Chromatin potential for gene regulation

金沢大学ナノ生命科学研究所本研究は、生命活動の根源である遺伝子発現を分子レベルで解明することを目的とし、高速原子間力顕微鏡（高速AFM）を用いて、クロマチン動態のナノ空間・リアルタイム撮影を試みる。本研究は、当該領域が掲げる、クロマチン構造が潜在的に持つ遺伝子発現制御能力「クロマチンポテンシャル」の実体を可視化することできる。具体的には、①ヒストンの翻訳後修飾を制御するヒストンメチル化酵素とクロマチンの動態、②クロマチンリモデリング複合体によるクロマチン構造の大規模な構造変化、③クロマチン構造が凝集するヘテロクロマチン形成過程を動画撮影し、クロマチン動態、および、その関連タンパク質の分子作動メカニズムを明らかにする。生命の根源である遺伝子発現には、クロマチン動態が重要な役割を果たすと考えられている。しかしながら、クロマチン動態や、その制御因子群がクロマチン上で起こす動的構造変化をナノ空間かつ、リアルタイムで可視化し解析した例はない。本研究は、高速原子間力顕微鏡（以下、高速AFM）を用い、クロマチンのナノ動態の実時空間イメージングを試み、クロマチン動態および、その関連タンパク質の分子作動機構を明らかにすることが目的である。最終年度は、初年度で得た高速AFM基板条件を用い（１）ATP依存性クロマチンリモデリング複合体（SNF2H）の高速AFM観察を行った。試験管内で、あらかじめSNF2Hとヌクレオソームを結合させ、ATP非存在下で高速AFM観察を行った。しかし、SNF2Hとヌクレオソームが結合した複合体は観察されなかった。次に、高速AFM観察中にATPをバッファー内に添加し、リモデリング反応を開始させた。その結果、数回の観察例ではあるが、ヌクレオソームの近傍にSNF2Hが存在し、ヌクレオソームのヒストンコアの位置が変わる様子（再配列）を可視化することができた。この時、SNF2Hとヌクレオソームはタイトに結合するわけではなく、数nm程度離れた場所でSNF2Hのひも状ドメインがヒストンコアに作用する様子が見られた。（２）領域内共同研究（胡桃坂計画班）として、H2AヒストンバリアントであるH2A.Bを含むヌクレオソームの高速AFM観察を行い、H2A.B-H2BダイマーがH2A-H2Bダイマーと自発的に交換する分子メカニズムの解明を行った。通常型のヒストンで構成されたヌクレオソームとH2A.Bヒストンバリアントを含むヒストンで構成されたヌクレオソームを比較すると、ヌクレオソーム構造が大きく異なり、開いた構造をとることを発見し、H2A.Bヒストンバリアントの自己交換反応のモデルを提唱した。研究課題/領域番号:19H05257, 研究期間(年度):2019-04-01 – 2021-03-31出典：研究課題「クロマチン動態の実時空間イメージング」課題番号19H05257（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PUBLICLY-19H05257/）を加工して作

High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin

金沢大学理工研究域数物科学系Dynamic changes in protein conformation in response to external stimuli are important in biological processes, but it has proved difficult to directly visualize such structural changes under physiological conditions1-10. Here, we show that high-speed atomic force microscopy7 can be used to visualize dynamic changes in stimulated proteins. High-resolution movies of a light-driven proton pump, bacteriorhodopsin, reveal that, upon illumination, a cytoplasmic portion of each bacteriorhodopsin monomer is brought into contact with adjacent trimers. The bacteriorhodopsin-bacteriorhodopsin11,12 interaction in the transiently formed assembly engenders both positive and negative cooperative effects in the decay kinetics as the initial bacteriorhodopsin recovers and, as a consequence, the turnover rate of the photocycle is maintained constant, on average, irrespective of the light intensity. These results confirm that high-resolution visualization is a powerful approach for studying elaborate biomolecular processes under realistic conditions. © 2010 Macmillan Publishers Limited. All rights reserved

Structural changes in bacteriorhodopsin in response to alternate illumination observed by high-speed atomic force microscopy

金沢大学理工研究域数物科学系Blue light, green light: High-speed atomic force microscopy visualized light-induced structural changes of the D96N bacteriorhodopsin (bR) mutant under alternate two-color illumination. With green light, each bR molecule is displaced outward from the trimer center. This activated structure is driven back to the ground state by the subsequent blue-light illumination (see picture). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Histone variant H2A.B-H2B dimers are spontaneously exchanged with canonical H2A-H2B in the nucleosome

精子形成に重要なヒストンによるDNAの新たな折りたたみを解明. 京都大学プレスリリース. 2021-02-22.H2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate “open conformation”, in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome

Wide-area scanner for high-speed atomic force microscopy

High-speed atomic force microscopy (HS-AFM) has recently been established. The dynamic processes and structural dynamics of protein molecules in action have been successfully visualized using HS-AFM. However, its maximum scan ranges in the X- and Y-directions have been limited to ∼1 μm and ∼4 μm, respectively, making it infeasible to observe the dynamics of much larger samples, including live cells. Here, we develop a wide-area scanner with a maximum XY scan range of ∼46 × 46 μm2 by magnifying the displacements of stack piezoelectric actuators using a leverage mechanism. Mechanical vibrations produced by fast displacement of the X-scanner are suppressed by a combination of feed-forward inverse compensation and the use of triangular scan signals with rounded vertices. As a result, the scan speed in the X-direction reaches 6.3 mm/s even for a scan size as large as ∼40 μm. The nonlinearity of the X- and Y-piezoelectric actuators\u27 displacements that arises from their hysteresis is eliminated by polynomial-approximation-based open-loop control. The interference between the X- and Y-scanners is also eliminated by the same technique. The usefulness of this wide-area scanner is demonstrated by video imaging of dynamic processes in live bacterial and eukaryotic cells. © 2013 AIP Publishing LLC

Role of trimer-trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy

Bacteriorhodopsin (bR) trimers form a two-dimensional hexagonal lattice in the purple membrane of Halobacterium salinarum. However, the physiological significance of forming the lattice has long been elusive. Here, we study this issue by comparing properties of assembled and non-assembled bR trimers using directed mutagenesis, high-speed atomic force microscopy (HS-AFM), optical spectroscopy, and a proton pumping assay. First, we show that the bonds formed between W12 and F135 amino acid residues are responsible for trimer-trimer association that leads to lattice assembly; the lattice is completely disrupted in both W12I and F135I mutants. HS-AFM imaging reveals that both crystallized D96N and non-crystallized D96N/W12I mutants undergo a large conformational change (i.e., outward E-F loop displacement) upon light-activation. However, lattice disruption significantly reduces the rate of conformational change under continuous light illumination. Nevertheless, the quantum yield of M-state formation, measured by low-temperature UV-visible spectroscopy, and proton pumping efficiency are unaffected by lattice disruption. From these results, we conclude that trimer-trimer association plays essential roles in providing bound retinal with an appropriate environment to maintain its full photo-reactivity and in maintaining the natural photo-reaction pathway. © 2013 Elsevier Inc. All rights reserved

Real-space and real-Time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

金沢大学新学術創成研究機構The CRISPR-Associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-Time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-Time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9. © 2017 The Author(s).出版社

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

新規光駆動型イオンチャネルの構造解明と高性能分子ツールの創出 --神経科学に光を当てる--. 京都大学プレスリリース. 2022-02-03.ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology

Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor.

金沢大学がん進展制御研究所Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF–MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.Embargo Period 6 month