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

从实验上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追踪的模型系统，使我们能够做到这一点，现在我们可以使用最先进的显微镜和遗传学工具来探索，随着细胞复杂性的实时发展，可以在特定的时间定位一个分子。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，形成代谢休眠的孢子，耐高温、化学应激和抗生素治疗；孢子经常与食物中毒和医院获得性感染有关。当杆状细胞不对称地分裂时，孢子形成就开始了，这与“正常”的中期细胞分裂相反，从而产生了大小不等的遗传上相同的子细胞。在隔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“前孔”中表达。母细胞吞噬了前海，在这种滋养的微环境中，保护性蛋白质层被沉积下来。在牺牲的行为中，母细胞裂解释放成熟的孢子，这些孢子可以无限期地存活并在有利条件恢复时萌发。孢子形成是机械细胞生物学的宝库：它包括饥饿感知和信号整合、极性细胞分裂、差异基因表达、吞噬和程序性细胞死亡。此外，由于形成枯草芽胞杆菌的模型孢子的遗传易感性，控制和执行孢子形成的蛋白质成分是相对已知的。使用MCL实验室设计和建造的先进光学显微镜，学生将观察活细菌细胞中的单个复合体，并确定它们的组成和化学计量以及它们组装和拆卸的动力学，并将在这些超分辨设备的应用方面接受宝贵的跨学科培训。AJW利用蛋白质生物化学和结晶学技术研究了这些蛋白质及其复合体的结构-功能关系，学生还将接触到这些技术并进行培训。这里，中心和独特的重点将是确定活的孢子形成细胞中功能分子的相互作用。为了进行这些研究，学生将学习使用基因工程方法来产生表达目标蛋白和荧光报告蛋白的新菌株。具体地说，我们对三个关键调控因子感兴趣，这三个调控因子被认为要么控制哪些产孢子基因的启动，要么在母细胞和子细胞之间形成迷人的通道结构。

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.