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

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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.