Development of single molecule techniques for nanoscale imaging of toxic protein species in vitro and in cells.

蛋白质是生命的基本组成部分之一，但为了正常工作，蛋白质需要保持正确的形状，或者用科学的语言来说，它们需要保持折叠。当蛋白质错误折叠时，它们会呈现异常的形状，并经常以不溶性沉积物的形式崩溃，这可能对大脑中的神经元有毒。事实上，像阿尔茨海默病或帕金森病（PD）这样的重大疾病，其特征是形成不溶性的蛋白质沉积物，这些蛋白质以长丝状的形式聚合，称为淀粉样原纤维。不幸的是，导致纤维形成和随后聚集的分子相互作用尚未阐明；因此，表征原纤维超微结构的高分辨率技术有很高的需求。电子显微镜被广泛用于此目的，但其侵入性使其无法及时跟踪原纤维的形成，并且电子显微镜需要详细的样品固定和安装方案。原子力显微镜也可以对纤维进行高分辨率成像，但它仅限于表面拓扑结构。虽然荧光显微镜可以补充这些技术，但传统的光学方法受到衍射的限制，并且提供的分辨率太粗，无法提供蛋白质原纤维/聚集体的信息。最近，单分子荧光技术的变体已经开发出来，提供10-20纳米的分辨率，即比探测光的波长小得多。它们是如何工作的？样品被标记上所谓的光激活荧光团，它可以用光脉冲打开和关闭；因此，我们可以把每个荧光团想象成一个可切换的灯泡。通过在任何时候打开几个单独的灯泡并拍摄图像，可以非常精确地确定有源灯泡的位置（10-20纳米）。通过重复这个过程数千次，每次激活不同的荧光团，并拍摄尽可能多的图像，可以确定每个荧光团的精确位置，并可以获得有关底层结构的信息（作为一个类比，人们可能会想到一棵圣诞树，“贴上”许多灯泡：如果准确地知道单个灯泡的位置，就可以获得有关树的底层形状/结构的信息）。正是通过这种所谓的单分子荧光技术，我们第一次想要获得由alphasynclein （AS）形成的纤维结构的大小和形状的动态信息，这是一种错误折叠的蛋白质，位于PD的核心。我们将用我们开发的其他光学技术来补充这项研究，这些技术也为我们提供了细胞和测试溶液中形成的蛋白质聚集体的分子尺度分辨率。如果成功的话，我们最终将获得一些工具，这些工具将为我们提供迄今为止可能获得的关于细胞聚集过程的更直接的信息。例如，如果我们看到用潜在的抗聚集药物治疗过的细胞与没有治疗过的细胞形成的AS蛋白沉积的差异，那将是令人兴奋的。无论如何，我们最终将获得新的知识和工具，用来研究蛋白质的功能和“不当行为”。这项提议的工作汇集了分子和化学生物学家、物理学家和工程师的专业知识，他们已经用新的光学工具解决了神经退行性疾病中的这一重要问题。

Proteins are one of the fundamental building blocks for life, but in order to work correctly, proteins need to be in the right shape, or, in scientific language, they need to maintain their fold. When proteins misfold, they can take on aberrant shapes and often collapse in the form of insoluble deposits, which can be toxic to neurons in the brain. Indeed, major diseases such as Alzheimer's or Parkinson's diseases (PD), are characterized by the formation of insoluble deposits of proteins which polymerize in the form of long filaments, called amyloid fibrils. Unfortunately, the molecular interactions that lead to fibril formation and subsequent aggregation are not yet elucidated; therefore, high resolution techniques that characterize the ultrastructure of fibrils are in high demand. Electron microscopy is widely used for this purpose, but its invasive nature prevents it from following fibril formation in time and EM requires elaborate sample fixation and mounting protocols. Atomic force microscopy can also image fibrils at high resolution, but it is limited to surface topology. Although fluorescence microscopy can complement these techniques, conventional optical methods are limited by diffraction and provide a resolution far too coarse to provide information in protein fibrils / aggregates. Recently, variants of single molecule fluorescence techniques have been developed that provide 10-20 nm resolution i.e. much smaller than the wavelength of the probing light. How do they work? A sample is tagged with so called photoactivatable fluorophores which can be switched on and off with pulses of light; one can thus think of each fluorophore as a switchable light bulb. By switching on a few individual light bulbs at any one time and taking an image, the positions of the active light bulbs can be determined with very high precision (10-20 nm). By repeating this process thousands of times, activating different fluorophores each time, and taking as many images the precise location of each fluorophore can be determined and information on the underlying structure can be obtained (as an analogy one may think of a Christmas tree, 'labelled' with lots of lightbulbs: If the positions of the individual lightbulbs are accurately known, information on the underlying shape/structure of the tree can be regained). It is with such so called single molecule fluorescence techniques we would like to gain, for the first time, dynamic information on the sizes and shapes of fibrillar structures formed by alphasynclein (AS), a protein that misfolds and lies at the heart of PD. We will complement this research with other optical techniques developed by us, which also give us molecular scale resolution of protein aggregates forming in cells, and also in test solutions. If successful we will end up with tools that give us much more direct information on the aggregation process in cells than has hitherto been possible. It would be exciting for example, if we were to see differences in the AS protein deposits formed in cells treated with potential anti-aggregation drugs compared to those which have not been treated. In any case we will end up with new knowledge and tools, with which to study protein function and 'misbehaviour'. The proposed work brings together the expertise of molecular and chemical biologists, physicists, and engineers who have put in place the building blocks to tackle this important problem in neurodegenerative disease with novel optical tools.