A new tool for quantifying the nanoscale dynamics of liquids at the interface with fluid biological membranes

生物膜是一种高度复杂的结构，在细胞功能中起着重要作用。除了作为细胞内外之间的物理屏障外，生物膜还控制着无数的生物过程，从分子运输到细胞信号传导、生物能量学和生化功能、内吞作用、表面区隔化和膜形状调节。因此，生物膜已经进化成动态的实体，可以主动重新调整其局部组成和物理性质，以最好地支持细胞的生物功能。众所周知，这种调节涉及到专门的蛋白质和标记，但越来越多的证据也指出，膜的机械性能，特别是刚度和流动性的局部变化受到了很好的控制。膜附近的液体（水和水合离子）也被认为在形成膜、调节生物分子动力学和控制膜上的相互作用和扩散方面起着重要作用。这种界面液体能与生物膜发生强烈的相互作用，局部改变生物膜的力学性能。迄今为止，在纳米尺度上解决界面液体或膜流动性性质的大多数结果要么是间接的，要么是从理论考虑中得出的。这在很大程度上是由于缺乏能够以足够的空间和时间分辨率在局部操作的实验技术。原子力显微镜（AFM）领域的最新进展使得探测包括生物膜表面在内的界面液体成为可能。值得注意的是，结果可以在几秒钟内局部获得，并且可以提供靠近膜的液体所采用的平衡结构的亚纳米精度图。然而，目前还不可能在这个尺度上推导出有关界面液体动力学的任何信息。例如，不可能确定液体在界面处采用的定向流动模式。部分问题在于无法量化生物膜的纳米级流动性，这是影响接触液体动力学的一个因素。此外，界面液体和生物膜力学性能之间的相互影响从未在这个尺度上被探索过。本建议旨在开发一种新的工具，涡耗散显微镜（VDM），它可以定量平衡溶剂化结构和水在生物界面上的横向动力学，局部和纳米精度。VDM建立在最近的AFM发展的基础上，将能够提供界面水在奇点附近自然采用的流动模式的纳米级图表，例如膜结构域或蛋白质组装的边缘。值得注意的是，该技术还将能够以相同的分辨率在相同的面积上量化膜内的横向流动性。VDM随后将应用于牛眼晶状体膜的研究。它将被用来量化蛋白质通道附近的流动模式，水通道蛋白0和连接蛋白负责水和小分子通过晶状体细胞膜的运输。除了为VDM的性能提供一个天然的测试平台外，研究结果还将为透镜膜老化问题提供新的见解。已知老化与膜力学性能的变化有关，但其潜在的分子机制尚不清楚。这一问题具有重要的医学和社会后果。

Biological membranes (biomembranes) are highly sophisticated structures that play a major role in the cell function. Aside from acting as a physical barrier between the inside from the outside of the cell, biomembranes control countless biological processes ranging from molecular trafficking to cell signalling, bioenergetics and biochemical function, endocytosis, surface compartmentalisation, and membrane shape regulation. Biomembranes have hence evolved into dynamical entities that can actively re-adjust their local composition and physical properties so as to best support the cell's biological function. This regulation is known to involve specialised proteins and markers, but increasing evidence also points to well controlled local variations in the membrane's mechanical properties, in particular stiffness and fluidity. The liquid (water and hydrated ions) adjacent to the membrane is also believed to play an important role in shaping the membrane, regulating biomolecules' dynamics and controlling interactions and diffusion along the membrane. This interfacial liquid can strongly interact with the biomembrane and locally alter its mechanical properties. To date, most results addressing the properties of the interfacial liquid or of the membrane fluidity at the nanoscale are either indirect or derived from theoretical consideration. This is largely due to the lack of experimental techniques able to operate locally with sufficient spatial and temporal resolution.Recent advances in the field of atomic force microscopy (AFM) have made it possible to probe the interfacial liquid, including at the surface of biomembranes. Significantly, results are acquired locally, in seconds, and can provide sub-nanometre precision maps of the equilibrium structure adopted by the liquid adjacent to the membrane. At the present time, however, it is not possible to deduce any information about the interfacial liquid dynamics on that scale. It is, for example, not possible to determine the directional flow patterns adopted by the liquid at the interface. Part of the problem comes from the impossibility to quantify the nanoscale fluidity of the biomembranes, a factor that influence the dynamics of the contacting liquid. Furthermore, the mutual influence that the interfacial liquid and the biomembrane mechanical properties have on each other has never been explored at that scaleThis proposal aims at developing a new tool, the vortex dissipation microscope (VDM) that can quantify both the equilibrium solvation structure and the lateral dynamics of water at biointerfaces, locally and with nanometre precision. The VDM builds on recent AFM developments and will be able to provide nanoscale charts of the flow patterns naturally adopted by the interfacial water near singularities such as edges of membrane domains or protein assemblies. Significantly, the technique will also be able to quantify the lateral fluidity within the membranes, over the same area and with the same resolution.The VDM will subsequently be applied to the study of bovine eye lens membranes. It will be used to quantify the flow patterns adjacent to the protein channels aquaporin 0 and connexins that are responsible for the transport of water and small molecules through lens cell membranes. Aside from providing a natural test platform for the VDM's capabilities, the results will bring new insight into the problem of lens membrane aging. Aging is known to be coupled with changes of the membrane mechanical properties, but the underlying molecular mechanisms are still poorly understood. This problem has important medical and hence societal consequences.