A novel super-resolution microscopy approach to investigate the role of actin filament branching in cancer cell migration.

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Most cancer patients die from metastasis which require cancer cells to migrate. The force for cell migration is provided by actin polymerisation. Electron microscopy showed that actin filaments are nucleated at the leading edge of fixed cells by the Arp2/3 complex from the side of existing filaments creating a branched array. However, this has not been proven in live migrating cells and could be used to provide crucial information on Arp2/3 activation by oncogenes during cancer cell migrationIn this project we are studying three novel binding partners of the Scar/WAVE complex. They co-localise with the Scar/WAVE complex at the leading edge of migrating cells. My research question is to determine whether these proteins regulate the recruitment and activity of the Scar/WAVE complex, which is known to control branching of actin networks in lamellipodia via the branch-nucleator, Arp2/3 complex. I am also seeking to determine how these proteins function in directed networks, and how these networks interact to generate oscillatory protrusion-retractions of the leading edge which has been seen in previous research. I hope to then show how changes to this regulation manifest as changes in the mechanisms of cell migration, and thus seek to determine the function and importance of these three proteins in the context of cell motility.A variety of functional and super-resolution microscopy techniques combined with leading-edge and single-cell tracking data will be used to address these questions and hypotheses. The latter will facilitate the analysis to move from the level of molecular interactions to the level of cell-migration mechanisms and phenotype.In conjunction with the biological-driven experimentation, novel techniques in super-resolution such as PAINT/IRIS (transient binding approaches to single molecule localisation microscopy) will be developed and recently established software for the analysis of highly dense super-resolved micrographs will be employed. New novel probes in both super-resolution and FRET-FLIM will be designed to probe both structural and signalling proteins, as well as determine their activity and interactions. Improvements will be made to the mathematical implementations of Matlab programs designed to analyse cell migration, with emphasis on determining parameters such as migratory confinement, speed, persistence, and stability. These parameters will better enable the characterisation of cell migration in a quantitative way, and will result in more statistically robust, unbiased conclusions.FRET-FLIM microscopy will be used to study the activity of the Arp2/3 complex using a novel Arp2/3 biosensor developed in our laboratory in cells in which the genes for the novel Scar/WAVE binding proteins are knocked out using the CRISPR/CAS9 technique. Re-expression of cDNA of these proteins mutant for Scar/WAVE binding sites will be employed to test the functional significance of this regulation. In addition to FRET-FLIM, a novel super-resolution technique will be developed to image the actin network and its branch points in live cells: the positions of the Arp2/3 complex will be tracked and corrected against the global motion of the actin. Furthermore, computational techniques will be employed to monitor the retraction-protrusion of leading edges, and correlate this with information ascertained from prior functional and super-resolution images to determine the molecular origin of leading-edge oscillations and stability. Finally, cell tracking analysis will determine how changes in leading edge dynamics result in changes to cell motility.Ultimately, the aim is to compile this information to build a picture of: the complex interactions of the signalling networks involving the three aforementioned proteins; how these proteins alter the physical structure of lamellipodia; and how this change in structure effects the characteristics of migration.