Frascogna, Crescenzo (2024) Cell's Sense of Slope: Unveiling the Mechanism of Focal Adhesion Mechanosensing. [Tesi di dottorato]

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Abstract

In this thesis, we aim to identify and characterize the fundamental mechanistic principles governing the assembly of FAs, which play a central role in the processes of mechanosensing and mechanotransduction. FAs are dynamic structures that act as the primary link between the ECM and the CSK, enabling cells to detect and respond to mechanical stimuli from their environment. The complex behavior of these structures is essential for various cellular processes, including migration, proliferation, and differentiation, making them critical for understanding how cells adapt to mechanical cues. The central objective of this work is to uncover the working principle of mechanosensing, which we hypothesize to be driven by a tilting mechanism of FAs. This tilting process involves the dynamic reorganization of FAs in response to mechanical forces, potentially explaining how cells adjust their adhesion and mechanical state depending on external conditions. Through a combination of experimental approaches and computational modeling, we identified the molecular dynamics and physical forces that regulate this mechanistic behavior. Experimentally, we investigated how FAs respond to different mechanical environments. Techniques like atomic force microscopy and confocal imaging allow us to visualize how paxillin and CSK components reorganize during force transmission, while biophysical methods enabled us to investigate the spatial configuration adopted by FA complexes. By integrating these experimental data with computational models, we aim to construct a more precise framework for understanding how mechanical forces propagate through FAs and how these forces modulate cell behavior. The theoretical side of our study is focused on developing a mathematical model of molecular dynamics that describes the tilting mechanism and the distribution of forces within FAs. This model predicts how FAs behave under varying conditions of substrate stiffness, and how they mediate cellular responses to different mechanical environments. To advance our understanding of mechanosensing, we developed a mechanical correlation matrix that establishes an equivalence in the cellular mechanical state induced by both topographical (curvature) and mechanical (stiffness) cues. This matrix enables us to quantify how cells perceive these two distinct signals as comparable inputs, allowing curvature and stiffness to substitute for each other in regulating cellular responses. One of the broader goals of this research is to apply the insights gained from studying FAs to the design of cell-instructive materials. By carefully controlling the substrate’s curvature, we aim to create microenvironments that sustain the stemness of stem cells, effectively preventing premature differentiation. Such engineered microenvironments could create the necessary mechanical and biochemical conditions for long-term maintenance of stem cell pluripotency. Furthermore, these materials could be used to control lineage-specific differentiation by modulating the mechanical signals that cells receive, opening up new possibilities in tissue engineering and regenerative medicine.

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