"The precise timing and spatial organisation of the cell cycle is essential to the survival of any organism. Caulobacter crescentus is a simple, easily synchronisable organism which provides an excellent model system for cell cycle regulation, where a complex interacting network of regulatory proteins controls the precise timing and position of cell cycle processes. Although significant work has been carried out to determine the temporal and spatial dynamics of these proteins, the biophysical mechanisms by which bacterial regulatory proteins localise to specific cellular structures is largely unknown, due to the experimental challenge of studying diffusion of high-copy-number proteins.
It has recently become possible to study the diffusion of high-copy-number proteins using single particle tracking photoactivated localisation microscopy (sptPALM). We will develop a high-throughput implementation of sptPALM (HT-PALM), capable of automatically recording sptPALM data for multiple cells over the duration of the cell cycle. We will use HT-PALM to study C. crescentus cell cycle regulatory proteins, determining the spatio-temporal variation in diffusion coefficient and molecular confinement (“molecular mobility”) and its effect on protein localisation dynamics.
This study will advance our biophysical understanding of the role of molecular mobility in the bacterial cell cycle and at the same time provide the technological basis for a broadly useful high-throughput modality of the PALM technique. Thus, the proposed work will enable high-throughput super-resolution microscopy studies in additional prokaryotic systems, and pave the way for eukaryotic high-throughput super-resolution microscopy."
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