CELL CYCLE PALM

Quantifying regulatory protein diffusion in the bacterial cell cycle by high-throughput single particle tracking PALM

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 Obiettivo del progetto (Objective)

'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.'

Introduzione (Teaser)

So far, the study of bacteria has been hampered by their small size. A European consortium aimed to revolutionise bacterial research through an innovative microscopy-based technology.

Descrizione progetto (Article)

Bacteria are an imminent threat to human health and, therefore, understanding their biology is central to the development of new therapies. The problem is exacerbated by the emergence of antibiotic-resistant bacterial species. With no new classes of antibiotics discovered since 1987, it is vital to identify novel antibiotic targets.

However, the small size of bacteria has impeded detailed investigation of the bacterial cell and the identification of proteins that are central to its survival. Targeting such vital proteins constitutes the basis of antibiotic design.

To address this issue, the EU-funded CELL CYCLE PALM project employed the recently developed single particle tracking photo activated localisation microscopy (sptPALM) technology that allows the visualisation of abundant proteins. This high-resolution fluorescence microscopy was used to study bacterial cell biology simultaneously on multiple cells over the duration of the cell cycle.

The consortium's activities focused on the cell division protein FtsZ, a key next-generation antibiotic target, and on how it is organised during the cell cycle. Results showed that during cell division FtsZ concentrates in the middle of the cell in a band, possibly recruiting other proteins to the division site, and not providing a constrictive force as previously considered. This finding has important consequences for understanding the mechanism of bacterial cell division, and is anticipated to trigger further research studies.

CELL CYCLE PALM also developed software for imaging analysis, which is http://leb.epfl.ch/software (available online). The generated high-throughput modality of the PALM technique was disseminated by blogging, YouTube videos and press releases. Overall, this technique is anticipated to support further investigation and understanding of medically relevant processes in bacterial cell biology.