Studying Focal Adhesions on Nanometric Platforms by Single-Molecule Localisation Microscopy

Abstract

Focal adhesions are integrin-based protein complexes that form at the interface between cells and the extracellular matrix (ECM). These adhesion structures connect the cell to the adhesive ligands presented on the ECM and serve as signalling hubs to help cells to sense and respond to signals from the ECM. It has been shown that cells require an average 50-70 nm spacing of adhesive ligands for the formation of stable adhesion structures. However, the molecular organisation of ligands and adhesion proteins is less documented. The aim of this project was to produce surfaces with varying spacing of fluorescently-labelled adhesive ligand nano-domains and obtain single-molecule images of both the adhesive ligands and the adhesion proteins to better understand how adhesive ligand distribution influences adhesion organisation. First, the di-block copolymer phase separation method was optimised to produce surfaces with nano-domains with different average inter-domain spacing ranging from 18 to 93 nm (Chapter 3). Next, the nano-domains were functionalised with fluorescently-tagged adhesive ligands to facilitate cell attachment to the surfaces and formation of focal adhesions. Direct stochastic optical reconstruction microscopy (dSTORM) and quantitative analysis of the fluorescently-labelled adhesive ligands, based on density-based spatial clustering of application with noise (DBSCAN), were successfully combined in order to map the molecular organisation of adhesive ligands on the fabricated surfaces. Then, mouse embryonic Fibroblasts (MEF) were cultured on the surfaces for various time points. Cell attachment was found to be independent of the adhesive ligand nano-domain spacing while cells exhibited different patterns for spreading and adhesion formation on the different surfaces over time. Finally, two-colour single-molecule localisation microscopy (SMLM) was combined with DBSCAN and colocalisation analysis to produce colocalisation maps of adhesion proteins and adhesive ligands on surfaces with different spacings of adhesive ligand nano-domains, and to evaluate the degree of co-localisation between them (Chapter 5). In conclusion, the results presented in this thesis provide new insights on how the nanoscale distribution of adhesive ligand nano-domains influences cell attachment and spreading as well as the formation and organisation of focal adhesions at the molecular level.