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

Experimentally studying the emergence of cellular complexity is really hard.
But, the process of 'bacterial sporulation' presents a fantastically tractable
model system to enable us to do so, which we can now probe using exciting
state-of-the-art microscopy and genetics tools to pinpoint one molecule at a
time as cell complexity develops in real time. To survive starvation and other
forms of stress bacteria such as Bacillus and Clostridia abandon growth and
instead form a metabolically dormant spore, resistant to heat, chemical
stresses and antibiotic treatment; spores are frequently associated with food

poisoning 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 by
compartment-specific factors, different genes are expressed in the larger
mother cell and the smaller 'forespore'. The mother cell engulfs the forespore
and in this nurturing microenvironment, protective protein layers are
deposited. In an act of sacrifice the mother cell lyses releasing the mature spore
which can survive indefinitely and germinate when favourable conditions are
restored.
Spore formation presents a treasure trove for mechanistic cell biology: it
encompasses 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 are
largely known thanks to the genetic tractability of the model spore forming
bacterium Bacillus subtilis. Using advanced light microscopes designed and built
in the laboratory of MCL, the student will observe individual complexes in living
bacterial cells and determine their composition and stoichiometry as well as
the dynamics of their assembly and disassembly, and will receive invaluable
interdisciplinary training in the application of these super-resolution devices.
AJW has investigated structure-function relationships in these proteins and
their 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 functional
molecular interactions in live sporulating cells. In order to carry out these
studies, the student will learn to use genetic engineering methods to generate
new strains expressing target proteins fused to fluorescent reporter proteins.
Specifically, we are interested in three key regulators that are believed to either
control which sporulation genes are switched on, or to form fascinating channel
structures between the mother and daughter cells.