A Multiscale Analysis of Cell Polarity Transitions in a Bacterium (1): reconstitution of a biochemical oscillator inside liquid droplets
Cell decisions are governed by complex regulatory networks that integrate changes in the environment and convey a cellular response. Understanding the functioning of these networks is a major challenge because they are generally obtained from fragmentary datasets that lack both quantitative and spatiotemporal information. As a result, the genetic pathways mostly consist of blueprints that capture the interactions between the components but generally fail to contain mechanistic and thus predictive value. Given that molecular interactions are in essence only amenable to low-throughput analyses, any first attempt in modeling dynamic networks must focus on highly tractable experimental systems. In this project, five CENTURI teams, Mignot and Michelot (proposal 1), and Habermann, Tichit and Barrat (proposal 2) will develop an interdisciplinary collaboration to elucidate how bacterial cells (Myxococcus xanthus) make directional decision using an evolutionarily-conserved G-protein cell polarity oscillator.
The two intertwined projects will combine biophysical cell mimetic assays, genetic live experiments and computational simulations of higher-order networks to provide a first spatiotemporal model of the protein-protein interactions that drive protein oscillations. In the long run, this work will serve as a framework to study single cell decisions in multicellular contexts, a question of general significance in higher organisms.
This first proposal is principally experimental.
G-protein, biochemical oscillator, motility, cell mimetic systems, higher order networks
Myxococcus xanthus cells change their direction of movement in response to environmental signals by a mechanism involving a polarity change under the control of receptor-activated signal transduction (herein called the Frz pathway). At the core of this mechanism, the biochemical oscillations of a small G-protein (MglA) govern cell polarity, which involves sequential and spatial interactions of MglA with regulators of its nucleotide state at opposite cell poles (ie the MglB GAP at the lagging pole and the RomR GEF, at the leading pole). However, how Frz control the period of the oscillations and which biochemical interactions it perturbs is currently unknown due to a lack of knowledge of the stoichiometry and time sequence of protein interactions. In the course of this project, we will first study protein interaction quantitatively in a cell-free biomimetic system and test their importance in vivo. Knowledge from these experiments will be used to construct a motility model, computing Higher-Order Networks (HON) from First Order Networks (FON) in close collaboration with the postdoc selected from proposal 2. The predictive power of the model will then be tested experimentally.
Proposed approach (experimental / theoretical / computational)
In the past interactions between MglA and its binding partners (MglB, RomR) have been inferred from cell extracts or in solution, assays that provide valuable inputs but have either low quantitative value or arguably do not capture the intracellular environment where local concentrations and stochastic fluctuations drive biochemical reactions constantly away from steady-state. In the course of this project, we will use a new cell mimetic system, in which MglA and MglB interactions will be reconstituted inside lipid droplets. Specifically, we have shown that MglB interacts with lipids and MglA could displace MglB from the membrane at the pole to initiate the switch with the help of other Frz factors. We will test this hypothesis directly in liquid droplets using purified fluorescently labeled proteins and test how specific mutations and Frz effectors affect the dynamics of this interaction. The relevance of any new interaction thus discovered in vitro will be tested in a cellular system where the effect of genetic mutations can be tested directly on single cell motility and protein subcellular oscillations and dynamics. All together these experiments should identify concurrent and consecutive protein binding events in the polarity oscillator complex, which we will use to feed a first HON constructed from available data in proposal 2.
The project is highly interdisciplinary as it links in vitro biophysics to genetics and high-end computational modeling. Specifically, two postdocs for 2 years will be recruited and shared between five CENTURI member teams (Michelot, Mignot, Habermann, Tichit, Barrat). The postdoc selected for the proposal 1 will perform most of the experimental work, and will work side-by-side with the members of the proposal 2 to implement the computational algorithm.
All the tools are available to start the project:
(i) The Michelot lab has developed the cell mimetic system and shown its power to study actin polymerization
(ii) The Mignot lab has developed the genetic/cell biology tools and all purified proteins are available and active for the project. In addition, crystal structures of MglA and MglB alone, in different nucleotide states and in complex are also available (J. Cherfils and T. Mignot in preparation).