Biology subjects

Takada, S.

Publications and source records attributed to Takada, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Disruption of CaMKII biomolecular condensation induces attention-deficit/hyperactivity disorder-like phenotypes

CaMKII is a multifunctional kinase essential for synaptic plasticity and memory formation. While its canonical role involves enzymatic phosphorylation, recent evidence suggests CaMKII also functions through liquid-liquid phase separation (LLPS) with substrate proteins, including GluN2B-containing NMDA receptors. However, the physiological significance remains unclear. Here, we generated CaMKII subtype knock-in (KI) mice carrying a point mutation (I205K) in the hydrophobic pocket, a key interface required for LLPS. These mice exhibited a complete loss of structural long-term potentiation (sLTP) despite normal spine morphology, marked hyperactivity and profound deficits in aversive memory formation. Atomoxetine, an approved attention-deficit/hyperactivity disorder (ADHD) treatment, ameliorated the hyperactive phenotype. Notably, we identified a patient carrying the I205N variant presenting with ADHD and mild intellectual disability, mirroring the behavioral features observed in I205K KI mice. Additional neurodevelopmental disorder-associated variants within the same hydrophobic pocket similarly disrupted LLPS in vitro. Molecular dynamics simulations revealed these variants destabilize the CaMKII-GluN2B interaction through distinct mechanisms that perturb the dynamic stability of the binding interface. These findings establish CaMKII-mediated phase separation as critical for linking synaptic molecular assembly to cognitive function and provide a unifying molecular basis for synaptic disorganization, hyperactivity, and memory deficits associated with neurodevelopmental disorders. SignificanceCaMKII is essential for synaptic plasticity and memory, yet the contribution of its non-catalytic functions, including liquid-liquid phase separation (LLPS), to brain function remains unclear. Here, we show that disrupting CaMKII LLPS with its synaptic partners impairs synaptic localization, abolishes LTP, and causes profound memory deficits in vivo. A knock-in mouse carrying the I205K mutation showed hyperactivity and impaired learning. This recapitulate the clinical phenotypes of a human patients with the I205N variant, which is associated with ADHD and intellectual disability. Together, these findings establish LLPS as a critical mechanism underlying CaMKII function in the brain and provide a disease-relevant framework for understanding synaptic dysfunction in neurodevelopmental disorders.

neuroscience↗

bioRxiv2026

Genomic imprinting of the metabolic regulator gene Klf14 is regulated by a paternal sub-TAD anchored at Mest and a shared enhancer in mice.

KLF14 acts as a master regulator of gene expression in adipose tissue and variants at the human KLF14 gene show strong and reproducible association with type 2 diabetes and metabolic syndrome. Risk alleles are only pathologic when maternally inherited, consistent with the observation that KLF14/Klf14 is a maternally-expressed imprinted gene in human and mouse. However, how genomic imprinting is regulated at this important locus is currently unknown. In both species, KLF14/Klf14 is located [~]200 kb away from the paternally imprinted gene MEST/Mest, which is regulated by a maternal gametic differentially methylated region (gDMR) at its CpG island (CGI) promoter. Although the Klf14 CGI is kept unmethylated in most tissues, maternally-inherited DNA methylation (DNAme) marks are paradoxically required for Klf14 expression in mouse. Here, we show that Mest and Klf14 reside within the same topologically associating domain (TAD) in both mouse embryonic stem cells (ESCs) and differentiated cells, defined by sites of biallelic CTCF binding at the boundaries. Using allele-specific 4C-seq in F1 hybrid ESCs, we show that CTCF binding specifically to the unmethylated Mest gDMR generates a paternal allele-specific sub-TAD encompassing Klf14. We further show through CRISPR-Cas9-mediated mutagenesis, that deletions of the paternal Mest promoter region in ESCs as well as in vivo in mutant mice, results in both loss of Mest expression and acquisition of biallelic expression at Klf14. By analyzing epigenetic marks and chromatin looping in the Klf14-expressing pituitary cell line AtT-20, we identify a putative enhancer element shared by Mest and Klf14, providing a mechanistic model for the regulation of Klf14 imprinting. This work defines a new role for the Mest maternally methylated gDMR, revealing that it exerts long-range effects via allele-specific modulation of TAD structures and consequently act as an imprinting control region (ICR) in the regulation of Klf14 imprinting. Conservation of this TAD organisation at the human locus suggests that a similar regulatory mechanism operates at KLF14.

developmental biology↗

bioRxiv2026

OpenCafeMol with 3SPN.2 DNA model: GPU Acceleration for Long-Time Coarse-Grained Chromatin Simulations

There is a growing demand for molecular dynamics simulations to explore longer timescale behavior of giant protein-DNA complexes such as chromatin. To address this need, we extended OpenCafeMol, a GPU-accelerated residue-level coarse-grained molecular dynamics simulator originally developed for proteins and lipids, to support 3SPN.2 and 3SPN.2C DNA models. We also implemented a hydrogen-bond-type many-body potential to model DNA-protein interactions more accurately. To further improve computational efficiency, we introduced a localized scheme for calculating base-pairing and cross-stacking interactions. Benchmark tests show that OpenCafeMol on a single GPU achieves up to 200-fold speed-up for DNA-only systems and up to 100-fold speed-up for DNA-protein complexes compared to CPU-based simulations. To demonstrate the capability of our implementation for long-timescale biological processes, we simulated an archaeal SMC-ScpA complex undergoing DNA translocation via segment capture (a proposed mechanism for DNA loop extrusion) in the presence of a DNA-bound obstacle. We observed continuous captured-loop growth accompanied by obstacle bypass within the segment capture framework.

biophysics↗

bioRxiv2025

Self-organized fingering instabilities drive the emergence of tissue morphogenesis in digit organoids.

The emergence of complex anatomical structures -such as the hands- from unstructured tissues remains a fundamental question in developmental biology. Turing-type reaction-diffusion models have provided a molecular explanation for the periodic pre-patterning of digits; however, the physical principles driving 3D morphogenesis remain incompletely understood. To identify the biophysical design principles leading to digit formation, we develop a limb-mesenchymal organoid system that spontaneously forms elongated, digit-like protrusions. Iterations between experiments and agent-based models at the cellular level identify sufficient microscopic mechanisms leading to morphogenesis of digit-like structures: symmetry-breaking and the elongation of digits result from a combination of differential cell adhesion and morphogen-induced chemotaxis and convergent-extension. Lastly, to describe tissue-scale deformations, we perform a coarse-graining analysis of the agent-based model and derive a continuum model that reveals a structural analogy to Cahn-Hilliard-type equations. These equations are typically used to describe fluid phase separation and so-called ''fingering instabilities'' in fluid physics. Here, we show that they also accurately describe organoid morphogenesis. These findings suggest that ''finger'' formation is driven by a mechanical fingering instability acting in concert with chemical patterning, shedding a new light on vertebrate limb morphogenesis.

developmental biology↗

bioRxiv2025

Emergence of cell polarity by reciprocal interactions between Wnt and core PCP components

Planar cell polarity (PCP) is established by asymmetric localization of core PCP components in each cell. Since some Wnt proteins can induce PCP in vertebrates, it has been proposed that Wnt concentration gradients provide spatial cues for polarization. Here, however, we present evidence that Wnt11 can regulate PCP in a gradient-independent manner. In the neural plate of Xenopus embryos, endogenous Wnt11 does not form an obvious gradient in the direction of PCP, but is polarized at cell boundaries together with core PCP components. Wnt11 polarization is dependent on core PCP components, while polarization of core PCP components can also be induced by Wnt11, indicating a mutual amplification loop between Wnt11 and core PCP components in PCP formation. Furthermore, Wnt11 and core PCP components compose an intercellular loop to coordinate the direction of polarity. Thus, we propose that local and reciprocal interactions between Wnt11 and core PCP components can generate PCP.

developmental biology↗

bioRxiv2025

Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation.

Planar cell polarity (PCP) on a tissue plane is established by asymmetric polarization of core PCP components in individual cells. Recently, we found that Wnt11 organizes PCP in Xenopus embryos by accumulating locally with core PCP components to be polarized, but not by acting as a directional cue forming a global gradient. Since heparan sulfate proteoglycans (HSPGs) associate with Wnt on cell membranes, how HSPGs are involved in Wnt polarization and PCP formation is a central issue. Here, we show that HSPGs are dynamically organized in a polarized manner, as with core PCP components and Wnt11, by associating with them. While HSPGs with HS chains enriched by N-sulfation and N-deacetylation are polarized, depending on Wnt and core PCP components, NDST1, which catalyzes these modifications, is required for Wnt11 polarization and PCP formation. Thus, mutual regulation among HS chains, Wnt11, and PCP components appears to be essential for PCP formation. HighlightsO_LIndst1 is required for Wnt11 localization and proper PCP formation. C_LIO_LINDST1-modified HS chains are polarized in the neural plate during development. C_LIO_LIPolarization of NDST1-modified HS chains depends on PCP formation. C_LIO_LIPositive feedback loop results in polarized distribution of HSPGs, Wnt11, and PCP. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/669988v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1db75adorg.highwire.dtl.DTLVardef@10aec47org.highwire.dtl.DTLVardef@1e50d5org.highwire.dtl.DTLVardef@4cd41c_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Physics-based nucleosome-resolution modeling of epigenetic-driven chromatin domain dynamics

Chromatin spatially organizes the Eukaryotic genome to support key cellular processes such as gene regulation, but the interplay between epigenetics, chromatin structure, and function is still poorly understood. We propose a nucleosome-resolution coarse-grained model that captures the essential features of chromatin organization over multiple scales: from nucleosome dynamics to chromatin fiber folding, and liquid-liquid phase separation. The model describes the effects of DNA linker length, histone tail acetylation, linker histone H1, and multi-bromodomain proteins such as BRD4. It is designed to be experimentally accurate but computationally efficient, allowing the study of 100kb genomic regions on a timescale of seconds with moderate resources. We apply this model to explore the structure and dynamics of two active loci of mouse embryonic stem cells, Pou5f1 and Sox2, as determined by solely their underlying epigenetic patterns. Our simulations reveal that chromatin folds into liquid-like domains characterized by similar histone modifications. These domains are highly dynamic, driving the formation of transient contacts between distant cis-regulatory regions. In silico mutation studies further clarify the distinct roles played by histone acetylation, linker histone, and BRD4. Overall, our physics-based modeling provides evidence that, in addition to other well-established mechanisms, epigenetic-dependent nucleosome-nucleosome interactions can play a key role in shaping the functional organization of genomic loci.

biophysics↗

bioRxiv2025

Mechanistic models of asymmetric hand-over-hand translocation and nucleosome navigation by CMG helicase

Faithful replication of eukaryotic chromatin requires the CMG helicase to translocate directionally along single-stranded DNA (ssDNA) while unwinding double-stranded DNA (dsDNA) and navigating nucleosomes. However, the mechanism by which CMG achieves processive translocation and deals with nucleosomal barriers remains incompletely understood. Here, using coarse-grained molecular dynamics simulations with ATP-driven conformational switching, we showed that asymmetric rotational transitions among four distinct ssDNA-binding states enable CMG to achieve directional translocation and DNA unwinding. We further demonstrated that the fork protection complex (Csm3/Tof1) and RPA enhance processivity through distinct mechanisms: Csm3/Tof1 grips the parental duplex to suppress backtracking, while RPA alleviates lagging-strand clogging. Upon nucleosome encounter, Csm3/Tof1 promoted partial unwrapping of the entry DNA, but further progression was energetically restricted near the nucleosomal dyad. The histone chaperone FACT lowered this barrier and simultaneously prevented inappropriate histone transfer to the lagging strand. Our results provide mechanistic insights into how the eukaryotic replisome coordinates helicase activity, nucleosome navigation, histone chaperon function, and histone recycling during eukaryotic DNA replication.

biophysics↗

bioRxiv2025

Membrane-enhanced repulsive interactions regulate protein diffusion in cell-size space

Intracellular molecular organization is often explained by attractive interactions driving clustering and phase separation. Although consideration of repulsive forces is essential in physics, their roles remain unclear in cellular contexts. Here, we demonstrated the fundamental role of repulsion in regulating protein diffusion within cell-size space. By analyzing negatively charged protein diffusion in bulk solutions and in cell-size spaces with membranes, we revealed that membrane-enhanced repulsion inhibited protein diffusion in cell-size spaces. This was due to the amplified electrostatic interactions among proteins because of the large membrane area-to-volume ratio. Notably, ATP, a cellular central energy source, further inhibited protein diffusion in cell-size spaces, whereas protein wave propagation on the membrane counteracted this inhibition. These findings suggest an active regulatory mechanism restoring molecular mobility by dynamically adjusting membrane-enhanced repulsive forces. Our study challenges the traditional emphasis on attractive interactions, highlighting repulsion as a critical tunable factor governing molecular transport and spatial organization in cells. TeaserProtein diffusion within crowded cell-size spaces is tuned by membrane-enhanced repulsion and its active regulation.

biophysics↗

bioRxiv2025

OpenCafeMol: A GPU-accelerated coarse-grained biomoleculardynamics simulator with OpenMM library

There has been an increasing demand for longer-timescale molecular dynamics (MD) simulations of larger biomolecular systems. To meet these demands, using the C++ API of OpenMM, we developed a fast and flexible MD software, OpenCafeMol, for residue-resolution protein and lipid models that shows high performance on graphics processing unit (GPU) machines. We validated OpenCafeMol for folding small proteins, lipid membrane dynamics, and membrane protein structures. Benchmark tests of the computation times showed that OpenCafeMol with one GPU for proteins and lipid membranes can be approximately 100 and 240 times faster than the corresponding simulations on a typical CPU machine (eight cores), respectively. Taking advantage of the high speed of OpenCafeMol, we applied it to two sets of vesicle fusion simulations; one driven by force and the other coupled with conformational dynamics of a SNARE complex. In the latter, a direct MD simulation at a high temperature resulted in vesicle docking, pore formation followed by fusion, which are coupled with local folding of linkers in the SNARE complex. This opens up new avenue to study membrane-fusion mechanisms via MD simulations. The source code for OpenCafeMol is fully available.

biophysics↗

bioRxiv2025

Multiphase separation in postsynaptic density regulated by membrane geometry via interaction valency and volume

Biomolecular condensates are found at various cellular locations, nucleus, cytoplasm, and membrane. These condensates often contain multiple components and can separate into multiple phases with various morphologies such as core-shell droplets, implicating functional roles. Demixing and arrangements of condensates are determined by competitive interactions and their locations. Recent studies reported a puzzling multiphase morphology in postsynaptic density components: AMPA-receptor, NMDA-receptor, PSD-95, and CaMKII. The multiphase morphology appears reversed when transitioning from the solution to the membrane. Using this system as a model, we study the multiphase behavior of condensates in solution (3D) and domain formation on and beneath the membrane (2D) and elucidate molecular mechanisms behind the puzzle. Our simulations reproduce the core-shell structure in 3D in vitro solution, where AMPA-receptor/PSD-95 form the core and NMDA-receptor/CaMKII form the shell, triggered by CaMKII activation. Then, we obtain a reversed morphology on the membrane. This reversal is primarily driven by CaMKIIs high valency and large volume. We find that, in solution, CaMKIIs non-specific volume interaction dominate, while on the membrane, specific multivalent interactions overcome the excluded volume interaction of CaMKII. The layered structures of receptors and CaMKIIs reduce the excluded volume effects of CaMKII on receptors, making the multivalent interaction dominant. These findings highlight the differences between condensate formation in solution and membrane domain formation, modulated by their layered arrangement. SignificanceMany proteins form condensates in cells, which are often further separated into two or more phases, implicating functional roles. Recent experiments on postsynaptic density proteins provide an interesting puzzle on a multiphasic morphology; while they form core-shell droplets with AMPA-receptor at the core and NMDA receptor in the shell in soluble constructs, the multiphase morphology is reversed when they form nanodomains on the membrane. This study identifies the mechanism behind the puzzle by using computer simulations with a mesoscale model. While active CaMKII, an inducer of the multiphase morphology, has dominant non-specific volume interactions in solution, its specific domain-domain multivalent interaction overcomes the volume interaction under the membrane with their layered arrangement.

biophysics↗

bioRxiv2025

KEAP1 retention in phase-separated p62 bodies drives liver damage in autophagy-deficient conditions

Phase-separated p62 bodies activate NRF2, a key transcription factor for antioxidant response, by sequestering KEAP1, which targets NRF2 for degradation. Although p62 bodies containing KEAP1 are degraded by autophagy, they accumulate in various liver disorders. Their precise disease role remains unclear. We show that excessive KEAP1 retention in p62 bodies and NRF2 activation are major causes of liver damage when autophagy is impaired. In mice with weakened or blocked p62-KEAP1 interactions, KEAP1 retention and NRF2 activation under autophagy-deficient conditions were suppressed. Transcriptome and proteome analyses revealed that NRF2 targets upregulated by autophagy suppression normalized in p62 mutants unable to bind KEAP1. Autophagy deficiency caused organelle accumulation, especially of the ER, regardless of p62 mutation. Liver damage and hepatomegaly from autophagy suppression markedly improved in p62 mutants, particularly those with blocked KEAP1 binding. These findings highlight excessive KEAP1 retention in p62 bodies and defective organelle turnover as key drivers of liver pathology, underscoring the significance of phase separation in vivo.

cell biology↗

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

Drosophila Trus, the orthologue of mammalian PDCD2L, is required for proper cell proliferation, larval developmental timing, and oogenesis

Toys are us (Trus) is the Drosophila melanogaster ortholog of mammalian Programmed Cell Death 2-Like (PDCD2L), a protein that has been implicated in ribosome biogenesis, cell cycle regulation, and oncogenesis. In this study, we examined the function of Trus during Drosophila development. CRISPR/Cas9 generated null mutations in trus lead to partial embryonic lethality, significant larval developmental delay, and complete pre-pupal lethality. In mutant larvae, we found decreased cell proliferation and growth defects in the brain and imaginal discs. Mapping relevant tissues for Trus function using trus RNAi and trus mutant rescue experiments revealed that imaginal disc defects are primarily responsible for the developmental delay, while the pre-pupal lethality is likely associated with faulty central nervous system (CNS) development. Examination of the molecular mechanism behind the developmental delay phenotype revealed that trus mutations induce the Xrp1-Dilp8 ribosomal stress-response in growth-impaired imaginal discs, and this signaling pathway attenuates production of the hormone ecdysone in the prothoracic gland. Additional Tap-tagging and mass spectrometry of components in Trus complexes isolated from Drosophila Kc cells identified Ribosomal protein subunit 2 (RpS2), which is coded by string of pearls (sop) in Drosophila, and Eukaryotic translation elongation factor 1 alpha 1 (eEF11) as interacting factors. We discuss the implication of these findings with respect to the similarity and differences in trus genetic null mutant phenotypes compared to the haplo-insufficiency phenotypes produced by heterozygosity for mutants in Minute genes and other genes involved in ribosome biogenesis. Authors SummaryRibosomes are essential macromolecular machines required for decoding mRNA to make proteins, the major biomolecules that carry out all central cellular functions. As such, their structural and operational integrity is critical to organismal survival, and mutations that disrupt proper stoichiometry or assembly of ribosomes produce serious pathological consequences during an organisms development and/or adult life. The ribosome assembly factor PDCD2L is highly conserved from yeast to man, yet its overall function and requirement during development is poorly understood. By examining the developmental consequences of null mutations in trus, which encodes the Drosophila PDCD2L homolog, we demonstrate an essential role for this factor in cell-cycle regulation. Furthermore, disruption of Trus function in mitotically dividing imaginal tissue activates the Xrp1-dilp8 stress response pathway which limits production of ecdysone, the major arthropod molting hormone, leading to severe developmental delay during larval stages. These studies provide new insights on the requirements of this highly conserved ribosome assemble factor during development.

developmental biology↗

bioRxiv2024

In silico nanoscope to study the interplay of genome organization and transcription regulation

In eukaryotic genomes, regulated access and communication between cis-regulatory elements (CREs) are necessary for enhancer-mediated transcription of genes. The molecular framework of the chromatin organization underlying such communication remains poorly understood. To better understand it, we develop a multiscale modeling pipeline to build near-atomistic models of the 200 kb Nanog gene locus in mouse embryonic stem cells comprising nucleosomes, transcription factors, co-activators, and RNA polymerase II-Mediator complexes. By integrating diverse experimental data, including protein localization, genomic interaction frequencies, cryo-electron microscopy, and single-molecule fluorescence studies, our model offers novel insights into chromatin organization and its role in enhancer-promoter communication. The models equilibrated by high-performance molecular dynamics simulations span a scale of [~]350 nm, revealing an experimentally consistent local and global organization of chromatin and transcriptional machinery. Our models elucidate that the sequence-regulated chromatin accessibility facilitates the recruitment of transcription regulatory proteins exclusively at CREs, guided by the contrasting nucleosome organization compared to other regions. By constructing an experimentally consistent near-atomic model of chromatin in the cellular environment, our approach provides a robust framework for future studies on nuclear compartmentalization, chromatin organization, and transcription regulation.

molecular biology↗

bioRxiv2024

Tension-induced suppression of allosteric conformational changes explains coordinated stepping of kinesin-1

The dimeric motor protein kinesin-1 walks along microtubules by alternating ATP hydrolysis and movement of its two motor domains ("head"). The detached head preferentially binds to the forward tubulin-binding site after ATP binds to the microtubule-bound head, but the mechanism preventing premature binding to the microtubule while the partner head awaits ATP remains unknown. Here, we examined the role of the neck linker, the segment connecting the two heads, in this mechanism. High-resolution structural analyses of the nucleotide-free head revealed a bulge just ahead of the neck linkers base that creates an asymmetric constraint on its mobility. While the neck linker can stretch freely backward, it must navigate around this bulge to extend forward. Based on this finding, we hypothesized that premature binding of the tethered head is suppressed by an intolerable increase in neck linker tension. Molecular dynamic simulations and single-molecule fluorescent assays supported this model. These findings demonstrate a tension-based regulation mechanism where off-pathway conformational transitions are thermodynamically suppressed through entropy loss associated with neck linker stretching, suggesting that neck linker tension influences the allosteric conformational transition rather than directly affecting the nucleotide state.

biophysics↗

bioRxiv2024

Molecular dynamics simulations of human cohesin subunits identify DNA binding sites and their potential roles in DNA loop extrusion

The SMC complex cohesin mediates interphase chromatin structural formation in eukaryotic cells through DNA loop extrusion. Here, we sought to investigate its mechanism using molecular dynamics simulations. To achieve this, we first constructed the amino-acid-residue-resolution structural models of the cohesin subunits, SMC1, SMC3, STAG1, and NIPBL. By simulating these subunits with double-stranded DNA molecules, we predicted DNA binding patches on each subunit and quantified the affinities of these patches to DNA using their dissociation rate constants as a proxy. Then, we constructed the structural model of the whole cohesin complex and mapped the predicted high-affinity DNA binding patches on the structure. From the spatial relations of the predicted patches, we identified that multiple patches on the SMC1, SMC3, STAG1, and NIPBL subunits form a DNA clamping patch group. The simulations of the whole complex with double-stranded DNA molecules suggest that this patch group facilitates DNA bending and helps capture a DNA segment in the cohesin ring formed by the SMC1 and SMC3 subunits. In previous studies, these have been identified as critical steps in DNA loop extrusion. Therefore, this study provides experimentally testable predictions of DNA binding sites implicated in previously proposed DNA loop extrusion mechanisms and highlights the essential roles of the accessory subunits STAG1 and NIPBL in the mechanism.

molecular biology↗

bioRxiv2024

Submolecular-scale Hairpin DNA Folding Dynamics Studied by High-Speed AFM with Optical Tweezers

Optical tweezers have contributed to elucidate the folding mechanisms associated with biomolecules. By combining single-molecule or super-resolution techniques, imaging can also be performed while measuring or inducing force coupling with biochemical reactions; however, they cannot capture structural information beyond the fluorophore spatial resolution. To overcome this problem, here, we developed a technique that combines optical tweezers with high-speed atomic force microscopy (AFM). To solve the problem of incompatible instrumental configurations, we developed a unique optical tweezers measurement system that is specialized for high-speed AFM. Upon applying an external force to a synthesized DNA secondary structure, we successfully visualized the dissociation of the duplex structure. Furthermore, we succeeded in observing spontaneous reannealing of the duplex structure upon releasing the force, which demonstrates that the folding reaction can be reversibly controlled. We also reveal that along with duplex unfolding, a metastable secondary structure is generated and its topology changes transiently over time. The results indicate that this technique provides structural insights that cannot be obtained by conventional fluorescence techniques.

biophysics↗