Synthetic Mimicry of Cellular Polarisation

Project Information

POLAR

Grant agreement ID: 705587

DOI

10.3030/705587

Project closed

EC signature date 4 March 2016

Start date 1 September 2017

End date 31 August 2019

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 171 460,80

EU contribution

€ 171 460,80

Coordinated by

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - POLAR (Synthetic Mimicry of Cellular Polarisation)

Reporting period: 2017-09-01 to 2019-08-31

Summary of the context and overall objectives of the project

A question of fundamental importance in biology is how order spontaneously emerges from homogenously distributed building blocks, for example in the formation of cells and the many biochemical processes occurring within them to spatially organise processes such as motility and division. This transformation from a homogenous state to one where components are spatially organised is often referred to as ‘symmetry breaking’. One process by which it is achieved is called self-organisation. One of the most fundamental biological self-organisation processes is cellular polarisation, for example as extensively studied in budding yeast, whereby a cell spontaneously orients molecules within it such that a single focus on the cell membrane is formed. This polarization process underlies both the mating behaviour of yeast as well as cell division, where daughter cells form and bud off from this focal point.

Some theoretical models suggest that cellular polarisation can spontaneously occur through a simple mechanism that requires: (i) reversible lipid membrane binding, (ii) diffusion while bound upon the membrane, and (iii) positive feedback, i.e. membrane-bound molecules recruit more molecules to bind the membrane. The goal of this project was to test these models by building a synthetic system from the bottom up that recapitulated these key features. As well as helping us understand the mechanism by which symmetry breaking, such as in cellular polarisation, occurs, achieving this goal would also provide useful tools for the creation of ‘proto-cells’—artificial, self-contained systems that exhibit life-like processes. Our strategy incorporated a key tenet of synthetic biology, namely that the system should be built in a modular manner from minimal components. In this case, the minimal modules were to be minimal peptides and protein domains. The project contained two planned milestones: first, to construct a reversible membrane-binding switch; and second, to subsequently add additional modules to generate positive feedback, and thereby generate symmetry breaking.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Initial efforts to build the reversible membrane binding switch were hampered by non-specific protein aggregation. We subsequently altered the design and further minimised the complexity of the system by replacing some of the modules that were based on protein domains with minimal, coiled-coil peptides. As well as reducing the complexity of the design, this change also allowed us to use chemical strategies, specifically solid phase peptide synthesis. In collaboration with the University of Bristol, we took advantage of this by rapidly prototyping and evaluating these minimal modules through chemical synthesis and a variety of biophysical measurements. These efforts resulted in a highly suitable coiled-coil peptide interaction module whose oligomeric state could be switched between monomer and dimer states by reversible phosphorylation and dephosphorylation. We then proceeded to incorporate this module into our designs to generate reversible membrane targeting.

We explored two designs for membrane targeting: (i) where one binding partner (the ‘anchor’) is permanently attached to the lipid membrane, with a ‘cargo’ molecule that reversible attaches to the anchor from solution via the minimal interaction module, and (ii) where both binding partners are free in solution and membrane binding is switched through changes in their oligomeric state, with monomers remaining in solution and heterodimers binding the membrane. We produced proteins formed from combinations of our minimal modules and tested them on model lipid membranes, successfully achieving both modes of membrane targeting. To achieve the second mode of binding, we had to seek alternative membrane-binding modules and carried out several rounds of design and optimisation to develop a suitable peptide motif.

Due to the additional stages of alteration and refinement required to implement our designs, we had little time to pursue the second milestone of the project, to incorporate additional modules for generating positive feedback. In testing our initial designs we identified several challenges that will need to be circumvented through further engineering cycles as described above for our other modules.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project has produced several new technologies with broad applicability in protein and bioengineering. The development of reversible, coiled-coil interaction modules opens up new applications that were not in the scope of this project, such as creating logic circuits and controlling the assembly and disassembly of higher order structures. Further, we coupled these interaction modules with additional functional modules to build reversible membrane-targeting switches that allow the localisation of a variety of molecules to be controlled in artificial membrane systems and potentially in vivo. These are foundational technologies that we envisage will be broadly beneficial to the synthetic biology community. Although we did not achieve our final milestone of incorporating positive feedback and hence building a synthetic system capable of symmetry breaking, the tools we have developed in this action will form the basis of further efforts towards this goal. We will shortly publish this work in a peer-reviewed journal, and have presented the results at several international conferences.

Reversible membrane targeting