Projects

Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter- and intra-molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin-membrane distance of 2.5 nm. We demonstrate that QD-FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub-nanometer accuracy.

We are implementing a completely new artificial cell model that will be used to reconstitute focal adhesions (FAs). We are proposing to investigate “inside-out” integrin-mediated FA assembly in this artificial cell model. We are reconstituting FA complexes using the microinjection of proteins into GUVs to better understand recruitment sequence and FA assembly mechanism. 

The project aims to develop Focal adhesion nanosensors that can measure forces at the pN level in living cells and identify the mechanosensing process.
Mechanosensing is the process of force transmission across the cell membrane, essential for cell to properly accomplish adhesion and migration processes required for tissue building and repair. Force transmission is mediated by focal adhesions (FAs), highly organised, self-assembled microstructures. Mechanosensors, that make up FAs, are specialised proteins capable of sensing tension applied by the cell contracting apparatus. In this way, mechanosensors receive both molecular and mechanical stimuli allowing them to rapidly re-model the FA. Because of the complexity of FA assembly, its mechanosensing mechanism remains unclear. Although mechanical forces play a crucial role in biology, imaging cellular force with sub-100-nm resolution remains a challenge. Thus, in this project we are developing new FRET mechanosensors for measuring forces in the pN range and imaging molecular interactions at the nanoscale.