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Designing molecular switches to build cell polarisation from the ground up

For many biochemical processes to take place in cells, components must organise within the space, a process known as symmetry breaking. European researchers investigated this phenomenon through a bottom-up synthetic biology approach using biological building blocks.

Fundamental Research

Cellular life is thought to have emerged when mixtures of precursor molecules became encapsulated into crude compartments to generate primitive cells called ‘protocells’. However, to exhibit the behaviours we associate with life, such as cell division and motility, cellular components must also self-organise in space and time.

Using biological building blocks to generate artificial molecular switches

One such self-organisation process is cellular polarisation, where homogeneously distributed molecules within cells become asymmetrically arranged. ‘Top-down’ methods are usually used to study this process, where the outcome of perturbations applied to a natural system is measured. With the support of the Marie Skłodowska-Curie programme, the POLAR project instead employed a complementary bottom-up synthetic biology approach. “We built minimal models from biological building blocks to produce synthetic systems that recapitulate a fundamental mechanistic aspect of cell polarisation and self-organisation,″ explains POLAR project fellow Leon Harrington, who led the project under the guidance of project coordinator Petra Schwille. To test mechanisms of self-organisation, the researchers built a synthetic membrane-targeting switch inspired by the Escherichia coli Min proteins, a well-studied pattern-forming system that facilitates bacterial cell division. Working with collaborators Jordan Fletcher and Dek Woolfson from the University of Bristol, Harrington designed and built a reversible membrane-targeting switch using minimal peptide motifs known as coiled coils. These molecules are much smaller than protein domains and have more tractable and predictable design rules. The team were able to switch the coiled coils between monomer and heterodimer states by reversible phosphorylation and dephosphorylation. The resulting ‘module’ was subsequently coupled to membrane-targeting motifs to build reversible membrane switches that can be used to study mechanisms of symmetry breaking. Biomolecules are prone to non-specifically stick together in synthetic contexts, rendering the engineering and control of protein-protein and protein-lipid interactions particularly difficult. Harrington admits it was technically challenging, but added: “The best proof of a theory or mechanism is to actually build it from the ground up and test it.″ The bottom-up approach of the POLAR project allows scientists to focus on the bare essentials required for living processes and enables the use of a broader range of measurement techniques than would be possible inside a living organism.

Future applications of the POLAR system

Future plans include introducing additional modules to generate positive and negative feedback, thereby decisively testing mechanisms of symmetry breaking. The scientists are also incorporating DNA-based components into the POLAR system. The ease of DNA manipulation and its predictable behaviour make these particularly attractive as biological building blocks. Applications of the molecular switches extend beyond the main aim of studying symmetry breaking. With collaborators, Harrington and co-workers are interested in expanding the scope of the POLAR molecular switches, for example by using other types of post-translational modification, or by designing switches with higher stoichiometries, such as trimers and tetramers. They also plan to integrate these switches within designed protein structures to generate dynamic behaviour inside cells. Overall, the development of reversible, coiled-coil interaction modules opens up many avenues for the design of dynamic molecular systems. Looking ahead, Harrington concludes: “These technologies will be broadly beneficial to the synthetic biology community.″

Keywords

POLAR, molecular switch, symmetry breaking, cell polarisation, coiled coil, synthetic biology, DNA

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