Muneyoshi Ichikawa

Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia assembly depends on intraflagellar transport (IFT) along doublet microtubules (doublets). Unlike dynein-1, which works on cytoplasmic singlet microtubules, dynein-2 works on the doublets inside cilia. Previous studies have shown that retrograde IFT, driven by dynein-2, occurs on the A-tubule of the doublet, suggesting an elusive mechanism by which dynein-2 recruits retrograde IFT to the A-tubule. Here, we investigated the molecular basis of this mechanism using cryo-electron tomography (cryo-ET), molecular dynamics (MD) simulations, and biochemical analysis. Our biochemical assays revealed that the microtubule-binding domain of dynein-2 exhibits a higher affinity for the ciliary doublets than dynein-1. Cryo-ET further visualized the preferential binding of dynein-2 to the A-tubule of the doublet. MD simulations suggest that dynein-2 prefers the tyrosinated tubulin lattice as is present in the A-tubule. These findings reveal a recruitment mechanism of retrograde IFT by dynein-2, providing new insights into the spatial and functional specialization of ciliary doublets.

The cortically anchored adaptor KANK1 organizes microtubules at focal adhesions through a long, intrinsically disordered linker (L2), yet how this linker spans the ∼35–50 nm membrane–microtubule gap is unclear. Here, we combine in-cell, biochemical, and biophysical assays, predictions of motif interaction and multivalent assembly using AlphaFold, and structural analysis by electron microscopy to show that the hub protein LC8, which binds more than 100 clients, converts the intrinsically disordered 600 amino acid L2 into an elongated, multivalent, rod-like assembly. In contrast, isolated motif peptides fail to bind LC8 at physiologically relevant concentrations, indicating that strong complex formation arises from cooperativity among multiple weak sites. These results establish LC8 as a molecular switch that rigidifies and extends KANK1 L2 via distributed weak motifs and short linkers. This interaction produces compositionally homogeneous yet conformationally adaptable rods, long enough to bridge the membrane–microtubule gap, resolving the paradox. This work expands the LC8 binding repertoire, reveals design principles for multivalent assembly, and suggests a generalizable strategy for tuning length, rigidity, and flexibility in large protein architectures.

The bacterial phosphotransferase system (PTS) is essential for carbohydrate uptake, and IIC transporters in the system facilitate intracellular sugar transport. Here, we report the crystal and cryo-EM structures of Leminorella grimontii GatC (LgGatC), a putative xylose transporter in the IIC family, in its outward-facing state. Structural analysis indicates that the outward-facing cavity recognizes the linear form of D-xylose. A structure-based homology model of an inward-facing state supports an elevator-like sugar transport mechanism. These findings provide insights into PTS-mediated xylose transport and its potential applications in microbial engineering.

Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia structures rely on intraflagellar transport (IFT), which involves dynein-2 for retrograde and kinesin-2 for anterograde movements along doublet microtubules. Unlike dynein-1, which works on singlet microtubules within the cytoplasm, dynein-2 specifically works on the doublet microtubules inside the cilia. Previous cryo-electron tomography studies have shown that retrograde IFT, driven by dynein-2, occurs on the A-tubule of the doublet, suggesting a specialized regulatory mechanism involving dynein-2. However, the molecular basis of this specificity remains unclear. Here, we investigated this mechanism using cryo-electron tomography (cryo-ET) with Volta Phase Plate (VPP), molecular dynamics (MD) simulations, and biochemical analysis. Our biochemical assay revealed that the microtubule-binding domain of dynein-2 exhibits a higher affinity for the ciliary doublet microtubule compared to dynein-1. Cryo-ET with VPP further visualized the preferential binding of dynein-2 to the A-tubule of the doublet microtubule. MD simulations suggest that the preferential binding of dynein-2 is attributed to the tyrosinated tubulin in the A-tubule. These findings uncover a tyrosination-dependent regulatory mechanism that governs the bidirectional transport of IFT on doublet microtubules, providing new insights into the spatial and functional specialization of ciliary transport systems.

Microtubules, a major component of the cytoskeleton consisting of tubulin dimers, are involved in various cellular functions, including forming axons and dendrites of neurons and retaining cell shapes by forming various accumulated superstructures such as bundles and doublets. Moreover, microtubule-accumulated structures like swarming microtubule assemblies are attractive components for dynamic materials, such as active matter and molecular robots. Thus, dynamic control of microtubule superstructures is an important topic. However, implementing stimulus-dependent control of superstructures remains challenging. This challenge can be resolved by developing designer protein approaches. We have previously developed a Tau-derived peptide (TP), which binds to the inner or outer surface of microtubules depending on the timing of the incubation. In this report, we designed the TP-fused photoswitchable protein Dronpa (TP-Dronpa) that reversibly photoconverts between monomeric and tetrameric states to photocontrol microtubule assemblies. The formation of microtubule superstructures, including bundles and doublets, was induced by tetrameric TP-Dronpa, whereas monomeric TP-Dronpa ensured that microtubules remained dispersed. Tetrameric TP-Dronpa also induced motile aster-like structures and swarming movement of microtubules on a kinesin-coated substrate. The formation/dissociation of these microtubule superstructures can be controlled by light irradiation. This system can generate and photocontrol various microtubule superstructures and provides an approach to facilitate the assembly of dynamic materials for various applications.

Protein translocation across cellular membranes is an essential and nano-scale dynamic process. In the bacterial cytoplasmic membrane, the core proteins in this process are a membrane protein complex, SecYEG, corresponding to the eukaryotic Sec61 complex, and a cytoplasmic protein, SecA ATPase. Despite more than three decades of extensive research on Sec proteins, from genetic experiments to cutting-edge single-molecule analyses, no study has visually demonstrated protein translocation. Here, we visualize the translocation, via one unit of a SecYEG-SecA-embedded nanodisc, of an unfolded substrate protein by high-speed atomic force microscopy (HS-AFM). Additionally, the uniform unidirectional distribution of nanodiscs on a mica substrate enables the HS-AFM image data analysis, revealing dynamic structural changes in the polypeptide-crosslinking domain of SecA between wide-open and closed states depending on nucleotides. The nanodisc-AFM approach will allow us to execute detailed analyses of Sec proteins as well as visualize nano-scale events of other membrane proteins.

Cys-loop receptors are a large superfamily of pentameric ligand-gated ion channels with various physiological roles, especially in neurotransmission in the central nervous system. Among them, zinc-activated channel (ZAC) is a Zn ²⁺ -activated ion channel that is widely expressed in the human body and is conserved among eukaryotes. Due to its gating by extracellular Zn ²⁺ , ZAC has been considered a Zn ²⁺ sensor, but it has undergone minimal structural and functional characterization since its molecular cloning. Among the families in the Cys-loop receptor superfamily, only the structure of ZAC has yet to be determined. Here, we determined the cryo-EM structure of ZAC in the apo state and performed structure-based mutation analyses. We identified a few residues in the extracellular domain whose mutations had a mild impact on Zn ²⁺ sensitivity. The constriction site in the ion-conducting pore differs from the one in other Cys-loop receptor structures, and further mutational analysis identified a key residue that is important for ion selectivity. In summary, our work provides a structural framework for understanding the ion-conducting mechanism of ZAC.

Microtubules, a major component of the cytoskeleton consisting of tubulin dimers, are involved in various cellular functions, including forming axons and dendrites of neurons and retaining cell shapes by forming various accumulated superstructures such as bundles and doublets. Moreover, microtubule-accumulated structures like swarming microtubule assemblies are attractive components for dynamic materials, such as active matter and molecular robots. Thus, dynamic control of microtubule superstructures is an important topic. However, implementing stimulus-dependent control of superstructures remains challenging. This challenge can be resolved by developing designer protein approaches. We have previously developed a Tau-derived peptide (TP), which binds to the inner or outer surface of microtubules depending on the timing of the incubation. In this report, we designed the TP-fused photo-switchable protein Dronpa (TP-Dronpa) that reversibly photoconverts between monomeric and tetrameric states to photocontrol microtubule assemblies. The formation of microtubule superstructures, including bundles and doublets, was induced by tetrameric TP-Dronpa, whereas monomeric TP-Dronpa ensured that microtubules remained dispersed. Tetrameric TP-Dronpa also induced motile aster-like structures and swarming movement of microtubules on a kinesin-coated substrate. The formation/dissociation of these microtubule superstructures can be controlled by light irradiation. This system can generate and photocontrol various microtubule superstructures and provides an approach to facilitate the assembly of dynamic materials for various applications.