Quantification modelling and analysis of molecular dynamics patterning and signalling in the NK synapse.

Lead Research Organisation: University of Warwick
Department Name: Mathematics


Biological systems function by coordinated signalling and communication between cells, signals which determine cell function and response. The immune system is one of the best studied as regards signalling between cells with both signalling via soluble mediators and cell:cell contact. Such signalling regulates and directs the immune system response to pathogen exposure where an inappropriate response can lead to disease and death. Signalling during cell:cell contact results in formation of a macroscopic signalling interface with patterning on the scale of microns of various receptors, the simplest pattern observed being a bull's-eye configuration. A theory for the mechanism of this patterning has been suggested and a mathematical model available for simulation which gives realistic results, a model that emphasises the role of the cell:cell interface environment on signalling. We will test this model by measuring a number of model parameters, varying from cell elasticity to chemical reaction affinities, and verify model predictions. This will be achieved by sophisticated imaging using green fluorescent jelly fish protein fused to proteins of interest, and leading edge image analysis. The system we will be studying is of immense importance in medicine and immunology.

Technical Summary

Experimental techniques to monitor and decouple processes in the immunological synapse are unravelling a vast complexity in the process and time orchestration of T-cell and NK cell activation; a process that involves physical processes such as molecule diffusion and membrane physics coupled with reaction kinetics, signalling and active cytoskeletal activity. We propose to test, develop and parametrise a model of receptor patternation and signalling that occurs in the Natural Killer cell synapse through a joint experiment-theory programme. Transformed cell lines expressing chimeric proteins will be generated, including gfp fusions and extracellular domain length variants. Sophisticated image analysis techniques will be used to analyse 3D high resolution scanning confocal images of synapse patterns in cell:cell conjugates and molecule movement in membranes. Model parameters, including 2D affinities in situ, will be measured by a variety of methods. The model will be tested through comparing predictions for key observables, such as the enrichment ratios, between the model and observed synapses over a range of ligand concentrations, and in perturbed systems where ligand localisation is predicted to alter under changes in bond length. The model will be used to analyse NK synapse development, signal dynamics and identify the key determinants of the inhibitory and cytotoxic synapses. More generally our methods will generate a flexible image analysis tool appropriate for analysis and parametrisation of enrichment domains and transport in cell membranes, whilst our experimental programme will probe the nature of the cell:cell environment and how it regulates signalling.
Description This project was one of the first efforts worldwide to directly combine mathematical models and quantitative imaging in testing specific hypotheses for formation and function of immunological synapses. As a result, we developed several new methods in quantitative imaging, image analysis and mathematical modeling. Specifically:

i. We discovered that receptor-ligand dimensions are important in NK cell recognition, and that optimal integration of activating and inhibitory receptor signals requires that their receptor-ligand complexes have similar dimensions. (PLoS ONE 5(11): e15374. doi:10.1371/journal.pone.0015374.).

ii. We developed a new statistical method to extract and model 3D spatial data on individual cells. The key challenge here was to model protein redistribution on the cell geometry and maximise pixel info. PLoS Comp Biol. 2011, DOI: 10.1371/journal.pcbi.1002076

iii. We demonstrated that the model predictions for size driven segregation between proteins are satisfied. Mutual exclusion follows a functional relationship and two key instability criteria required for patternation were verified to hold. PLoS Comp Biol. 2011, DOI: 10.1371/journal.pcbi.1002076
Exploitation Route The fact that ligand size is so important to signalling in the immune system, this potentially can provide a basis for development of immune modulation techniques.
Sectors Pharmaceuticals and Medical Biotechnology