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构件之一，但为了正常工作，蛋白质需要处于正确的形状，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，它们可能会呈现出异常的形状，并经常以不溶沉积的形式崩溃，这可能会对大脑中的神经元产生毒性。事实上，阿尔茨海默氏症或帕金森氏病(PD)等重大疾病的特征是形成不溶于水的蛋白质沉积，这些沉积以长丝的形式聚合，称为淀粉样纤维。不幸的是，导致纤维形成和随后聚集的分子相互作用尚不清楚；因此，表征纤维超微结构的高分辨率技术是非常必要的。电子显微镜被广泛用于这一目的，但其侵入性使其无法及时跟随原纤维的形成，EM需要精心设计的样品固定和安装方案。原子力显微镜也可以以高分辨率成像纤维，但它仅限于表面拓扑。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，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被开发出来，它们提供了10-20 nm的分辨率，即远远小于探测光的波长。它们是如何工作的？样品被标记了所谓的可光激活的荧光团，这种荧光团可以通过光脉冲来开启和关闭；因此，人们可以将每个荧光团视为一个可切换的灯泡。通过一次打开几个单独的灯泡并拍摄图像，可以非常高的精度(10-20 nm)确定活动灯泡的位置。通过重复这一过程数千次，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准确位置，并可以获得关于底层结构的信息(作为一个类比，人们可能会想到一棵圣诞树，用许多灯泡‘标记’：如果准确知道单个灯泡的位置，就可以重新获得关于树的潜在形状/结构的信息)。正是通过这种所谓的单分子荧光技术，我们希望第一次获得由阿尔法同步蛋白(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，位于帕金森病的心脏。我们将用我们开发的其他光学技术来补充这项研究，这些技术也可以让我们在细胞和测试溶液中形成蛋白质聚集体的分子级分辨率。如果成功，我们最终将获得比迄今可能的更多关于细胞内聚合过程的直接信息的工具。例如，如果我们看到接受潜在抗聚集药物治疗的细胞中形成的AS蛋白沉积与未治疗的细胞相比存在差异，那将是令人兴奋的。在任何情况下，我们最终都会有新的知识和工具，用来研究蛋白质的功能和“不端行为”。这项拟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，他们已经建立了基础，用新的光学工具解决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.