High-resolution, large scanning atomic force microscope (AFM) for capturing cellular processes in action

高分辨率、大扫描原子力显微镜 (AFM)，用于捕获活动中的细胞过程

• 批准号: EP/M022536/1
• 负责人: Sergi Garcia-Manyes
• 金额: $ 0.28万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FM022536%2F1
• 关键词: resolution; large; scanning; atomic; force

Atomic force microscopy (AFM) has become, in the last recent years, a key analytic tool to investigate the topographical properties of a wide variety of substrates, at the nanometer scale. While initial applications were basically focused on surface science and tribological applications, this technique has now matured enough to evolve and take on new challenges, such as the understanding of the physics underlying the molecular mechanisms governing a number of fundamental biological processes occurring within the core of an individual cell. Due to their large size, spanning up to ca. 30 micrometers in height, these cell measurements have been severely hampered by the (limited) imaging size affordable by current AFM instrumentation. Here we aim to acquire a high-resolution, fast, large scanning atomic force microscope (AFM) that will circumvent these technical limitations, thus enabling us to visualise and quantify molecular interactions on whole living cells and tissues at high spatial, temporal, and force resolution. Its unique combination with high-resolution optical microscopy will allow coupling single molecule nanomechanics with single molecule biophotonics. Since Scanning Probe techniques have gained experimental access to the molecular/atomic level, many crucial questions that remained unexplored can now be experimentally attacked. For example, while general thermodynamics laws were deducted for large ensembles of molecules, many key biological processes require only a few individual molecules to occur. Therefore, new single molecule experiments, often occurring under non-equilibrium conditions, will probe the extent and validity of classical thermodynamics laws to describe out-of-equilibrium biological processes occurring in real time within the framework of a living cell. Moreover, by pushing forward the instrumental limits, topographic sub-nanometer resolution will allow direct observation and measurement of the physical properties of distinct bio-molecular interfaces with key in-vivo implications. The novel combination with optical microscopy will enable to combine the strengths of both microscopy techniques and capture the single molecule processes occurring on the cell substrate (AFM) and those occurring in the cell interior, using fluorescence microscopy. Combined, these experiments will allow a comprehensive vista on individual processes occurring within a cell with unprecedented single molecule detection. The research enabled by this novel instrumentation is open ended. In particular, it will help elucidate the molecular mechanisms underlying cell mechanics, and the mechanical feedback mechanism by which substrate stiffness dictates the fate of individual stem cells. It will also allow to directly probe the hypothesis that several genes are mechano-activated, and that mechanical forces can transmit from the extracellular matrix down to the cell nucleus in an efficient way that does not rely on simple damped diffusion. These experiments will put a strong accent on the mechanisms governing mechanostranduction and cell adhestion, thus greatly complementing and expanding world-leading research being currently conducted in King's College London and other leading institutions in the London Area (Oxford, Francis Crick Institute). Moreover, the technical developments allowed by this new instrument will enable new cell-based nanotechnological applications, of particular interest for the London Centre for Nanotechnology (LCN). Altogether, this equipment will foster and encourage fruitful collaborations with other London- (and UK-) based institutions working on the intense and prolific research fields of mechanobiology and biophysics, allowing a cross-disciplinary approach and dwelling from the single cell to the single molecule level.

在近年来，原子力显微镜（AFM）已成为一种关键分析工具，用于以纳米尺度研究各种底物的地形特性。尽管最初的应用基本上集中在表面科学和摩擦学应用上，但该技术现在已经足够成熟，可以发展和应对新的挑战，例如对分子机制基础的理解，这些物理学的基础机制管理了许多基本生物学过程，这些生物过程发生在单个细胞的核心内。由于它们的尺寸较大，跨越大约。 30微米的高度，这些细胞测量已被当前AFM仪器负担得起的（有限）成像尺寸严重阻碍。在这里，我们旨在获得高分辨率，快速，大型扫描原子力显微镜（AFM），该原子显微镜（AFM）将避免这些技术局限性，从而使我们能够在高空间，时间和力分辨率下可视化和量化整个活细胞和组织上的分子相互作用。它与高分辨率光学显微镜的独特组合将允许将单分子纳米力学与单分子生物素化合物耦合。由于扫描探针技术已经获得了对分子/原子水平的实验访问，因此现在可以实验攻击许多尚未探索的关键问题。例如，虽然对大型分子扣除了一般热力学定律，但许多关键的生物过程仅需要几个单独的分子。因此，通常在非平衡条件下发生的新单分子实验将探测经典热力学定律的程度和有效性，以描述活细胞框架内实时实时发生的平衡生物学过程。此外，通过推动仪器限制，地形子纳米分辨率将允许直接观察和测量具有关键的体内含义的不同生物分子界面的物理特性。与光学显微镜的新型组合将使两种显微镜技术的强度结合起来，并使用荧光显微镜捕获细胞底物（AFM）和细胞内部发生的单分子过程。这些实验结合在一起，将允许全面地远景对具有前所未有的单分子检测的细胞内发生的单个过程。这项新型仪器启用了这项研究。特别是，它将有助于阐明细胞力学的分子机制，以及底物刚度决定单个干细胞命运的机械反馈机制。它还将允许直接探测几个基因被机械激活的假设，并且机械力可以以有效的方式从细胞外基质传播到细胞核，而不依赖简单阻尼的扩散。这些实验将对有关机械施工和细胞处理的机制产生强烈的重音，因此目前在伦敦国王学院和伦敦地区的其他领先机构（牛津，弗朗西斯·克里克学院）进行了极大的补充和扩大世界领先的研究。此外，该新工具允许的技术发展将使新的基于细胞的纳米技术应用，特别是伦敦纳米技术中心（LCN）。总的来说，该设备将促进和鼓励与其他伦敦（和英国）基于伦敦（和英国）的机构合作，从事机械生物学和生物物理学的激烈而多产的研究领域，从而允许跨学科的方法并从单个细胞到单分子水平。

项目成果

Loxl2 is dispensable for dermal development, homeostasis and tumour stroma formation.

• DOI: 10.1371/journal.pone.0199679
• 发表时间: 2018
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Kober KI;Cano A;Géraud C;Sipilä K;Mobasseri SA;Philippeos C;Pisco AO;Stannard A;Martin A;Salvador F;Santos V;Boutros M;Rognoni E;Watt FM
• 通讯作者: Watt FM

Controlling Anomalous Diffusion in Lipid Membranes

• DOI: 10.1016/j.bpj.2018.12.024
• 发表时间: 2019-03-19
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Coker, Helena L. E.;Cheetham, Matthew R.;Wallace, Mark, I
• 通讯作者: Wallace, Mark, I

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair.

• DOI: 10.1016/j.devcel.2021.10.022
• 发表时间: 2021-12-06
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Wallis SS;Ventimiglia LN;Otigbah E;Infante E;Cuesta-Geijo MA;Kidiyoor GR;Carbajal MA;Fleck RA;Foiani M;Garcia-Manyes S;Martin-Serrano J;Agromayor M
• 通讯作者: Agromayor M

Forcing the reversibility of a mechanochemical reaction.

• DOI: 10.1038/s41467-018-05115-6
• 发表时间: 2018-08-08
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Beedle AEM;Mora M;Davis CT;Snijders AP;Stirnemann G;Garcia-Manyes S
• 通讯作者: Garcia-Manyes S

Protein nanomechanics: The power of stretching

蛋白质纳米力学：拉伸的力量

• DOI: 10.1051/epn/2020503
• 发表时间: 2020
• 期刊: Europhysics News
• 作者: Mora M