Towards higher resolution and cellular imaging using cryo-electron tomography

使用冷冻电子断层扫描实现更高分辨率和细胞成像

• 批准号: RGPIN-2016-04954
• 负责人: Bui, KhanhHuy
• 金额: $ 2.77万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687234
• 关键词: Towards; higher; resolution; cellular; imaging

Studying the structure of macromolecular complexes is essential for understanding how they function. Furthermore, studying the structure of the macromolecular complexes within their cellular context provides the most reliable information about their molecular function. Cryo-electron tomography (cryo-ET) is a powerful imaging technique that provides three-dimensional information about cellular architecture devoid of chemical fixation and staining artifacts. Cryo-ET can reach ~10 nm resolution and up to 3 nm with subtomogram averaging in silico. The limiting factor in cryo-ET resolution is the electron dose-limiting radiation damage and thick specimen. This resolution gives the information about the overall shape of the proteins but is not able to provide any molecular mechanism. My research focus is to push the resolution of cryo-ET to sub-nanometer resolution using the microtubule doublet as the test sample. At such resolutions, secondary structures are clearly identifiable and consequently, atomic models of subunits can be docked into the experimental tomographic maps to gain novel insights into protein-protein interactions and molecular mechanism. My research will also push cryo-ET to work on thicker sample i.e. looking at part of the cells by improving the signal-to noise ratio using the phase plate, direct electron detector and improved acquisition scheme. This will provide us with a unique ability to open a window into the cell to look at cellular process as unprecedented resolution. Furthermore, to address the dynamic nature of the cellular process, my laboratory will develop correlative light-electron microscopy technique, which combines the power of cryo-ET to resolve ultrastructure with the specific identification power of fluorescence light microscopy to precisely localize proteins of interest in the context of their surroundings to yield insights into fundamental biological mechanisms. ***The test sample for our method development will be the eukaryotic cilia, which are present in almost all cells in human. We plan to obtain the sub-nanometer structure of the microtubule doublet from the cilia using cryo-ET and the in situ structure of the intraflagellar transport complex, which is involved in the ciliary assembly within intact cells using CLEM. The technological developments outlined here will bridge multi-scale resolutions from isolated macromolecular complexes to in vivo cellular structures. The technology will provide important insights into the inner workings of cells and how they are altered in disease states. Our high-resolution structure will shed light onto the fundamental principals behind the biophysical properties of microtubule doublet, and lead to the mechanism of its function and potentially new ideas in biomaterial research.**

研究大分子复合物的结构对于理解它们的功能至关重要。此外，研究细胞环境中大分子复合物的结构可以提供有关其分子功能的最可靠信息。冷冻电子断层扫描 (cryo-ET) 是一种强大的成像技术，可提供有关细胞结构的三维信息，而无需化学固定和染色伪影。 Cryo-ET 分辨率可达到约 10 nm，在计算机中进行亚断层图平均时分辨率可达 3 nm。冷冻电子断层扫描分辨率的限制因素是电子剂量限制辐射损伤和厚样本。该分辨率提供了有关蛋白质整体形状的信息，但无法提供任何分子机制。我的研究重点是使用微管双联体作为测试样本，将冷冻电子断层扫描的分辨率提高到亚纳米分辨率。在这样的分辨率下，二级结构是清晰可辨的，因此，亚基的原子模型可以对接到实验断层扫描图中，以获得对蛋白质-蛋白质相互作用和分子机制的新见解。我的研究还将推动冷冻电子断层扫描技术应用于更厚的样品，即通过使用相位板、直接电子探测器和改进的采集方案提高信噪比来观察部分细胞。这将为我们提供一种独特的能力，打开一扇进入细胞的窗口，以前所未有的分辨率观察细胞过程。此外，为了解决细胞过程的动态性质，我的实验室将开发相关的光电子显微镜技术，该技术将冷冻电子显微镜解析超微结构的能力与荧光光学显微镜的特异性识别能力相结合，以在其周围环境中精确定位感兴趣的蛋白质，从而深入了解基本的生物机制。 ***我们方法开发的测试样本将是真核纤毛，它存在于几乎所有人类细胞中。我们计划使用cryo-ET获得纤毛的微管双联体的亚纳米结构，并使用CLEM获得鞭毛内运输复合物的原位结构，该复合物参与完整细胞内的纤毛组装。这里概述的技术发展将连接从孤立的大分子复合物到体内细胞结构的多尺度分辨率。该技术将为细胞的内部运作及其在疾病状态下如何改变提供重要的见解。我们的高分辨率结构将揭示微管双联体生物物理特性背后的基本原理，并导致其功能机制和生物材料研究中的潜在新想法。 **