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

通过实验研究细胞复杂性的出现确实很难。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进行探测，随着细胞复杂性在真实的时间内的发展，一次精确定位一个分子。为了在饥饿和其他形式的压力下生存，细菌如芽孢杆菌和梭菌放弃生长，而是形成代谢休眠的孢子，耐热，耐化学压力和抗生素治疗;孢子经常与食物中毒和医院获得性感染有关。当杆状细胞不对称分裂时，孢子形成开始，而不是“正常”的中间细胞分裂，产生大小不等的遗传相同的子细胞。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“前孢子”中表达。母细胞吞噬了前孢子，在这个微环境中，保护性的蛋白质层被沉积下来。在一种牺牲行为中，母细胞溶解，释放出成熟的孢子，这些孢子可以无限期地存活，并在有利的条件恢复时发芽。孢子的形成为机械细胞生物学提供了一个宝库：它包括饥饿感测和信号整合，极性细胞分裂，差异基因表达，吞噬作用和程序性细胞死亡。此外，控制和执行孢子形成的蛋白质组分由于模式孢子形成细菌枯草芽孢杆菌的遗传易处理性而在很大程度上是已知的。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物，并确定其组成和化学计量以及组装和拆卸的动力学，并将在这些超分辨率设备的应用中接受宝贵的跨学科培训。AJW研究了结构-使用蛋白质生物化学和晶体学技术，这些蛋白质及其复合物中的功能关系，学生也将接触这些技术并接受这些技术的培训。将是活孢子形成细胞中功能分子相互作用的测定。为了进行这些研究，学生将学习使用基因工程的方法来产生新的菌株，这些菌株表达与荧光报告蛋白融合的靶蛋白。具体地说，我们对三个关键的调节因子感兴趣，这三个因子被认为是控制孢子形成基因的开启，或者是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.