Cellular machinery in situ by correlative microscopy

细胞中的分子机器对生命至关重要。这些机器的故障会导致癌症和痴呆等疾病。相反，病原体中的机械阻塞可用于应对全球健康威胁，包括抗生素耐药性和寄生虫病。虽然这些机器在原子水平上运行，但它们的功能和功能失调的后果在细胞和组织中表现出来，这些细胞和组织比机器本身大很多个数量级，也更复杂。因此，需要在不同生物尺度上的成像能力，包括光学显微镜和电子显微镜，以充分了解分子机制的运作和功能。低温电子显微镜（cryo-EM）是一项获得诺贝尔奖的变革性技术，对研究分子机器至关重要。Cryo-EM样品使用低温液体快速冷冻，并在非常低的温度下进行检测。有了这种类型的制备，样品可以在其自然的水合状态下成像。然而，在冷冻电镜数据中没有单独的关于成像样品的分子身份的信息。在复杂的细胞和组织样本中，使用光学显微镜观察到的荧光标记可以定位感兴趣的单个成分。这意味着细胞或组织样本的特定区域可以有效地针对低温电镜数据收集。ISMB是国际公认的低温电子显微镜卓越中心。我们希望申请一种新的冷冻荧光显微镜的资金，这种显微镜可以在冷冻样品中进行高精度的荧光定位，从而用冷冻电镜识别感兴趣的目标区域。冷冻荧光显微镜将允许在不同的成像系统之间无缝转移易碎样品，并将在荧光和冷冻电镜成像模式中整合感兴趣区域的定位。加上它的技术能力，我们计划购买的下一代显微镜将提高吞吐量，并方便非专业人员访问。受益于该新设备的具体项目包括：1)细菌共轭转移的分子机制研究，这是抗生素抗性基因在细菌间传播的主要途径；2)淀粉样蛋白纤维组装和拆卸的研究将揭示细胞的质量控制机制如何处理与神经退行性疾病相关的蛋白质错误折叠；3)研究控制货物和代谢物通过细胞运输的分子机制，当这种机制被破坏时，可能会产生有害的生理后果，包括心功能受损；4)研究神经元微管细胞骨架的结构和调控，揭示它们在大脑发育过程中如何适应其动态，以及这种动态在人类疾病中如何被破坏；5)调节蛋白的研究，当被自然发生的突变破坏时，会引起蛋白质聚合，破坏肝脏和肺细胞功能，分别导致肝硬化和肺病。

Molecular machines in cells are essential for life. Malfunctions of these machines cause diseases such as cancer and dementia. Conversely, machinery blockage in pathogens can be used to tackle global health threats, including antibiotic resistance and parasitic diseases. While these machines operate at the atomic level, the consequences of their function and dysfunction manifest in cells and tissues that are many orders of magnitude larger and more complex than the machines themselves. Imaging capabilities at different biological scales including light and electron microscopies are therefore required to fully understand molecular machinery operation and function. Cryo-electron microscopy (cryo-EM) is a Nobel Prize-winning transformative technology that is essential for studying molecular machines. Cryo-EM samples are rapidly frozen using cryogenic liquids and examined at very low temperature. With this type of preparation, samples can be imaged in their natural, hydrated state. However, no information is available in cryo-EM data alone about the molecular identity of the imaged sample. Within complex cellular and tissue samples, the use of fluorescent labels observed using light microscopy allows individual components of interest to be localised. This means that specific regions of cell or tissue samples can be efficiently targeted for cryo-EM data collection. The ISMB is internationally recognised as a centre of excellence for cryo-EM. We wish to apply for funds for a new cryo-fluorescence microscope that enables high-precision fluorescence localisation in frozen samples to identify regions of interest to target with cryo-EM. The cryo-fluorescence microscope would allow seamless transfer of fragile samples between different imaging systems and would integrate localisation of regions of interest in both fluorescence and cryo-EM imaging modalities. Together with its technical capabilities, our planned purchase of a next-generation microscope will enhance throughput and facilitate non-expert access. Specific projects that will benefit from this new equipment include: 1) Studies of the molecular mechanisms of bacterial conjugative transfer, the main means by which antibiotic resistance genes spread among bacteria; 2) Studies of amyloid fibre assembly and disassembly that will reveal how the cell's quality control machinery handles protein misfolding associated with neurodegenerative disease; 3) Studies of the molecular machinery that controls the trafficking of cargo and metabolites through cells, which when disrupted can have detrimental physiological consequences including compromised cardiac function; 4) Studies of the structure and regulation of the neuronal microtubule cytoskeleton that will reveal how they adapt their dynamics during brain development and how this is disrupted in human diseases; 5) Studies of regulatory proteins which when disrupted by naturally occurring mutations, cause protein polymerisation that disrupts liver and lung cell function, resulting in cirrhosis and lung disease respectively.