changes in salt concentration and temperature, mirroring the dynamic behaviour of natural systems. NIPAD combines rational protein design with biophysical insights, leading to the creation of biomimetic, adaptable, and reversible higher-order assemblies. This approach deepens our understanding of protein assembly design and complex biological structures. Concurrently, it broadens the horizons of synthetic biology and material science, holding significant implications for unravelling life’s fundamental processes and pioneering new applications.