Mechanosensitive protein-protein interactions in nascent focal adhesions determined by three-colour FRET sensing using Multiphoton Fluorescence Lifetime Imaging Microscopy

Student thesis: Doctoral ThesisDoctor of Philosophy


Focal Adhesions (FA) are large sub-cellular structures comprised of macro-molecular 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 and pathology.

In this project, we have used a tension-sensitive biosensor to detect real-time changes in applied force across the mechanosensitive FA protein vinculin. The biosensor contains two fluorescent proteins joined by a short-coiled linker that extends when force is applied. When the biosensor is under tension, the two fluorescent proteins separate, decreasing the amount of Fo¨rster Resonance Energy Transfer (FRET) observed. By measuring FRET using time-correlated single-photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM), we observe the loss of FRET, compared to a control, as a direct consequence of an applied intracellular force across the biosensor. By transiently transfected vinculin null mouse embryonic fibroblasts (MEFs) with a vinculin construct encoding the Tension Sensing Module (TSM), we can demonstrate how force-transduction changes within maturing adhesions in both fixed and live cells.

In addition, we have also shown through biochemical and biophysical assays that RIAM (Rap1-interacting Adapter Molecule) interacts with vinculin through an N-terminal binding domain. Furthermore, by developing a novel three-colour FRET sensing methodology, we have been able to describe the spa-tiotemporal relationship between several components assembled in nascent adhesions, namely talin, vinculin and RIAM. We have combined live cell fluo-rescence lifetime imaging with three-colour FRET sensing to determine the order of assembly for a multimeric complex as a function of time.
Date of Award1 Feb 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorSimon Ameer-Beg (Supervisor) & Madeline Parsons (Supervisor)

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