記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell Reports  

Cardiomyocyte (CM) nuclei are constantly exposed to mechanical stress, but how they maintain their nuclear shape remains unknown. In this study, we found that ventricular CM nuclei acquire characteristic prominent spatial organizations of heterochromatin (SOH), which are disrupted by high-level expression of H2B-mCherry in mice. SOH disruption was associated with nuclear softening, leading to extreme elongation and rupture under unidirectional mechanical stress. Loosened chromatin then leaks into the cytosol, causing severe inflammation and cardiac dysfunction. Although SOH disruption was accompanied by loosened higher-order genomic structures, the change in gene expression before nuclear deformation was mild, suggesting that SOH play major roles in nuclear structural integrity. Aged CM nuclei consistently exhibited scattered SOH and marked elongation. Furthermore, we provide mechanistic insight into the development and maintenance of SOH driven by chromatin compaction and condensate formation. These results highlight SOH as a safeguard of nuclear shape and genomic integrity against mechanical stress.

DOI： 10.1016/j.celrep.2024.115048

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Iscience  

How actin filaments (F-actins) are dynamically reorganized in motile cells at the level of individual filaments is an open question. To find the answer, a high-speed atomic force microscopy (HS-AFM) system has been developed to live-imagine intracellular dynamics of the individual F-actins. However, noise and low resolution made it difficult to fully recognize individual F-actins in the HS-AFM images. To tackle this problem, we developed a new machine learning method that quantitatively recognizes individual F-actins. The method estimates F-actin orientation from the image while improving the resolution. We found that F-actins were oriented at ±35° toward the membrane in the lamellipodia, which is consistent with Arp2/3 complex-induced branching. Furthermore, in the cell cortex our results showed non-random orientation at four specific angles, suggesting a new mechanism for F-actin organization demonstrating the potential of our newly developed method to fundamentally improve our understanding of the structural dynamics of F-actin networks.

DOI： 10.1016/j.isci.2024.110907

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Cell Science  

Liquid–liquid phase separation (LLPS) has increasingly been found to play pivotal roles in a number of intracellular events and reactions, and has introduced a new paradigm in cell biology to explain protein–protein and enzyme–ligand interactions beyond conventional molecular and biochemical theories. LLPS is driven by the cumulative effects of weak and promiscuous interactions, including electrostatic, hydrophobic and cation–π interactions, among polypeptides containing intrinsically disordered regions (IDRs) and describes the macroscopic behaviours of IDR-containing proteins in an intracellular milieu. Recent studies have revealed that interactions between ‘charge blocks’ – clusters of like charges along the polypeptide chain – strongly induce LLPS and play fundamental roles in its spatiotemporal regulation. Introducing a new parameter, termed ‘charge blockiness’, into physicochemical models of disordered polypeptides has yielded a better understanding of how the intrinsic amino acid sequence of a polypeptide determines the spatiotemporal occurrence of LLPS within a cell. Charge blockiness might also explain why some post-translational modifications segregate within IDRs and how they regulate LLPS. In this Review, we summarise recent progress towards understanding the mechanism and biological roles of charge block-driven LLPS and discuss how this new characteristic parameter of polypeptides offers new possibilities in the fields of structural biology and cell biology.

DOI： 10.1242/jcs.261394

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Iscience  

Most mammalian cells prevent viral infection and proliferation by expressing various restriction factors and sensors that activate the immune system. Several host restriction factors that inhibit human immunodeficiency virus type 1 (HIV-1) have been identified, but most of them are antagonized by viral proteins. Here, we describe CCHC-type zinc-finger-containing protein 3 (ZCCHC3) as a novel HIV-1 restriction factor that suppresses the production of HIV-1 and other retroviruses, but does not appear to be directly antagonized by viral proteins. It acts by binding to Gag nucleocapsid (GagNC) via zinc-finger motifs, which inhibits viral genome recruitment and results in genome-deficient virion production. ZCCHC3 also binds to the long terminal repeat on the viral genome via the middle-folded domain, sequestering the viral genome to P-bodies, which leads to decreased viral replication and production. This distinct, dual-acting antiviral mechanism makes upregulation of ZCCHC3 a novel potential therapeutic strategy.

DOI： 10.1016/j.isci.2024.109107

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記述言語：英語   出版者・発行元：Scientific Reports  

Correction to: Scientific Reports, published online 18 April 2023 The original version of this Article contained errors in Figures. 2, 3 and 5 and the legends where the statistical analyses were incorrect. The original Figures 2, 3 and 5 and accompanying legends appear below. (Figure presented.) (Figure presented.) (Figure presented.) Dissociation of RIG-I/poly (I:C) complex in the presence of ATP. (a) Recombinant RIG-I (insect cells) was incubated with LMW or HMW poly (I:C) at 37 °C for 60 min as in Fig. 1C. RIG-I/poly (I:C) complexes on magnetic beads were collected. After washing the beads three times, ATP was added to the beads at final concentration of 1 mM and incubated at 37 °C for different times. RIG-I/dsRNA complexes remaining on the beads were isolated and analyzed by immunoblotting using anti-Flag (top). The dissociation kinetics was determining by quantification of the band intensities (bottom). The uncropped original blots are shown in Supplementary Fig. S8a. (b) AFM observation of RIG-I/HMW poly (I:C) complex. RIG-I and HMW poly (I:C) were incubated at 37 °C for 30 min, then further incubated in the presence of 1 mM ATP and subjected to AFM analysis. Four representative fields are shown. For size comparison, see AFM image of naïve RIG-I in Fig. 3A. (c) The effect of ATP on the dissociation of RIG-I/LMW poly (I:C) complex was analyzed. The RIG-I/LMW poly (I:C) complexes formed on the magnetic beads were isolated and incubated in the absence or presence of 1 mM ATP at 37 °C for different times. RIG-I bound to poly (I:C) (beads) was analyzed by immunoblotting as in (a). The uncropped original blots are shown in Supplementary Fig. S8b. (d) The effect of AMP-PNP on dissociation of RIG-I/LMW poly (I:C) complex. RIG-I/LMW poly (I:C) complex was incubated in the absence or presence of 1 mM AMP-PNP or ATP and analyzed as in (a). The uncropped original blot is shown in Supplementary Fig. S8c. The percentage of association and dissociation was calculated from band intensities (bottom). Error bars represent standard deviation (n = 3). *P < 0.05,. **P < 0.01, unpaired Student’s t test. n.s, not significant. Morphological analysis of RIG-I protein by AFM. (a) Purified Flag-tagged RIG-I expressed in 293 T cells (naïve RIG-I, METHODS) was analyzed by AFM (top). Naïve RIG-I was incubated with 1 mM ATP (middle). Naïve RIG-I was bound to immobilized LMW poly (I:C) and incubated with 1 mM ATP for 30 min and released RIG-I (dissociated RIG-I) was recovered and subjected to AFM analysis (bottom). Enlarged images are shown as inset (500 × 500 nm2). (b) The diameter of objects observed by AFM were measured and plotted as histogram. Average diameter (c), height (d) and volume (e) were similarly determined. Error bars represent standard deviation (n = 50). ***P < 0.001, unpaired Student’s t test. n.s, not significant. Biological activity of dissociated RIG-I in cells. (a) 293 T cells were transfected with p-55C1BLuc, reporter gene containing repetitive IRF3 binding sites, and pRL-TK as an internal control vector. Twenty-four hours after transfection, cells were transfected with 1 µg of naïve or dissociated RIG-I protein, isolated from MDA5 KO 293 T cells, by protein transfection reagent for 2 h (METHODS). After 4 h cultivation with fresh medium, cell extracts were prepared for dual-luciferase assay. Relative firefly luciferase activity normalized by Renilla luciferase activity is shown. Luciferase activity data are the average of 2 independent experiments. Error bars represent standard deviation (n = 2). *P < 0.05, unpaired Student’s t-test. n.s, not significant. (b) The cell extracts were analyzed by native PAGE for IRF-3 dimer formation. The positions of monomers and dimers are indicated. The uncropped original blot is shown in Supplementary Fig. S10. (c) Naïve or dissociated RIG-I protein, isolated from MDA5 KO 293 T cells, was transfected into HeLa by after 30 min incubation with RNase III. The expression level of IFNB was analyzed by qPCR. Error bars represent standard deviation (n = 2). *P < 0.05, unpaired Student’s t-test. In addition, the Supplementary Information file published with this Article contained an error in the Supplementary Figure S1. The original Supplementary Information file is provided below. The original Article and the Supplementary Information file have been corrected.

DOI： 10.1038/s41598-023-50644-w

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：一般社団法人 日本生物物理学会  

Phosphorylation regulates protein function by modulating stereospecific interactions between protein-protein or enzyme-ligand. On the other hand, many bioinformatics studies have demonstrated that phosphorylation preferably occurs in intrinsically disordered regions (IDRs), which do not have any secondary and tertiary structures. Although studies have demonstrated that phosphorylation changes the phase behavior of IDRs, the mechanism, which is distinct from the “stereospecific” effect, had not been elucidated. Here, we describe how phosphorylation in IDRs regulates the protein function by modulating phase behavior. Mitotic phosphorylation in the IDRs of Ki-67 and NPM1 promotes or suppresses liquid-liquid phase separation, respectively, by altering the “charge blockiness” along the polypeptide chain. The phosphorylation-mediated regulation of liquid-liquid phase separation by enhancing or suppressing "charge blockiness," rather than by modulating stereospecific interactions, may provide one of the general mechanisms of protein regulation by posttranslational modifications and the role of multiple phosphorylations.

DOI： 10.2142/biophysico.bppb-v21.0012

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：FASEB Journal  

Yes-associated protein (YAP) is a transcriptional co-activator that controls the transcription of target genes and modulates the structures of various cytoskeletal architecture as mechanical responses. Although it has been known that YAP regulates actin-regulatory proteins, the detailed molecular mechanism of how they control and coordinate intracellular actin architecture remains elusive. Herein, we aimed to examine the structure and dynamics of intracellular actin architecture from molecular to cellular scales in normal and YAP-knockout (YAP-KO) cells utilizing high-speed atomic force microscopy (HS-AFM) for live-cell imaging and other microscope-based mechanical manipulation and measurement techniques. YAP-KO Madin–Darby canine kidney cells had a higher density and turnover of actin filaments in the cell cortex and a higher elastic modulus. Laser aberration assay demonstrated that YAP-KO cells were more resistant to damage than normal cells. We also found that Rho GTPase-activating protein 18 (ARHGAP18), a downstream factor of YAP, translocated from the cortex to the edge of sparsely cultured YAP-KO cells. It resulted in high RhoA activity and promotion of actin polymerization in the cell cortex and their reductions at the edge. HS-AFM imaging of live cell edge and a cell-migration assay demonstrated lower membrane dynamics and motility of YAP-KO cells than those of normal cells, suggesting lower actin dynamics at the edge. Together, these results demonstrate that a YAP-dependent pathway changes the intracellular distribution of RhoGAP and modulates actin dynamics in different parts of the cell, providing a mechanistic insight into how a mechano-sensitive transcription cofactor regulates multiple intracellular actin architecture and coordinates mechano-responses.

DOI： 10.1096/fj.202201992R

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記述言語：英語   出版者・発行元：Nature Communications  

Clathrin-mediated endocytosis is pivotal to signal transduction pathways between the extracellular environment and the intracellular space. Evidence from live-cell imaging and super-resolution microscopy of mammalian cells suggests an asymmetric distribution of actin fibres near the clathrin-coated pit, which induces asymmetric pit-closing rather than radial constriction. However, detailed molecular mechanisms of this ‘asymmetricity’ remain elusive. Herein, we used high-speed atomic force microscopy to demonstrate that CIP4, a multi-domain protein with a classic F-BAR domain and intrinsically disordered regions, is necessary for asymmetric pit-closing. Strong self-assembly of CIP4 via intrinsically disordered regions, together with stereospecific interactions with the curved membrane and actin-regulating proteins, generates a small actin-rich environment near the pit, which deforms the membrane and closes the pit. Our results provide mechanistic insights into how disordered and structured domain collaboration promotes spatio-temporal actin polymerisation near the plasma membrane.

DOI： 10.1038/s41467-023-40390-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Signal Processing  

The detrending moving average (DMA) analysis demonstrates excellent performance for the characterization of long-range correlations and fractal scaling and is performed in various research fields. The conventional DMA with a simple moving average can remove linear trends embedded in the observed time series. To improve the detrending ability of the DMA, higher-order DMA including a higher order polynomial detrending was also introduced using the Savitzky-Golay filter and its fast implementation algorithm was developed. However, the higher-order DMA applicable to higher dimensional data is yet to be well established. As the data dimension increases, an increase in the computational cost becomes a problem that needs to be resolved. Further, the implementation of the higher order DMA is a time-consuming procedure. To resolve this problem, we here proposed a fast algorithm for multidimensional DMA with higher order polynomial detrending. In the proposed algorithm, to reduce the computational complexity, parallel translation and recurrence techniques are introduced. Monte Carlo experiments for two-dimensional data show that the computational time of the proposed algorithm is approximately proportional to the cubic of the data length, whereas the computational time of the conventional implementation is approximately proportional to the quartic of the data length. Moreover, we evaluate the estimation accuracy of the Hurst exponent of the proposed method. Finally, we demonstrate the possible application of the proposed method by estimating the Hurst exponent of images.

DOI： 10.1016/j.sigpro.2023.108997

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

Retinoic acid-inducible gene I (RIG-I) is the most front-line cytoplasmic viral RNA sensor and induces antiviral immune responses. RIG-I recognizes short double-stranded (dsRNA) (< 500 bp), but not long dsRNA (> 500 bp) to trigger antiviral signaling. Since RIG-I is capable of binding with dsRNA irrespective of size, length-dependent RIG-I signaling remains elusive. Here, we demonstrated that RIG-I bound to long dsRNA with slow kinetics. Remarkably, RIG-I/short dsRNA complex efficiently dissociated in an ATP hydrolysis-dependent manner, whereas RIG-I/long dsRNA was stable and did not dissociate. Our study suggests that the dissociation of RIG-I from RIG-I/dsRNA complex could be a step for efficient antiviral signaling. Dissociated RIG-I exhibited homo-oligomerization, acquiring ability to physically associate with MAVS, and biological activity upon introduction into living cells. We herein discuss common and unique mechanisms of viral dsRNA recognition by RIG-I and MDA5.

DOI： 10.1038/s41598-023-33208-w

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記述言語：日本語   出版者・発行元：公益社団法人日本生化学会  

リン酸化をはじめとするタンパク質翻訳後修飾は，「立体構造特異的」にタンパク質機能を制御すると考えられてきた．しかし，近年のバイオインフォマティクス解析からは，翻訳後修飾の多くは，立体構造を持たないタンパク質領域（天然変性領域）に生じることが示されている．リン酸化が天然変性領域の液–液相分離を制御する事例が報告されているが，そのメカニズムは不明であった．ここでは，近年報告されたリン酸化の「電荷ブロック」型の液–液相分離制御機構に焦点を当て，従来の「立体構造特異的」制御との相違点を交えながらその特徴を解説する．

DOI： 10.14952/seikagaku.2023.950029

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

<p>タンパク質のリン酸化は，非構造領域（IDR）で高頻度に生じる．この事実は，リン酸化によるタンパク質機能制御が，従来の「立体構造特異性」のみでは説明しきれないことを示す．本稿では，IDRにおけるリン酸化が，電荷ブロックを形成・消失させることで液－液相分離を促進・抑制するという新しい仕組みを概説する．</p>

DOI： 10.2142/biophys.63.153

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Molecular Neurodegeneration  

Background: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Methods: We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. Results: We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. Conclusion: Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration. Graphical Abstract: [Figure not available: see fulltext.].

DOI： 10.1186/s13024-022-00585-1

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記述言語：英語   出版者・発行元：Springer Nature  

Dynamic morphological changes of intracellular organelles are often regulated by protein phosphorylation or dephosphorylation^1–6. Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown. Here we determined the cell cycle-specific behaviour of Ki-67, which localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis. Mitotic hyperphosphorylation of disordered repeat domains of Ki-67 generates alternating charge blocks in these domains and increases their propensity for liquid–liquid phase separation (LLPS). A phosphomimetic sequence and the sequences with enhanced charge blockiness underwent strong LLPS in vitro and induced chromosome periphery formation in vivo. Conversely, mitotic hyperphosphorylation of NPM1 diminished a charge block and suppressed LLPS, resulting in nucleolar dissolution. Cell cycle-specific phase separation can be modulated via phosphorylation by enhancing or reducing the charge blockiness of disordered regions, rather than by attaching phosphate groups to specific sites.

DOI： 10.1038/s41556-022-00903-1

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2022.0.2s25.0

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1101/2022.11.21.517438

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

Dynamic morphological changes of intracellular organelles are often regulated by protein phosphorylation or dephosphorylation^1–6. Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown. Here we determined the cell cycle-specific behaviour of Ki-67, which localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis. Mitotic hyperphosphorylation of disordered repeat domains of Ki-67 generates alternating charge blocks in these domains and increases their propensity for liquid–liquid phase separation (LLPS). A phosphomimetic sequence and the sequences with enhanced charge blockiness underwent strong LLPS in vitro and induced chromosome periphery formation in vivo. Conversely, mitotic hyperphosphorylation of NPM1 diminished a charge block and suppressed LLPS, resulting in nucleolar dissolution. Cell cycle-specific phase separation can be modulated via phosphorylation by enhancing or reducing the charge blockiness of disordered regions, rather than by attaching phosphate groups to specific sites.

その他リンク： https://www.nature.com/articles/s41556-022-00903-1

記述言語：日本語  

記述言語：日本語  

記述言語：日本語   会議種別：口頭発表（招待・特別）  

記述言語：日本語   会議種別：口頭発表（招待・特別）  

記述言語：日本語   会議種別：口頭発表（招待・特別）  

記述言語：日本語   会議種別：口頭発表（招待・特別）  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語   会議種別：口頭発表（招待・特別）  

記述言語：英語  

記述言語：英語  

記述言語：英語