Biology subjects

Kodera, N.

Publications and source records attributed to Kodera, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Dual role of the RIG-I 2CARD as a signaling module and a brake on dsRNA translocation

RIG-I is an innate immune receptor for viral double-stranded RNA (dsRNA). Upon binding dsRNA, RIG-I nucleates MAVS filaments through its two caspase activation and recruitment domains (2CARD). However, how the 2CARD behaves during activation has remained unclear. Using high-speed atomic force microscopy, we visualized the 2CARD in full-length RIG-I across its apo, dsRNA-bound, translocating, multimeric, and polyubiquitin-bound states. Protrusions consistent with the 2CARD appeared only transiently in the apo state, whereas a mobile 2CARD emerged upon RNA binding. During ATP-driven translocation, wild-type RIG-I moved approximately twofold slower and showed more frequent pauses and multimers than RIG-I {Delta}2CARD. Within multimers, 2CARDs formed a complex that polyubiquitin stabilized into larger, dsRNA-deforming assemblies. These findings reveal the 2CARD as a brake on the RIG-I motor, beyond signaling.

biophysics↗

bioRxiv2026

Characterization of the trimeric TOM complex by HS-AFM single-molecule analysis

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for mitochondrial proteins. Approximately 99 % of mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and subsequently translocated into mitochondria through the TOM complex. The TOM complex exists in a dynamic equilibrium among multiple assembly states through spatial rearrangements of its subunits. The recent cryo-electron microscopy (cryo-EM) studies revealed near-atomic structures of the TOM core dimer, whereas previous biochemical studies indicated the TOM complex functions as a trimer in intact mitochondria. However, the relationship between the core dimer and the functional trimer remains unclear. In the present study, we analyzed the dynamics of the TOM complex using high-speed atomic force microscopy (HS-AFM) to investigate the assembly states and conformation transitions of the TOM complexes. We demonstrated that purified yeast TOM complexes predominantly adopt a trimeric organization but dynamically dissociate into dimeric and monomeric states during HS-AFM observation. The trimeric particles observed by HS-AFM exhibited spherical molecular shapes consistent with a trimeric structural model proposed from previous crosslinking analyses. In contrast, the dissociated dimeric particles closely resembled the dimensions of the TOM core-dimer structures determined by cryo-EM. Furthermore, HS-AFM analyses provided insight into the spatial arrangement of the Tom20 receptor, consistent with previous models of the trimeric TOM complex. These observations enabled characterization of the trimeric TOM complex in vitro and provide a foundation for future structural and functional analyses of TOM complex assembly. Significance statementThe translocase of the mitochondrial outer membrane (TOM) complex is the universal entry gate for almost all mitochondrial proteins, yet its native organization has remained elusive. By combining high-speed atomic force microscopy with cryo-electron microscopy, we provide evidence that the yeast TOM complex predominantly adopts a trimeric assembly with three protein-conducting channels, rather than the dimeric architecture proposed by previous structural studies. Furthermore, single-molecule imaging directly visualized interactions with mitochondrial precursor proteins and receptor-specific antibodies, enabling the experimental identification of receptor subunits within the trimeric TOM complex. Together, these findings uncover previously unrecognized structural and dynamic features of the TOM complex and provide a framework for understanding the molecular mechanism of mitochondrial protein import.

biochemistry↗

bioRxiv2026

Maturation-dependent structural dynamics and nanomechanical properties of dengue virus revealed by high-speed AFM and 3D force mapping

Although the maturation state of dengue virus (DENV) particles is a key determinant of their infectivity, maturation is unusually inefficient. Fully mature and immature DENV particles are well-studied; however, little is known about partially mature particles. Moreover, single-particle structural dynamics and nanomechanical properties are unknown. Here, we observe wildtype and immature DENV particles using a single-particle approach combining high-speed AFM (HS-AFM) and 3D force mapping (3DFM). HS-AFM shows that the conformations of each morphotype are heterogeneous and dynamic in liquid, particularly partially mature virions. Tracking immature prM-E spikes elucidates their dynamic movements, which show intraviral variation and constrained independence. 3DFM measurements suggest that internal DENV structure is also heterogeneous and undergoes maturation-dependent changes, with the nucleocapsid core not occupying the full internal volume of immature virions. This approach complements current structural virology techniques and adds a new dimension to our understanding of the structural properties of viruses.

biophysics↗

bioRxiv2026

CaMKIIα holoenzymes self-organize into worm-like mesoscale clusters

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in dendritic spines at concentrations comparable to those of cytoskeletal proteins and plays a central role in synaptic plasticity. During long-term potentiation (LTP), CaMKII further accumulates in spines. However, the mechanisms governing its higher-order organization remain poorly understood. Here, we use high-speed atomic force microscopy to visualize inter-holoenzyme interaction of CaMKII at mesoscopic scales (5-500 nm). Under freely diffusible conditions, CaMKII holoenzymes do not form stable clusters. In contrast, when spatially confined, they assemble into worm-like chain clusters mediated by kinase-domain interactions. These clusters expand upon activation, concomitant with the dissociation of the regulatory segment. Notably, the CaMKII P212L mutant associated with neurodevelopmental disorders, forms extensive clusters even in the basal state. Together, our findings demonstrate that CaMKII-CaMKII interactions drive mesoscale cluster formation and that precise regulation of cluster size and activation-dependent growth might be critical for synaptic signaling.

neuroscience↗

bioRxiv2025

HS-AFM visualizes stepwise condensation dynamics in RNA-driven LLPS

Liquid-liquid phase separation (LLPS) is a fundamental mechanism of intracellular biomolecular condensation formation. While RNA self-assembly has been implicated in condensate formation, it remains unclear how RNA condensation translates into distinct physical behaviors at the nanoscale. Utilizing HS-AFM, this study characterizes RNA condensation dynamics with single-molecule resolution. Imaging captured the transition from individual RNA folding to intermolecular clustering, ultimately leading to progressive condensate assembly. Beyond morphological description, condensate behavior was further examined through fusion dynamics and mechanical response. Post-fusion shape evolution quantified how merged condensates recover circular morphology over time, providing a dynamic readout of material behavior. Nanomechanical properties were independently assessed through force-curve measurements. Together, these analyses consistently distinguish the droplet-like poly A condensates from fractal-shaped assemblies of total RNA. Taking together, these findings directly link RNA folding dynamics, condensate assembly, and emergent physical properties, establishing a quantitative framework for defining condensate material states at the nanoscale.

biophysics↗

bioRxiv2025

Flexible Motion of T7 bacteriophage Tail Fibers Suggest a Dynamic Viral Infection Mechanism

Viruses are nanoscale infectious agents capable of specifically targeting and reprograming host cells. A unique group of viruses, bacteriophages, have regained popularity in research partly due to the rising number of multidrug-resistant bacterial infections. Phages could potentially replace antibiotics, but only if we understand every detail of their structure and infection cycle. T7 bacteriophages are a group of dsDNA viruses, which infect E. coli bacteria. T7 virions are comprised of an icosahedral protein shell which encapsulates the genomic DNA, and a tail-fiber complex which is primarily used for target recognition and DNA injection. The virus has six" L"-shaped, [~]40 nm long fibers (gp17 protein trimers) attached to the tail-tube, which are thought to be essential for initial host recognition and possibly surface exploration. Using high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations combined with small angle X-ray scattering (SAXS) we observed the molecular structure and movements of isolated tail fibers. Firstly, we have identified a hinge region within the fibers, which makes them highly flexible, allowing the bending of their distal region. Furthermore, we have observed the dynamic triple helical coiled coil structure of the proximal region, which would allow fiber rotation. These two points of flexibility allow a more efficient and highly dynamic host recognition and virus anchoring process. The observed flexibility might allow host surface exploration by walking. Such flexibility in the host recognition machinery may not be unique to T7 bacteriophages, getting us one step closer to understanding the intricate details of virus-host interactions.

biophysics↗

bioRxiv2025

Energy conversion mechanism revealed by ATP-free and ATP-1 dependent walking of myosin V motor

The mechanical processes of ATPase protein motors are coupled to their ATPase reactions. The free energy released at a given step(s) of the ATPase cycle is converted into mechanical work. Therefore, it has long been thought that ATP is the energy source for mechanical work. Here, we show that the double-headed myosin V (M5) motor can perform ATP-free walking. In the presence of ADP, M5 bound to F-actin by both heads makes a forward step when the trailing head is detached by an external force. This forward step harnesses the strain energy in the leading (L) head, provided from the strong binding energy between the L-head and actin. Consequently, strain generation reduces the binding affinity, which manifests as sporadic, brief L-head detachment. Strain generation and brief L-head detachment also occur during ATP-dependent walking to similar extents as during ATP-free walking. These results suggest that the strong binding energy is also converted into mechanical work during ATP-dependent walking.

biophysics↗

bioRxiv2025

Submolecular video-imaging of the Smc5/6 complex topologically bound to DNA

Structural maintenance of chromosomes (SMC) complexes are ring-shaped ATPases that hold distinct DNA segments together to regulate various chromosome structures and functions. However, their dynamic mechanisms of DNA binding and processing remain poorly understood. Here, using high-speed atomic force microscopy, we directly visualized the dynamics of the Smc5/6 complex on DNA at submolecular resolution, resolving its individual domains. ATP-bound Smc5/6 stably binds DNA via the ATPase head domain, whereas following ATP hydrolysis, the DNA is entrapped within the SMC compartment and positioned around the opposite hinge domain. Furthermore, Smc5/6 topologically entraps two DNA segments together and stabilizes the DNA twist structures, promoting DNA compaction. Our findings provide a visual demonstration of how an SMC complex employs its ring architecture to facilitate distinct DNA binding modes.

biophysics↗

bioRxiv2025

Actin monomers influence the interaction between Xenopus cyclase-associated protein 1 and actin filaments

Cyclase-associated protein (CAP) binds to both actin monomers and filaments and regulates multiple aspects of the actin dynamics including polymerization and depolymerization. CAP has been isolated from multiple species as a stable equimolar complex with actin monomer. However, functional significance of the CAP-actin complex is unknown. We previously demonstrated that native Xenopus cyclase-associated protein 1 (XCAP1) forms a 4:4 complex with actin. Here, we characterized how actin-free XCAP1 and the XCAP1-actin complex interact with actin filaments using high-speed atomic force microscopy and found that XCAP1-bound actin monomers influence dwell time and positional preference of XCAP1 on actin filaments. Actin-free XCAP1 bound to actin filaments transiently with a dwell time of [~]0.2 seconds. The XCAP1-actin complex also bound to actin filaments transiently but with a 3-to 5-fold longer dwell time than actin-free XCAP1. Actin-free XCAP1 bound to both side and ends of actin filaments with moderate preference to ends. However, the XCAP1-actin complex bound preferentially to the side of actin filaments. These results indicate that binding of actin monomers to XCAP1 affects its binding modes to actin filaments, suggesting that this might be a novel mechanism to regulate the effects of CAP on actin filaments.

biochemistry↗

bioRxiv2025

High-Speed AFM Reveals IDR-Mediated Structural Plasticity of EML4-ALK Modulated by ALK Inhibitors

EML4-ALK is a key oncogenic driver in lung cancer, but variant-specific chemoresistance limits the efficacy of current ALK inhibitors. Because the N-terminus contains an intrinsically disordered region (IDR), how ALK inhibitors affect the structure and dynamics of full-length EML4-ALK remains unclear. Here, using high-speed atomic force microscopy (HS-AFM), we visualize the overall structures of three full-length EML4-ALK variants (v1, v3, and v5) at the single-molecule level. We identified a transient globular subdomain (residues 191-217) within the v3 IDR that contributes to distinct oligomerization patterns. Notably, ALK inhibitors compact the IDR subdomain and reduce oligomerization, whereas this effect is abolished by the resistance mutation ALKG1202R, suggesting that ALK inhibitors not only affect kinase activity but also modulate IDR dynamics. Our findings shed new light on the structural regulation of EML4-ALK and provide a foundation for developing next-generation targeted therapies that account for IDR-driven molecular behavior. TeaserHigh-speed AFM revealed the full-length EML4-ALK structures and their IDR-mediated plasticity caused by ALK kinase inhibitors

cancer biology↗

bioRxiv2025

Mycobacterial DNA-binding protein 1 (MDP1) induces RNA condensation as revealed by high-speed AFM

Mycobacterial DNA-binding protein 1 (MDP1) is a histone-like protein in Mycobacterium tuberculosis (Mtb) that contributes to genome organization and dormancy adaptation. MDP1 consists of a structured HU-like region (HUR), and a post-translational modifications (PTMs)-enriched intrinsically disordered region (IDR), that regulate its function. While its role in DNA condensation is well established, how MDP1 interacts with RNA remains unclear, despite growing recognition of transcriptional regulation during dormancy. Here, by using total RNA from E. coli as a model substrate, we investigated whether MDP1 induces RNA condensation. Our high-speed AFM and optical microscopy analysis shows that native MDP1 from Mtb, rich in PTMs, forms globular RNA condensates. In contrast, MDP1 expressed in E. coli, which lacks PTMs, induces chain-like RNA condensates. Domain-specific analysis revealed that the IDR, the synergy between the IDR and HUR, and PTMs are essential for MDP1-induced RNA condensation. These findings suggest that MDP1 mediates RNA organization during dormancy.

biophysics↗

bioRxiv2025

Flexible fitting to infer atomistic-precision models of large-amplitude conformational dynamics in biomolecules from high-speed atomic force microscopy imaging

High-speed atomic force microscopy (HS-AFM) experiments allow direct observation of biomolecular dynamics at the single-molecule level, acquiring a large amount of topographic imaging data that visualizes changes of the molecular surface during functional activity over an extended period of time. Since images have no atomistic resolution, a major challenge has been to develop post-experimental computational methods to infer atomistic information from measurements. The recently developed NMFF-AFM flexible fitting method provides a computationally efficient approach promising to infer atomistic-precision models of conformational dynamics from resolution-limited AFM imaging data. We report the software integration of this method into the well-established BioAFMviewer platform and demonstrate first applications to experimental HS-AFM imaging data. To facilitate applications, we developed a direct workflow from raw experimental AFM data to visualization and analysis of fitting results. The presented applications to experimental data of a single protein domain, a protein complex, and a megadalton size protein filament demonstrate versatility of NMFF-AFM modelling to reproduce large-amplitude conformational motions of biomolecular dynamics from HS-AFM imaging. As a first step towards automated large-scale analysis of AFM imaging data, we furthermore demonstrate reconstruction of an atomistic molecular movie of protein dynamics, involving large-amplitude conformational transitions, from a measured HS-AFM movie sequence. Implementation of flexible fitting within the standalone user-friendly interactive BioAFMviewer software opens the opportunity for broad range applications to facilitate the understanding of resolution-limited HS-AFM measurements.

biophysics↗

bioRxiv2025

Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM

CaMKII predominantly assembles into a 12-meric ring assembly, primarily consisting of CaMKII and CaMKII{beta} variants in the brain. Previous biochemical studies have reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKII/{beta} heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employed high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKII/{beta} at a 3:1 ratio. Our findings revealed that the CaMKII and CaMKII{beta} subunits are intermixed within the 12-meric ring assembly, with more than 83% probability that CaMKII{beta} subunits adjacent to one another. Furthermore, in the activated state, CaMKII/{beta} heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKII{beta} subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKII{beta} subunits within the CaMKII/{beta} heterododecamer.

neuroscience↗

bioRxiv2025

Solution AFM imaging and coarse-grained molecular modeling of yeast condensin structural variation coupled to the ATP hydrolysis cycle

Condensin is a protein complex that regulates chromatin structural changes during mitosis. It varies the molecular conformation through the ATP hydrolysis cycle and extrudes DNA loops into its ring-like structure as a molecular motor. Condensin contains Smc2 and Smc4, in which a coiled-coil arm tethers the hinge and head domains and dimerizes at the hinge. ATPs bind between the heads, induce their engagement, and are hydrolyzed to promote their disengagement. In the previous study, we performed solution atomic force microscopy (AFM) imaging of yeast condensin holo-complex in the presence of ATP and conducted flexible molecular fitting to the AFM image, obtaining the hinge structure with open conformation. However, it has yet to be clarified how the opening/closing of the hinge is coupled to the ATP hydrolysis cycle. In this study, we performed solution AFM imaging in the presence and absence of varying nucleotides, including AMP-PNP, ATP{gamma}S, and ADP. Furthermore, we conducted coarse-grained molecular dynamics simulations of a Smc2/4 heterodimer and selected the molecular structure that best represented each AFM image. Our results suggested that head engagement upon ATP binding is coupled to hinge opening. Also, the results indicated that the N-terminal region of Brn1, one of the accessory subunits, re-associates to the Smc2 head after ADP release. This study deepens our understanding of the conformational variation of yeast condensin driven by the ATP hydrolysis cycle.

biophysics↗

bioRxiv2024

From Monomers to Oligomers: Structural Mechanism of Receptor-Triggered MyD88 Assembly in Innate Immune Signaling

MyD88 plays a pivotal role in Toll-like receptor (TLR) and interleukin-1 family signaling through its oligomerization upon receptor activation, leading to downstream protein recruitment. The Toll/interleukin-1 receptor domain of MyD88 (TIRMyD88) is responsible for this receptor-mediated oligomerization, but the detailed mechanism involved remains elusive. We investigated the structure of TIRMyD88 oligomers and their interactions with TLRs. Cryoelectron microscopy revealed that tandemly arrayed TIRMyD88 subunits formed an antiparallel double-stranded filament that could further form rings and cylindrical filaments. Moreover, the self-assembly of TIRMyD88 in vitro was markedly accelerated by dimeric rather than monomeric receptor TIRs, possibly reflecting the signal initiation step in vivo. High-speed atomic force microscopy further captured the dynamic processes of oligomerization of TIRMyD88, in addition to its direct interaction with the receptor TIRs. Based on these results, a novel regulatory mechanism of TIRMyD88 oligomerization underlying the signal initiation step was revealed.

biophysics↗

bioRxiv2023

High-speed AFM reveals fluctuations and dimer splitting of the N-terminal domain of GluA2-γ2

AMPA glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which participate in AMPAR clustering at synapses. Nevertheless, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit with TARP {gamma}2 in lipid environments. HS-AFM videos of GluA2-{gamma}2 in the resting and open states revealed fluctuations in NTD dimers. Conversely, in the desensitized state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we visualized individual NTD dimers transitioning into monomers. Furthermore, this NTD-dimer splitting resulted in intersubunit exchange between NTD dimers and an increased number of binding sites with the synaptic protein neuronal pentraxin 1. Therefore, our findings illuminate the significance of NTD dynamics in the synaptic clustering of AMPARs.

neuroscience↗

bioRxiv2023

Deciphering the actin structure-dependent preferential cooperative binding of cofilin

The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0-6.3 nm) than the MAD within typical helices (4.3-5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.

molecular biology↗

bioRxiv2023

Atom filtering algorithm and GPU-accelerated calculation of simulation atomic force microscopy images

Simulation atomic force microscopy computationally emulates experimental scanning of a biomolecular structure to produce topographic images that can be correlated with measured images. Its application to the enormous amount of available high-resolution structures, as well as to molecular dynamics modelling data, facilitates the quantitative interpretation of experimental observations by inferring atomistic information from resolution-limited measured topographies. The computation required to generate a simulated AFM image generally includes the calculation of contacts between the scanning tip and all atoms from the biomolecular structure. However, since only contacts with surface atoms are relevant, a filtering method shall highly improve the efficiency of simulation AFM computations. In this report we address this issue and present an elegant solution based on graphics processing unit (GPU) computations that significantly accelerates the computation of simulation AFM images. The method not only allows for visualization of biomolecular structures combined with ultra-fast synchronized calculation and graphical representation of corresponding simulated AFM images (live simulation AFM), but, as we demonstrate, can also reduce the computational effort during automatized fitting of atomistic structures into measured AFM topographies by orders of magnitude. Hence, the developed method will play an important role in post-experimental computational analysis involving simulation AFM. Implementation is realized in our BioAFMviewer software package for simulation AFM of biomolecular structures and dynamics.

biophysics↗