The role of Piezo1 in cell-extracellular matrix interactions

Tanribil, Patrick Selcuk (2025): The role of Piezo1 in cell-extracellular matrix interactions. Dissertation, LMU München: Faculty of Medicine

Abstract

One of the most fundamental aspects of physiology is the interaction between cells and their physical microenvironment. Sensing the forces at play, adapting to them, and remodelling of the extracellular matrix are essential for processes such as development, tissue homeostasis, and injury response. In the cardiovascular system, cells constantly encounter biomechanical forces from their surroundings, which play a pivotal role in their behaviour. These physical stimuli encompass a range of active cues, such as shear stress related stimuli as well as passive cues, including matrix composition, substrate stiffness and spatial patterning. Modern therapeutic advances, like regenerative medicine, rely on a comprehensive understanding of how cells respond to these mechanical forces. The field of mechanobiology is dedicated to studying how cells process and adapt to such signals. The discovery of the mechanically sensitive, non-selective cation channel Piezo1 has revealed a principal transducer of mechanical signals across the plasma membrane, and this thesis aims to elucidate Piezo1's role in cell-matrix interactions. While Piezo1s' involvement in the physiology and pathology of various organs and tissues has been established, its underlying role in cell-extracellular matrix (ECM) interactions remains an active area of investigation. Cellular adhesion to the extracellular substrate is a fundamental requirement for physiological remodelling of the ECM and forms the basis for a multitude of processes across the body including e.g. morphogenesis, fibrosis and hemostasis. The deciding factors involved in determining whether cells commit to adhering to a substrate remain not yet fully understood. This study demonstrates that Piezo1 is a crucial component of the initial adhesion process. The findings suggest that Piezo1 is fundamentally necessary for maintaining physiological levels of adhesion across diverse substrate conditions. In order to explore the impact of Piezo1 on cellular response to substrates with different rigidities murine embryonic fibroblasts (MEFs) were established as a model system. Genetically modified cell lines expressing fluorescent reporters were generated for microscopic imaging. The peptide D-GsMTx-4, a selective inhibitor of cationic mechanosensitive channels, was utilized to inhibit the function of Piezo1. Experiments on fibrin-coated glass revealed that D-GsMTx-4 impaired the dynamics of cellular adhesion by prolonging the time required for successful cell adhesion without affecting the ability to interact with and contract the ECM. Further experiments using surfaces functionalized with Arg-Gly-Asp (RGD) cell adhesion sequence confirmed that D-GsMTx-4 reduced adhesion to the substrate. This effect could be rescued by chemical integrin activation with Mn2+, indicating it may be dependent on integrins. Generation of Piezo1-knockout cells and gene rescue experiments corroborated these results. To further elucidate the role of ECM rigidity, tension gauge tethers (TGTs)- dsDNA tethers of defined strength that irreversibly break under tensile force, were employed. Adhesion on TGTs was also impaired in Piezo1-knockout cells and under D-GsMTx-4 treatment, at a ligand density of at least 10% and ≥43 pN resistance per tether. Therefore the observed effect appears to be a process dependent on the rigidity of the extracellular matrix. This phenotype could also be rescued by addition of Mn2+ illustrating the inter-influence of integrins and the ECM. Subjecting MEFs to shear stress to further test the impact of Piezo1 inhibition on stable early cell-substrate bonds, additionally supported the involvement of Piezo1 in early adhesion processes. Collectively, these results suggest that Piezo1 plays an integral role in initial cell adhesion, potentially by acting as an amplifier of integrin ligand affinity and clustering through a proposed positive feedback loop involving Piezo1-mediated Ca2+ influx. The precise mechanisms regulating initial adhesion formation remain unclear and warrant further investigation. The findings presented here demonstrate that Piezo1 is a strong candidate for facilitating the mechanosensory capacity in these adhesion sites, underscoring the importance of understanding the role of Piezo1 in physiology as well as pathology on a fundamental cellular level.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 600 Technology, Medicine; 600 Technology, Medicine > 610 Medical sciences and medicine
• Faculties: Faculty of Medicine
• Language: English
• Date of oral examination: 3. April 2025
• 1. Referee: Gärtner, Florian
• Signature of the printed copy: 0700/UMD 22735