Biology subjects

Kubo, S.

Publications and source records attributed to Kubo, S..

At least 19 recordsLinked to original sources

bioRxiv2026

The native structure of the Trichonympha centriole cartwheel reveals a zigzag stacking pattern

Centrioles are essential cellular organelles with complex architectures. Their earliest assembly intermediate--the cartwheel--has a 9-fold symmetry established by Spindle assembly abnormal protein 6 (SAS-6). We used cryo-electron tomography and sub-tomogram averaging to resolve the native structure of the SAS-6 rings within the cartwheel of the exceptionally long Trichonympha proximal centriole. A 16-nm axial periodicity was formed by stacked rings of V-shaped SAS-6 tetramers, which formed the fundamental unit of the cartwheels central hub. SAS-6 head domains adopted a zigzag stacking pattern. Furthermore, the improved resolution provides new insights into the function of the previously observed central inner domain, which forms nine asymmetric densities that bridge adjacent tetramers, thereby imparting polarity and enhancing structural stability. Molecular dynamics simulations indicate increased rigidity in inter-tetramer interactions, supporting efficient ring formation and revealing how cartwheel architecture and polarity are established and stabilized.

cell biology↗

bioRxiv2025

Cellular and molecular fine mapping pinpoints new immunopathology of lupus

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with an unknown etiology. To pinpoint new disease-relevant cell states and their molecular profiles, we performed an in-depth investigation of multimodal single-cell datasets comprising [~]2.1 million peripheral blood mononuclear cells from 346 donors. By resolving 123 fine-grained cell states across 27 cell types, we identified previously uncharacterized populations distinctively associated with clinical severity and treatment status, including GZMK+GZMH+HLA-DR+ effector memory CD8+ T cells (double-positive [DP] EMCD8) and FOXO1+ARHGAP15+ T cells. Through extensive statistical frameworks and multimodal approaches, we delineated their aberrant immune signaling networks, transcriptional regulators, key surface proteins, T cell receptor repertoires, and genetic/epigenetic landscapes, underscoring them as candidate drivers of SLE immunopathology. These findings provide new insights into therapeutic target discovery in SLE.

immunology↗

bioRxiv2025

Membrane constriction by dynamin through GTP-driven conformational changes from coarse-grained molecular dynamics simulations

Dynamin, a GTPase involved in membrane remodeling processes, plays a pivotal role in membrane fission during endocytosis. Understanding the molecular mechanisms of dynamin assembly and its conformational change triggered by GTP hydrolysis on lipid membranes is crucial for elucidating the intricate details of membrane constriction and fission events. However, the size and complexity of the dynamin assembly prevent us from obtaining comprehensive molecular mechanisms. In this study, we performed coarse-grained molecular dynamics (CG-MD) simulations to elucidate the chemo-mechanical coupling mechanism of dynamin membrane constriction. We set up a CG Martini simulation system that contains dynamin rings on a tubular membrane with realistic molecular structures. Controlling the length of the tubular membrane with a pressure coupling, we simulate membrane constriction through the nucleotide-state-dependent conformational change of dynamin. The simulation results suggest that the conformational change to the GDP state loosens and expands the dynamin rings, resulting in membrane constriction in the protein-uncoated region. This study lays the groundwork for simulating the chemo-mechanical coupling of dynamin-mediated membrane constriction. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/675253v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@3080b3org.highwire.dtl.DTLVardef@519814org.highwire.dtl.DTLVardef@d76b0forg.highwire.dtl.DTLVardef@b5e4ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Structural Basis for Efficient Fo Motor Rotation Revealed by MCMD simulation and Structural Analysis

Fo domain of ATP synthase functions as a rotary molecular motor, coupling proton translocation with the rotation of the c-ring rotor. This process involves proton uptake at the entry half channel, rotor rotation, and proton release to the exit half channel. While the overall coupling mechanism is established, the design principle for efficient rotation remains unclear. Here, we employed hybrid molecular simulations--combining coarse-grained modeling and Monte Carlo methods--to investigate the roles of side chain flexibility at proton-binding residues and the angular mismatch between the proton uptake process and the proton release process. Our results indicate that both factors promote rotational activity, with side chain flexibility playing a more significant role. Comparable analysis of Fo structures from different species revealed that the key residue geometry is conserved, and that the asymmetric geometry of the two half channels aligns with the mechanism suggested by simulation. These findings highlight a conserved design principle that enhances rotational efficiency and offer a mechanistic basis for engineering synthetic rotary systems. Significance StatementFo motor is the proton-conducting unit of ATP synthase and a rotary molecular motor driven by proton translocation across the membrane. Previous biochemical and structural studies have established the "half-channel model," which explains how proton translocation is coupled with rotation. However, it remains unclear what structural features of Fo enable the smooth coordination of proton flow and rotation for kinetically efficient motion. In this study, we conducted a hybrid molecular simulation combining coarse-grained model calculations with Monte Carlo simulations. The present study found that the conformational flexibility of side chains in amino acid residues involved in proton translocation is one of the critical factors for kinetically efficient rotation. Furthermore, it is also found that the fundamental structural features are conserved across different species, suggesting that the principal mechanism and the design principles of Fo motor are well conserved across species.

biophysics↗

bioRxiv2025

Dynein-2 is tuned for the A-tubule of the ciliary doublet through tubulin tyrosination

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.

biophysics↗

bioRxiv2024

Theoretical insights into rotary mechanism of MotAB in the bacterial flagellar motor

Many bacteria enable locomotion by rotating their flagellum. It has been suggested that this rotation is realized by the rotary motion of the stator unit, MotAB, which is driven by proton transfer across the membrane. Recent cryo-electron microscopy studies have revealed a 5:2 MotAB configuration, in which a MotB dimer is encircled by a ring-shaped MotA pentamer. While the structure implicates the rotary motion of the MotA wheel around the MotB axle, the molecular mechanisms of rotary motion and how they are coupled with proton transfer across the membrane remain elusive. In this study, we built a structure-based computational model for Campylobacter jejuni MotAB, conducted comprehensive protonation state-dependent molecular dynamics simulations, and revealed a plausible proton-transfer coupled rotation pathway. The model assumes rotation-dependent proton transfer, in which proton uptake from the periplasmic side to the conserved aspartic acid in MotB is followed by proton hopping to the MotA proton-carrying site, followed by proton export to the cytoplasm. We suggest that, by maintaining two of the proton-carrying sites of MotA in the deprotonated state, the MotA pentamer robustly rotates by [~]36{degrees} per proton transfer across the membrane. Our results provide a structure-based mechanistic model of the rotary motion of MotAB in bacterial flagellar motors and provide insights into various ion-driven rotary molecular motors. Significance StatementThis study aims to elucidate the mechanism by which bacteria move by rotating their flagella. The driving force for flagellar rotation is predicted to be driven by protons passing through the transmembrane protein MotAB, but the actual rotation mechanism has not yet been elucidated. Using advanced computational modeling and molecular dynamics simulations, we have elucidated the detailed processes by which proton translocation achieves the rotation of the bacterial flagellar motor. This work not only sheds light on the fundamental mechanisms of bacterial motility but also provides a framework for understanding similar ion-driven rotation mechanisms in other biological systems, potentially paving the way for new bioinspired technologies.

biophysics↗

bioRxiv2024

Investigating EMT-mediated resistance to EGFR tyrosine kinase inhibitors in NSCLC using innovative organoid models

BackgroundEpithelial-mesenchymal transition (EMT) has emerged as a key mechanism underlying resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, the intricacies of EMT-mediated resistance, driven by tumor microenvironment (TME) interactions, remain enigmatic. This study aimed to probe EMT-induced resistance in NSCLC using innovative in vitro organoid models. MethodsWe generated organoids by co-culturing EGFR-mutant NSCLC cells (HCC827, H1975), mesenchymal stem cells and endothelial cells. Drug susceptibility was compared between organoids and spheroids (cancer cells only) using EGFR TKIs - Gefitinib, Afatinib, Osimertinib. EMT marker (E-cadherin, ZEB1) expression was analyzed via immunofluorescence and western blotting. The effects of Bevacizumab and miR200c on overcoming resistance were also investigated. ResultsThe study identified a significant link between EMT and EGFR-TKI resistance. Notable findings included the decrease of E-cadherin and an increase in ZEB1, both of which influenced EMT and resistance to treatment. Bevacizumab showed promise in improving drug resistance and mitigating EMT, suggesting an involvement of the VEGF cascade. Transfection with miR200c was associated with improved EMT and drug resistance, further highlighting the role of EMT in TKI resistance. ConclusionsThis study offered vital insights into EMT-driven EGFR TKI resistance, highlighting the utility of organoid models in evaluating resistance modulated by TME interactions. Our findings reveal promising directions for overcoming EMT-mediated resistance involving Bevacizumab and miR200c, warranting further in vivo validation.

cancer biology↗

bioRxiv2023

Effect of alpha-tubulin acetylation on the doublet microtubule structure

Acetylation of -tubulin at the lysine 40 residue (K40) by ATAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage-resistant. K40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that K40 acetylation exerts a small-scale effect on the doublet microtubule structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between K40 acetylation and phosphorylation in cilia.

cell biology↗

bioRxiv2023

Inference of Selective Force on House Mice Genomes during Secondary Contact in East Asia

The house mouse (Mus musculus), commensal to humans, has spread globally via human activities, leading to secondary contact between genetically divergent subspecies. This pattern of genetic admixture can provide insights into the selective forces at play in this well-studied model organism. Our analysis of 163 house mouse genomes, with a particular focus on East Asia, revealed substantial admixture between the subspecies castaneus and musculus, particularly in Japan and southern China. We revealed, despite the admixture, all Y chromosomes in the East Asian samples belonged to the musculus-type haplogroup, potentially explained by genomic conflict under sex ratio distortion due to varying copy numbers of ampliconic genes on sex chromosomes. We also investigated the influence of selection on the post-hybridization of the subspecies castaneus and musculus in Japan. Even though the genetic background of most Japanese samples closely resembles the subspecies musculus, certain genomic regions overrepresented the castaneus-like genetic components, particularly in immune-related genes. Furthermore, a large genomic block containing a vomeronasal/olfactory receptor gene cluster predominantly harbored castaneus-type haplotypes in the Japanese samples, highlighting the crucial role of olfaction-based recognition in shaping hybrid genomes.

evolutionary biology↗

bioRxiv2022

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 38 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

biophysics↗

bioRxiv2022

Key residue on cytoplasmic dynein for asymmetric unbinding and unidirectional movement along microtubule

Cytoplasmic dynein 1 is almost exclusively responsible for intracellular transport toward the minus-end of microtubules in animal cells. One of the key factors for the unidirectional movement of dynein is the asymmetry of the unbinding of the motor from the microtubule when an external load is applied; it dissociates more easily from microtubules with minus-end directed loading than with plus-end directed loading. To elucidate the molecular basis for this property, we performed molecular dynamics simulations to identify the key residues responsible for asymmetry, which were then examined experimentally. First, we reproduced asymmetry in the unbinding behavior of dynein using coarse-grained simulations. Then, data analysis together with mutational analysis in silico predicted the specific residues that may be responsible for the asymmetry in unbinding. To examine this prediction, we expressed and purified recombinant dynein with mutations in either of the identified key residues. Consistent with the simulations, one of the mutants did not exhibit asymmetry in the in vitro unbinding assay. Moreover, the mutant dynein was able to bind and move diffusely along a microtubule but was unable to restrict its movement to the minus-end direction. Our results demonstrate both experimentally and theoretically how the key residue on the microtubule-binding domain generates asymmetry in unbinding, which is a critical mechanism for the unidirectional movement of dynein along a microtubule track. Significance StatementCytoplasmic dynein moves to the minus end of microtubules. This unidirectional dynein motility provides the driving force for various cellular activities including vesicle transport, organelle positioning and cell division. One of the key factors for dynein to exhibit unidirectional movement is the asymmetry of unbinding of dynein from the microtubule depending on the direction of external load. By combining computational simulations and in vitro experiments, we identified a residue responsible for the asymmetry. A point mutation at the residue indeed abolished unidirectional motility, highlighting the importance of the asymmetric unbinding property in dyneins unidirectional movement.

biophysics↗

bioRxiv2022

Regulatory mechanisms of the dynein-2 motility by post-translational modification revealed by molecular dynamic simulation

Intraflagellar transport for ciliary assembly and maintenance is driven by dynein and kinesins specific for the cilia. It has been shown that anterograde and retrograde transports run on different regions of the doublet microtubule, i.e., separate train tracks. However, little is known about the regulatory mechanism of this selective process. Since the doublet microtubule is known to display specific post-translational modifications of tubulins, i.e. "tubulin code", for molecular motor regulations, we investigated the motility of ciliary specific dynein-2 under different post-translational modification by coarse-grained molecular dynamics. Our setup allows us to simulate the stochastic stepping behaviors of dynein-2 on un-modified, detyrosinated, poly-glutamylated and poly-glycylated microtubules in silico. Our study revealed that poly-glutamylation can play an inhibitory effect on dynein-2 motility. Our result indicates that poly-glutamylation of the B-tubule of the doublet microtubule can be used as an efficient means to target retrograde intraflagellar transport onto the A-tubule.

biophysics↗

bioRxiv2021

F0-F1 coupling and symmetry mismatch in ATP synthase resolved in every F0 rotation step

The F0F1 ATP synthase, essential for cellular energy production, is composed of the F0 and F1 rotary motors. While both F0 and F1 have pseudo-symmetric structures, their symmetries do not match. How the symmetry mismatch is solved remains elusive due to missing intermediate structures of rotational steps. Here, for ATP synthases with 3- and 10-fold symmetries in F1 and F0, respectively, we uncovered the mechanical couplings between F0 and F1 at every 36{degrees} rotation step via molecular dynamics simulations and comparison of cryo-electron microscopy structures from three species. We found that the frustration is shared by several elements. The F1 stator partially rotates relative to the F0 stator via elastic distortion of the b-subunits. The rotor can be distorted. The c-ring rotary angles can be deviated from symmetric ones. Additionally, the F1 motor may take non-canonical structures relieving stronger frustration. Together, we provide comprehensive understanding to solve the symmetry mismatch.

biophysics↗

bioRxiv2021

Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation

In FoF1-ATP synthase, proton translocation through Fo drives rotation of the c-subunit oligomeric ring relative to the a-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c-subunits contribute to proton release to and proton uptake from the a-subunit. However, no studies have demonstrated cooperativity among c-subunits toward FoF1-ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring c-ring with various combinations of wild-type and cE56D, enabled by genetically fused single-chain c-ring. ATP synthesis and proton pump activities were decreased by a single cE56D mutation and further decreased by double cE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. Simulations revealed that prolonged proton uptake in mutated c-subunits is shared between two c-subunits, explaining the cooperation observed in biochemical assays.

biochemistry↗

bioRxiv2020

Remodeling and activation mechanisms of outer arm dynein revealed by cryo-electron microscopy

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryoelectron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with molecular dynamics simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

cell biology↗

bioRxiv2020

Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother

High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement of the instruments, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie, compared to that mimics the raw AFM movie, and the Kalman filter result. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhAc and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.

biophysics↗

bioRxiv2019

Structural basis for two-way communication between dynein and microtubules

The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain of dynein via an a-helical coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulated MTBD either in high-affinity or low-affinity states by introducing a disulfide bond between the coiled-coils and analyzed the resulting structures by NMRand cryo-EM. In the MT-unbound state, the affinity changes of MTBD were achieved by sliding of the N-terminal -helix by one half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, cryo-EM analysis showed that MT binding induced further sliding of the N-terminal -helix even without the disulfide bond, which suggests the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.

biophysics↗

bioRxiv2019

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

Microtubules are cytoskeletal structures involved in structural support, microtubule-based transport and the organization of organelles in the cells. The building blocks of the microtubule, the - and {beta}-tubulin heterodimers, polymerize into protofilaments, that associate laterally to form the hollow microtubule. There exists a specific type of microtubule structures in the cilia, termed doublet microtubules, where high stability is required for ciliary beating and function. The doublet microtubule, consisting of a complete A-tubule and a partial B-tubule maintains its stability through unique interactions at its outer and inner junctions, where the A- and B-tubules meet.\n\nUsing cryo-electron microscopy, we present the answer to the long-standing question regarding the identities, localizations and structures of the Chlamydomonas doublet microtubule inner junction proteins. Using a combination of sequence bioinformatics and mass spectrometry, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction associated protein FAP126. We show that inner junction proteins PACRG and FAP20, together with FAP52, previously unidentified FAP276, FAP106 and FAP126, form an interaction hub at the inner junction, which involves tubulin sites for post-translational modifications. We further compare the Chlamydomonas and Tetrahymena doublet microtubule structures to understand the common and species-specific features of the inner junction.

biophysics↗