Advanced quantitative microscopy for functional mapping of protein interaction networks.
Lead Research Organisation:
King's College London
Department Name: Randall Div of Cell and Molecular Biophy
Abstract
The project will develop new super-resolution fluorescence techniques and apply time-resolved fluorescence imaging and spectroscopy to the study of the dynamic interaction between fascin and actin in live cells. In particular, we will investigate the dynamic binding of actin and fascin at filopodia in response to external perturbation (i.e. chemogradient, inhibition etc). We will develop a new paradigm of super-resolved fluorescence microscopies whereby single-molecule spectroscopic techniques are applied to both spatially resolve molecules and determine their photophysical properties. Our recent work has shown irect association of actin and fascin to be transient and we propose to investigate the degree of correlation between the known dynamic behaviour of actin and that of fascin using a combination of imaging correlation, particle velocimetry and fluorescence lifetime resolved super-resolution imaging. Development of key technologies for super-resolved functional imaging will have a marked impact on our ability to probe perturbations of the actin/fascin interactome.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/N509498/1 | 01/10/2016 | 30/09/2021 | |||
| 1951109 | Studentship | EP/N509498/1 | 01/10/2017 | 31/03/2022 | Conor Treacy |
| EP/R513064/1 | 01/10/2018 | 30/09/2023 | |||
| 1951109 | Studentship | EP/R513064/1 | 01/10/2017 | 31/03/2022 | Conor Treacy |
| Description | Focal Adhesions (FAs) are large sub-cellular structures comprised of macromolecular assemblies that anchor cells to the extracellular matrix and play a key role in force transduction and intracellular mechano-signalling pathways. Understanding how mechanical signalling influences specific molecular and cellular mechanisms is crucial in progressing our knowledge of how the external environment affects normal cell physiology. In this project, we used two tension sensitive biosensors to detect changes in force across two mechanosensitive FA proteins - Vinculin and Talin. These FA proteins both have a Tension Sensing Module (TSM), which contains two fluorescent proteins joined by a short-coiled linker that extends when force is applied. If the biosensors are not under tension, the two fluorescent proteins are in close enough proximity to undergo FRET (Förster Resonance Energy Transfer). By measuring FRET using multiphoton TCSPC (time-correlated single-photon counting) fluorescence lifetime imaging we observe the loss of FRET, compared to a control, as a direct consequence of an applied intracellular force across the biosensor. These force-dependent conformational changes enable us to describe the role that tension plays within specific protein-protein interactions found in FAs. These interactions affect FA assembly, maturation and degradation through force-dependent pathways and mechanisms. Ultimately, these pathways affect the adherence of cells to the extracellular matrix, which is of critical importance in understanding tumour growth and metastasis. |
| Exploitation Route | We will publish the findings and apply this work in the long term to migration of cells in 3D. Understanding cellular mechanosensing and force transduction is key to elucidating the mechanisms of cellular migration |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Research collaboration with Prof Madeline Parsons |
| Organisation | King's College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Provide Fluorescence lifetime imaging expertise |
| Collaborator Contribution | Helped and supported student with regards to basic cell-biology training, sample preparations and advice with experimental design. |
| Impact | This is a multi-disciplinary collaboration however there are no outcomes or outputs as of yet. |
| Start Year | 2017 |