Dissecting molecular complexity in single living cells using state-of-the-art super-resolution microscopy and biophysical chemistry

使用最先进的超分辨率显微镜和生物物理化学剖析单个活细胞中的分子复杂性

• 批准号: 2116421
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2116421
• 关键词: Dissecting; molecular; complexity; single; living

Experimentally studying the emergence of cellular complexity is really hard.But, the process of 'bacterial sporulation' presents a fantastically tractablemodel system to enable us to do so, which we can now probe using excitingstate-of-the-art microscopy and genetics tools to pinpoint one molecule at atime as cell complexity develops in real time. To survive starvation and otherforms of stress bacteria such as Bacillus and Clostridia abandon growth andinstead form a metabolically dormant spore, resistant to heat, chemicalstresses and antibiotic treatment; spores are frequently associated with foodpoisoning and hospital acquired infections. Sporulation begins when the rod-shaped cell divides asymmetrically, as opposed to 'normal' mid-cell division,giving rise to genetically identical daughter cells of unequal size. Directed bycompartment-specific factors, different genes are expressed in the largermother cell and the smaller 'forespore'. The mother cell engulfs the foresporeand in this nurturing microenvironment, protective protein layers aredeposited. In an act of sacrifice the mother cell lyses releasing the mature sporewhich can survive indefinitely and germinate when favourable conditions arerestored.Spore formation presents a treasure trove for mechanistic cell biology: itencompasses starvation sensing and signal integration, polar cell division,differential gene expression, phagocytosis and programmed cell death.Moreover, the protein components that control and execute sporulation arelargely known thanks to the genetic tractability of the model spore formingbacterium Bacillus subtilis. Using advanced light microscopes designed and builtin the laboratory of MCL, the student will observe individual complexes in livingbacterial cells and determine their composition and stoichiometry as well asthe dynamics of their assembly and disassembly, and will receive invaluableinterdisciplinary training in the application of these super-resolution devices.AJW has investigated structure-function relationships in these proteins andtheir complexes using protein biochemistry and crystallography techniques,and the student will also gain exposure to, and training in, these techniques.The central and distinct focus, here, will be the determination of functionalmolecular interactions in live sporulating cells. In order to carry out thesestudies, the student will learn to use genetic engineering methods to generatenew strains expressing target proteins fused to fluorescent reporter proteins.Specifically, we are interested in three key regulators that are believed to eithercontrol which sporulation genes are switched on, or to form fascinating channelstructures between the mother and daughter cells.

通过实验研究细胞复杂性的出现是非常困难的。但是，“细菌孢子形成”的过程提供了一个非常容易处理的模型系统，使我们能够做到这一点，我们现在可以使用令人兴奋的最先进的显微镜和遗传学工具来探测，每次精确定位一个分子，因为细胞的复杂性是实时发展的。为了在饥饿和其他形式的压力下生存，芽孢杆菌和梭状芽孢杆菌等细菌放弃生长，转而形成代谢休眠孢子，抵抗高温、化学胁迫和抗生素治疗；孢子常与食物中毒和医院获得性感染有关。当棒状细胞不对称分裂时，孢子形成开始，而不是“正常的”中细胞分裂，产生大小不等的基因相同的子细胞。在室特异性因子的指导下，不同的基因在较大的母细胞和较小的“前孢子”中表达。母细胞吞噬前孢子，在这个滋养的微环境中，保护性蛋白层沉积下来。在一种牺牲行为中，母细胞裂解，释放出成熟的孢子，成熟的孢子可以无限期地存活，并在有利条件恢复时发芽。孢子形成是机制细胞生物学的宝库：它包括饥饿感知和信号整合、极性细胞分裂、差异基因表达、吞噬和细胞程序性死亡。此外，由于模式孢子形成细菌枯草芽孢杆菌的遗传易变性，控制和执行孢子形成的蛋白质成分在很大程度上是已知的。使用先进的光学显微镜，学生将观察活细菌细胞中的单个复合物，并确定它们的组成和化学计量，以及它们的组装和拆卸的动力学，并将在这些超分辨率设备的应用方面接受宝贵的跨学科培训。AJW使用蛋白质生物化学和晶体学技术研究了这些蛋白质及其复合物的结构-功能关系，学生也将获得这些技术的接触和培训。在这里，中心和独特的焦点将是确定活孢子细胞中的功能分子相互作用。为了开展这些研究，学生将学习使用基因工程方法来产生表达目标蛋白与荧光报告蛋白融合的新菌株。具体来说，我们感兴趣的是三个关键的调节因子，它们被认为可以控制哪些产孢基因被打开，或者在母细胞和子细胞之间形成迷人的通道结构。