Biology subjects

Takada, S.

Publications and source records attributed to Takada, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

MitoNEET reduces the mitochondrial oxidative phosphorylation during epithelial-mesenchymal transition

Mitochondrial functions range from catabolic to anabolic, which are tightly coordinated to meet cellular demands for proliferation and motility. MitoNEET is a mitochondrial outer membrane protein with a CDGSH domain and is involved in mitochondrial function. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose their epithelial characteristics and acquire mesenchymal traits, such as motility, which is a vital step for organism development and wound-healing. Cellular motility is associated with high ATP consumption owing to lamellipodia formation, which is supported by upregulated oxidative phosphorylation (OXPHOS) capacity. However, how mitoNEET is involved in the regulation of OXPHOS capacity and subsequent cellular motility remains unclear. Here we show that loss of mitoNEET regulation during EMT impairs both OXPHOS enhancement and cell motility in non-transformed NMuMG mouse mammary gland epithelial cells. We found that mitoNEET is downregulated during EMT, and that the aberrant expression of mitoNEET abolishes the upregulation of OXPHOS, leading to the inhibition of cell motility. Furthermore, we found that mitoNEET topology may be crucial for the regulation of the mitochondrial electron transfer chain, suggesting an additional regulatory pathway for OXPHOS capacity. Our results demonstrate that mitochondrial OXPHOS capacity during EMT is partly regulated by the dynamics of the outer membrane protein. We believe that our findings are the first step towards understanding the mechanisms by which mitochondrial outer membrane protein topology affects organelle functions.

cell biology↗

bioRxiv2024

Heterogenous organization in condensates of multiple transcription factors in embryonic stem cells

Biomolecular condensates formed via liquid-liquid phase separation are ubiquitous in cells, especially in the nucleus. While condensates containing one or two kinds of biomolecules have been relatively well characterized, those with more heterogenous biomolecular components and interactions between biomolecules inside are largely unknown. This study used residue-resolution molecular dynamics simulations to investigate heterogeneous protein assemblies that include four master transcription factors in mammalian embryonic stem cells: Oct4, Sox2, Klf4, and Nanog. Simulations of the mixture systems showed highly heterogeneous and dynamic behaviors; the condensates mainly contained Sox2, Klf4, and Nanog, while Oct4 was dissolved into the dilute phase. Condensates consisted of loosely interacting clusters in which Klf4 was the most abundant in the cores. We suggest that Klf4 serves as a scaffold of the condensate where Sox2 and Nanog are bound to stabilize the condensate, whereas Oct4 is moderately recruited to the condensate, serving as a client mainly via its interaction with Sox2. Biological significanceIn eukaryotes, transcription is known to be regulated by many regulatory factors such as transcription factors, co-activators, and RNA polymerases, but precise molecular mechanisms of regulation remain obscured. A recently proposed model suggests that transcription-related proteins condense via liquid-liquid phase separation (LLPS) using their intrinsically disordered regions, which serves to control transcription. Master transcription factors in mammalian embryonic stem cells have been a model system. It was revealed that several master transcription factors exhibit LLPS by themselves, but dynamics and molecular structure of these proteins in their mixture have not been well addressed. In this study, we study molecular structures of condensates in a mixture of four master transcription factors, Oct4, Sox2, Klf4, and Nanog, via molecular dynamics simulation. We found that the three transcription factors Sox2, Klf4, and Nanog form mixed condensates, while Oct4 was largely dissolved. Klf4 mainly served as a scaffold of the condensate. The three proteins formed micelle-like structures as was recently found in the Nanog condensate. The condensates weakly recruited Oct4. Formation of heterogeneous condensates may provide fertile local environments in cells.

biophysics↗

bioRxiv2024

Chromosomal domain formation by archaeal SMC, a roadblock protein, and DNA structure

Structural maintenance of chromosomes (SMC) complexes fold genomes by extruding DNA loops. In eukaryotes, loop-extruding SMC complexes form topologically associating domains (TADs) by being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using high-resolution chromosome conformation capture sequencing, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We also find that TrmBL2, a nucleoid- associated protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif. TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with structural properties of underlying DNA sequences. Our study illuminates not only a eukaryotic-like mechanism of domain formation in archaea, but also an unforeseen role of intrinsic DNA structure in large-scale genome organization.

molecular biology↗

bioRxiv2024

SMC complex unidirectionally translocates DNA by coupling segment capture with an asymmetric kleisin path

SMC (structural maintenance of chromosomes) protein complexes are ring-shaped molecular motors essential for genome folding. Despite recent progress, the detailed molecular mechanism of DNA translocation in concert with the ATP-driven conformational changes of the complex remains to be clarified. In this study, we elucidated the mechanisms of SMC action on DNA using all-atom and coarse-grained molecular dynamics simulations. We first created a near-atomic full-length model of a prokaryotic SMC-kleisin complex based on experimental structures and implemented ATP-dependent conformational changes using a structure-based coarse-grained model. We further incorporated key protein-DNA hydrogen bond interactions derived from fully atomistic simulations. Extensive simulations of the SMC complex with 800 base pairs of duplex DNA over the ATP cycle observed unidirectional DNA translocation by the SMC complex. The process exhibited a step size of [~]200 base pairs, wherein the SMC complex captured a DNA segment of about the same size within the SMC ring in the engaged state, followed by its pumping into the kleisin ring as ATP was hydrolyzed. Analysis of trajectories identified the asymmetric path of the kleisin as a critical factor for the observed unidirectionality. SIGNIFICANCE STATEMENTRing-shaped SMC (structural maintenance of chromosomes) protein complexes, which are highly conserved across all three domains of life, play an essential role in chromosome organization through a process called DNA loop extrusion. However, the molecular mechanism underlying the ATP-dependent motor activity of SMC complexes remains unclear. Using all-atom and residue-resolution coarse-grained molecular dynamics simulations, we revealed that prokaryotic SMC complexes translocate unidirectionally along DNA via a segment capture mechanism. We found that the unidirectionality arises from the kleisin subunit breaking the symmetry of the ring-shaped SMC complex structure. Our findings provide insights into the molecular motor mechanisms shared by SMC complexes.

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↗

bioRxiv2023

Multiscale Bayesian Simulations Reveal Functional Chromatin Condensation of Gene Loci

Chromatin, the complex assembly of DNA and associated proteins, plays a pivotal role in orchestrating various genomic functions. To aid our understanding of the principles underlying chromatin organization, we introduce Hi-C metainference, a Bayesian approach that integrates Hi-C contact frequencies into multiscale prior models of chromatin. This approach combines both bottom-up (the physics-based prior) and top-down (the data-driven posterior) strategies to characterize the 3D organization of a target genomic locus. We first demonstrate the capability of this method to accurately reconstruct the structural ensemble and the dynamics of a system from contact information. We then apply the approach to investigate the Sox2, Pou5f1, and Nanog loci of mouse embryonic stem cells using a bottom-up chromatin model at 1kb resolution. We observe that the studied loci are conformationally heterogeneous and organized as crumpled globules, favoring contacts between distant enhancers and promoters. Using nucleosome-resolution simulations, we then reveal how the Nanog gene is functionally organized across the multiple scales of chromatin. At the local level, diverse chromatin folding motifs correlate with epigenetics, with open chromatin predominantly observed at cis-regulatory elements and compact tetranucleosomes in between. At the larger scale, we find that enhancer-promoter contacts are driven by the transient condensation of chromatin into compact domains stabilized by extensive inter-nucleosome interactions. Overall, this work highlights the condensed, but dynamic nature of chromatin in vivo, contributing to a deeper understanding of gene structure-function relationships.

biophysics↗

bioRxiv2023

Micelle-like clusters in phase-separated Nanog condensates: A molecular simulation study

The phase separation model for transcription suggests that transcription factors (TFs), coactivators, and RNA polymerases form biomolecular condensates around active gene loci and regulate transcription. However, the structural details of condensates remain elusive. In this study, for Nanog, a master TF in mammalian embryonic stem cells known to form protein condensates in vitro, we examined protein structures in the condensates using residue-level coarse-grained molecular simulations. Human Nanog formed micelle-like clusters in the condensate. In the micelle-like cluster, the C-terminal disordered domains, including the tryptophan repeat (WR) regions, interacted with each other near the cluster center primarily via hydrophobic interaction. In contrast, hydrophilic disordered N-terminal and DNA-binding domains were exposed on the surface of the clusters. Electrostatic attractions of these surface residues were responsible for bridging multiple micelle-like structures in the condensate. The micelle-like structure and condensate were dynamic and liquid-like. Mutation of tryptophan residues in the WR region which was implicated to be important for a Nanog function resulted in dissolution of the Nanog condensate. Finally, to examine the impact of Nanog cluster to DNA, we added DNA fragments to the Nanog condensate. Nanog DNA-binding domains exposed to the surface of the micelle-like cluster could recruit more than one DNA fragments, making DNA-DNA distance shorter. Author summaryIn eukaryotic transcription regulation, enhancer elements far from the promoter are known to modulate transcriptional activity, but the molecular mechanism remains elusive. One of these models, the phase separation model, suggests that transcription factors (TFs), coactivators, and transcription machinery form biomolecular condensates that include enhancer and promoter elements, making enhancer-promoter communication possible via the protein network in the condensate. Nanog, one of the core TFs involved in mammalian embryonic stem cells, has been reported to have the ability to form condensates. In this study, we addressed the structural details of Nanog condensates by performing residue-level coarse-grained molecular simulations. We found that Nanog formed micelle-like clusters via primarily hydrophobic interactions between tryptophan repeat regions in the C-terminal disordered domains within the condensate. On the other hand, highly charged N-terminal and DNA-binding domains were exposed to the surface of the micelle and were responsible for bridging many micelles into a condensate. The micelle-like clusters and condensate were dynamic and liquid-like. In addition, Nanog condensates could induce DNA-DNA attraction mediated by micelle-like structures.

biophysics↗

bioRxiv2023

Postsynaptic protein assembly in three- and two-dimensions studied by mesoscopic simulations

Recently, cellular biomolecular condensates formed via phase separation have received considerable attention. While they can be formed either in cytosol (denoted as 3D) or beneath the membrane (2D), the underlying difference between the two has not been well clarified. To compare the phase behaviors in 3D and 2D, postsynaptic density (PSD) serves as a model system. PSD is a protein condensate located under the postsynaptic membrane that influences the localization of glutamate receptors and thus contributes to synaptic plasticity. Recent in vitro studies have revealed the formation of droplets of various soluble PSD proteins via liquid-liquid phase separation. However, it is unclear how these protein condensates are formed beneath the membrane and how they specifically affect the localization of glutamate receptors in the membrane. In this study, focusing on the mixture of a glutamate receptor complex, AMPAR-TARP, and a ubiquitous scaffolding protein, PSD-95, we constructed a mesoscopic model of protein-domain interactions in PSD and performed comparative molecular simulations. The results showed a sharp contrast in the phase behaviors of protein assemblies in 3D and those under the membrane (2D). A mixture of a soluble variant of the AMPAR-TARP complex and PSD-95 in the 3D system resulted in a phase-separated condensate, which was consistent with the experimental results. However, with identical domain interactions, AMPAR-TARP embedded in the membrane formed clusters with PSD-95, but did not form a stable separated phase. Thus, the cluster formation behaviors of PSD proteins in the 3D and 2D systems were distinct. The current study suggests that, more generally, stable phase separation can be more difficult to achieve in and beneath the membrane than in 3D systems. SIGNIFICANCESynaptic plasticity is a key factor in memory and learning. Upon learning, protein condensates that form beneath the postsynaptic membrane are known to change their nature. Recent studies have suggested that condensate formation is related to liquid-liquid phase separation based on in vitro experiments of soluble parts. However, the phase behavior can be strongly dependent on physical dimensions. The mechanism by which condensate grows beneath the membrane is not well characterized. Taking advantage of the ease of systematic comparison using computer simulations, we investigated the phase behaviors of postsynaptic protein assemblies in 3D and 2D systems. The results revealed that even when a 3D system exhibited clear phase separation, the corresponding 2D system did not exhibit it stably.

biophysics↗

bioRxiv2023

DNA methylation signature in NSD2 loss-of-function variants appeared similar to that in Wolf-Hirschhorn syndrome

PurposeWolf-Hirschhorn syndrome (WHS), a contiguous gene syndrome caused by the hemizygous deletion of the distal short arm of chromosome 4 where NSD2 is, reportedly exhibits specific DNA methylation signatures in peripheral blood cells. However, responsible genomic loci for signatures are unreported. The objective of the study is to define the loci of WHS-related DNA methylation signatures and to explore the role of NSD2 for the signatures. MethodsWe conducted genome-wide methylation analysis of individuals with WHS or NSD2 variants using array. We studied genome-edited knock in mice or induced pluripotent stem cells to explore the function of NSD2 variants which are observed in congenital anomaly cases. ResultsThree undiagnosed cases with NSD2 variants showed WHS-related DNA methylation signatures. These variants were validated to be NSD2 loss-of-function in induced pluripotent stem cells or genome-edited knock-in mice. p.Pro905Leu variant decreased Nsd2 protein levels, and changed Histone H3-Lysine 36 demethylation levels in similar way in the same genomic regions as Nsd2 knock out mice regulated. Nsd2 knock out mice exhibited common DNA methylation changes. ConclusionThese results revealed that WHS-related DNA methylation signatures are dependent on NSD2 dysfunction and are useful in diagnosing NSD2 variants of unknown significance.

genetics↗

bioRxiv2022

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of phosphorylation remain unclear. Herein, we identify ULK1 as a kinase responsible for phosphorylation of p62. ULK1 co-localizes with p62 bodies, and directly interacts with p62. This phosphorylation allows KEAP1 to be retained within p62 bodies, activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351 corresponding to human Ser349 is replaced by Glu. These mice, but not phosphodefective p62S351A/S351A mice, exhibit NRF2 hyperactivation and growth retardation, the latter caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis. p62S351E/+ mice are a phenocopy of systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insight into the role of phase separation in this process.

cell biology↗

bioRxiv2022

End-to-End Differentiable Blind Tip Reconstruction for Noisy Atomic Force Microscopy Images

Observing the structural dynamics of biomolecules is vital to deepening our understanding of biomolecular functions. High-speed (HS) atomic force microscopy (AFM) is a powerful method to measure biomolecular behavior at near physiological conditions. In the AFM, measured image profiles on a molecular surface are distorted by the tip shape through the interactions between the tip and molecule. Once the tip shape is known, AFM images can be approximately deconvolved to reconstruct the surface geometry of the sample molecule. Thus, knowing the correct tip shape is an important issue in the AFM image analysis. The blind tip reconstruction (BTR) method developed by Villarrubia1 is an algorithm that estimates tip shape only from AFM images using mathematical morphology operators. While the BTR works perfectly for noise-free AFM images, the algorithm is susceptible to noise, or it is difficult to determine a threshold parameter against noise. To overcome this issue, we here propose an alternative BTR method, called end-to-end differentiable BTR, based on a modern machine learning approach. In the method, we introduce a loss function and a regularization term to prevent overfitting to noise, and the tip shape is optimized with automatic differentiation and backpropagations developed in deep learning frameworks. Using noisy pseudo-AFM images of myosin V motor domain as test cases, we show that our end-to-end differentiable BTR is robust against noise in AFM images. The method can also detect a double-tip shape and deconvolve doubled molecular images. Finally, application to real HS-AFM data of myosin V walking on an actin filament shows that the method can reconstruct the accurate surface geometry of actomyosin consistent with the structural model. Our method serves as a general post-processing for reconstructing hidden molecular surfaces from any AFM images. Codes are available at https://github.com/matsunagalab/differentiable_BTR.

biophysics↗

bioRxiv2022

Conservation and divergence of canonical and non-canonical imprinting in murids

BackgroundGenomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behaviour. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we used low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extra-embryonic tissues at allele-specific resolution. ResultsWe identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse revealed that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extra-embryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extra-embryonic imprinting. Meta-analysis of novel imprinted genes revealed multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the birth of ZFP57 binding motifs and the insertion of endogenous retroviral promoters. ConclusionsIn summary, we provide a comprehensive list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.

genomics↗

bioRxiv2022

The stoichiometric interaction model for mesoscopic molecular dynamics simulations of liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) has received considerable attention in recent years for explaining the formation of cellular biomolecular condensates. The fluidity and the complexity of their components make molecular simulation approaches indispensable for gaining structural insights. Domain-resolution mesoscopic model simulations have been explored for case in which condensates are formed by multivalent proteins with tandem domains. One problem with this approach is that interdomain pairwise interactions cannot regulate the valency of the binding domains. To overcome this problem, we propose a new potential, the stoichiometric interaction (SI) potential. First, we verified that the SI potential maintained the valency of the interacting domains for the test systems. We then examined a well-studied LLPS model system containing tandem repeats of SH3 domains and proline-rich motifs. We found that the SI potential alone cannot reproduce the phase diagram of LLPS quantitatively. We had to combine the SI and a pairwise interaction; the former and the latter represent the specific and non-specific interactions, respectively. Biomolecular condensates with the mixed SI and pairwise interaction exhibited fluidity, whereas those with the pairwise interaction alone showed no detectable diffusion. We also compared the phase diagrams of the systems containing different numbers of tandem domains with those obtained from the experiments, and found quantitative agreement in all but one case. SIGNIFICANCECells organize their interior structures as not only membrane-bound organelles but also as membrane-less organelles. Membrane-less organelles, such as stress granules, Cajal bodies, and postsynaptic density, are biomolecular condensates in which many biomolecules are gathered owing to their interactions. In some cases, biomolecular condensates are formed by tandemly connected multidomain proteins. In such cases, a mesoscopic simulation model representing each domain as a particle is effective; however, the problem with this approach is that a domain-domain pairwise interaction cannot regulate the well-defined valency. To overcome this problem, in this study, we have developed a new potential, viz. the stoichiometric interaction potential, and confirmed that this potential can reproduce the liquid-liquid phase separation of multidomain proteins, a hallmark of the membrane-less organelles.

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

Mode selection mechanism in traveling and standing waves revealed by Min wave reconstituted in artificial cells

Reaction-diffusion coupling (RDc) generates spatiotemporal patterns, including two dynamic wave modes: traveling and standing waves. Although mode selection plays a significant role in the spatiotemporal organization of living cell molecules, the mechanism for selecting each wave mode remains elusive. Here, we investigated a wave mode selection mechanism using Min waves reconstituted in artificial cells, emerged by the RDc of MinD and MinE. Our experiments and theoretical analysis revealed that the balance of membrane binding and dissociation from the membrane of MinD determines the mode selection of the Min wave. We successfully demonstrated that the transition of the wave modes can be regulated by controlling this balance and found hysteresis characteristics in the wave mode transition. These findings highlight a novel role of the balance between activators and inhibitors as a determinant of the mode selection of waves by RDc and depict a novel mechanism in intracellular spatiotemporal pattern formations. TeaserActivator-inhibitor balance determines whether a nonlinear wave in live cells becomes a traveling wave or standing wave.

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

Implementation of residue-level coarse-grained models in GENESIS for large-scale molecular dynamics simulations

Residue-level coarse-grained (CG) models have become one of the most popular tools in biomolecular simulations in the trade-off between modeling accuracy and computational efficiency. To investigate large-scale biological phenomena in molecular dynamics (MD) simulations with CG models, unified treatments of proteins and nucleic acids, as well as efficient parallel computations, are indispensable. In the GENESIS MD software, we implement several residue-level CG models, covering structure-based and context-based potentials for both well-folded biomolecules and intrinsically disordered regions. An amino acid residue in protein is represented as a single CG particle centered at the C atom position, while a nucleotide in RNA or DNA is modeled with three beads. Then, a single CG particle represents around ten heavy atoms in both proteins and nucleic acids. The input data in CG MD simulations are treated as GROMACS-style input files generated from a newly developed toolbox, GENESIS-CG-tool. To optimize the performance in CG MD simulations, we utilize multiple neighbor lists, each of which is attached to a different nonbonded interaction potential in the cell-linked list method. We found that random number generations for Gaussian distributions in the Langevin thermostat are one of the bottlenecks in CG MD simulations. Therefore, we parallelize the computations with message-passing-interface (MPI) to improve the performance on PC clusters or supercomputers. We simulate Herpes simplex virus (HSV) type 2 B-capsid and chromatin models containing more than 1,000 nucleosomes in GENESIS as examples of large-scale biomolecular simulations with residue-level CG models. This framework extends accessible spatial and temporal scales by multi-scale simulations to study biologically relevant phenomena, such as genome-scale chromatin folding or phase-separated membrane-less condensations. Author summaryMolecular dynamics (MD) simulations have been widely used to investigate biological phenomena that are difficult to study only with experiments. Since all-atom MD simulations of large biomolecular complexes are computationally expensive, coarse-grained (CG) models based on different approximations and interaction potentials have been developed so far. There are two practical issues in biological MD simulations with CG models. The first issue is the input file generations of highly heterogeneous systems. In contrast to well-established all-atom models, specific features are introduced in each CG model, making it difficult to generate input data for the systems containing different types of biomolecules. The second issue is how to improve the computational performance in CG MD simulations of heterogeneous biological systems. Here, we introduce a user-friendly toolbox to generate input files of residue-level CG models containing folded and disordered proteins, RNAs, and DNAs using a unified format and optimize the performance of CG MD simulations via efficient parallelization in GENESIS software. Our implementation will serve as a framework to develop novel CG models and investigate various biological phenomena in the cell.

biophysics↗

bioRxiv2021

Molecular atlas of the human brain vasculature at the single-cell level

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

neuroscience↗