The predominant signal transduction systems in bacteria, two-component systems are essential to enable microorganisms to adapt to changes of their environment. They regulate important developmental programs including bacterial virulence. Typically, they are composed of a transmembrane sensor-kinase protein and a cytoplasmic response regulator. Perception of a chemical or physical signal by the sensor leads to kinase activation and autophosphorylation, and then transfer of the phosphoryl group to the response regulator. Thus activated, the latter mediates a specific, frequently transcriptional, cellular response. The whooping cough agent Bordetella pertussis colonizes the upper respiratory tract of humans. Its virulence regulon is controlled by the two-component system BvgAS. At 37°C and in laboratory growth conditions, the BvgAS system is activated, leading to the transcription of the virulence regulon, including genes for B. pertussis’s adhesins and toxins. The virulent Bvg+ phase of B. pertussis is necessary for infection. Switching to the avirulent Bvg- phase is triggered by the addition of negative modulators. Thus, BvgS might be active by default and inactivated by antagonists at specific stages of the bacterium’s life cycle. BvgS is a hybrid sensor-kinase harbouring several cytoplasmic domains that mediate a complex phospho-transfer cascade. It also contains two periplasmic ‘Venus flytrap’ (VFT) domains in tandem and a cytoplasmic PAS domain before the kinase. Ubiquitous in nature, VFT domains usually function along a clamshell model, with two lobes that enclose specific ligands between them. Conformational changes of VFTs between open and closed forms upon ligand binding are widely used for transport, or also for signalling such as in the ion-channel coupled Glu receptors of higher eukaryotes. BvgS is the prototype for a large family of multi-domain sensor-kinases that harbour periplasmic VFT perception domains, which represents a new paradigm of VFT receptors involved in signal transduction. The molecular mechanisms of signal perception and transduction by these proteins remain unexplored. We have recently obtained the crystal structure of the entire periplasmic domain of BvgS, showing a novel architecture for VFT receptors. The periplasmic domain of BvgS is dimeric, with extensive interfaces between protomers. Our preliminary data have indicated that the conformations of the VFT domains determine BvgS activity and that the interprotomer interfaces are critical for function. This new structure and the tools that we have developed for the functional analysis of BvgS put us in a good position to decipher the molecular mechanisms of signalling by using an integrated approach that combines functional analyses, biochemistry, structural biology and in silico modelling. We will identify critical regions of the VFT domains that determine the active conformation of BvgS and the mechanisms by which negative signals are perceived and transmitted, using site-directed mutagenesis and a functional assay in bacterio. The conformation and dynamics of BvgS and its mutants will be investigated by crystallography and by modelling, using molecular dynamics simulations and coarse-grained approaches. Simulations will also be performed of the VFT domains with the transmembrane segment in a lipid environment. This will be combined with biochemical approaches to explore the topology and dynamics of the segment linking the periplasmic and cytoplasmic moieties, and of the linker between the PAS and kinase domains. The function and topology of the PAS domain will be characterized. Our program will enable us to propose a model that describes the molecular mechanisms of signal transduction in this family of VFT sensor-kinases. This will enlarge our knowledge on two-component-mediated signal transduction in bacteria, as well as lay the bases for new avenues of targeted therapeutic intervention.

The aim of the project is to theoretically model the effects of fluctuations in Structured Coulomb fluids (SCF’s) and to develop the computational means to apply implicit solvent models to biological systems. SCF’s are composed of solvated ions, charged molecules like DNA, proteins or protein complexes, or larger biomolecular structures such as membranes. Since often such biological systems are highly charged, both fluctuations of the electrostatic potential or electric field and the nature of the polarizable surrounding medium, water, need to be taken into account in order to describe these systems in a proper way. In the first part of the project we will extend an established generalization of the mean-field Poisson-Boltzmann theory of SCF’s based on the electrostatic potential to include fluctuations. The second part of the project will see the development a Poisson-Boltzmann theory for an SCF in terms of a local constrained functional of the electric field, which due to its convexity property has an essential computational advantage and will allow applications of our approach to still larger systems.

A human body performs about 10,000 trillion cell divisions in a lifetime. Deciphering how is precisely controlled the “decision” to enter into mitosis during each cell cycle is a major challenge in cell biology that will create new therapeutic perspectives. Irreversible entry into mitosis is under the control of checkpoint mechanisms; a G2 DNA damage checkpoint that will arrest cells in G2 phase in the presence of DNA lesions and an antephase checkpoint sensing stress conditions up to early prophase. These control mechanisms delay Cyclin B1-Cdk1 activation, the master kinase orchestrating entry into mitosis, and prevent genetic instability as a consequence of chromosome separation with unreplicated or damaged DNA. Experiments performed in yeast to human showed that deregulation of Cyclin B-Cdk1 activity or overexpression of its activator(s) can trigger entry into mitosis of S phase cells still containing unreplicated DNA, enlightening the importance of the tight control of entry into mitosis for genomic stability. When the checkpoint mechanisms are satisfied, Cyclin B1-Cdk1 activation takes place and trigger entry into mitosis. We previously developed a FRET (Förster Resonance Energy Transfer)-based specific CyclinB1-Cdk1 activity biosensor to demonstrate that its initial activation is taking place at a very reproducible set time in each individual living cell. What are the immediate upstream mechanisms that reproductively trigger its initial activation during each G2 phase is still a fundamental unanswered question. More generally, the core molecular machinery taking place after the completion of DNA replication during a normal G2 phase progression and ultimately leading to Cyclin B1-Cdk1 activation is poorly understood. In the present project, we aim to investigate the spatio-temporal regulation and roles of ERK (Extracellular Regulated Kinases) 1&2 and Plk1 (Polo-like kinase 1) in the cell cycle progression from early G2 to mitosis. Recent reports suggest that ERK1&2 activities regulate a gene expression program in early G2 specifically in epithelial versus fibroblast cells. Because temporal and intensity-modulated ERK1&2 activities strongly affect their ability to activate downstream events, we will analyze in real time their activation signature during G2 progression using our recently developed FRET-based specific activity reporter. We will next combine the use of this biosensor with ERK inhibitor(s) to visualize in each individual living cell the extent of ERK1&2 inhibition and the consequences for G2 phase progression and/or mitotic entry in cells from different tissue origins. We thus expect to clarify their involvement in the regulation of G2/M progression. Plk1 is known to participate in the regulation of entry into mitosis in mammals but the underlying mechanisms are not fully understood. We identified a main target, among the Cyclin B1-Cdk1 regulators, and observed that its phosphorylation is taking place from late G2 cells, mostly at centrosomes. We will determine if a burst of Plk1 kinase activity is taking place just before entry into mitosis during each cell cycle and if Plk1 dependent phosphorylation of the Cyclin B1-Cdk1 regulator triggers entry into mitosis. We will evaluate if the centrosome is a "platform" facilitating the initial activation of CyclinB1-Cdk1 and entry into mitosis using optogenetic inducible recruitment of this regulator. Finally, an ambitious aim of this project will be to analyze the conservation of our findings concerning the regulation and roles of ERK1&2 and Plk1 during G2 to mitosis progression in Xenopus embryonic epithelial tissues as a model of cell proliferation in vertebrate tissues using our corresponding FRET-based activity reporters and implemented frequency domain FLIM FRET microscopy approaches. We thus expect to significantly improve our knowledge of the successive molecular steps taking place during G2 to mitosis progression and which are potential therapeutic targets.