Examining the ligand-induced change in cell adhesion using the quartz crystal microbalance with dissipation monitoring

Marcela Del Pilar Garcia

doi:10.17918/etd-6023

Cell adhesion is a highly regulated process involving initial receptor-ligand binding, and subsequent clustering of these receptors and rapid association with the actin cytoskeleton as focal adhesions is assembled. Focal adhesions enhance adhesion, working as structural links between the cytoskeleton and the extracellular matrix, to finally trigger signaling pathways that direct cell function. In the past two decades, the quartz crystal microbalance (QCM) has become a powerful bio-analytical tool that is capable of studying complex biological processes such as cell adhesion. The work presented investigates the use of the quartz crystal microbalance with dissipation monitoring (QCM-D) to track ligand-induced changes in cell adhesion of a monolayer of cells by means of real-time changes in energy dissipation factor, [delta]D, of the cells. Here I report the first QCM-D study that examines the restructuring of focal adhesions induced by the epidermal growth factor (EGF) in MCF-10A cells. The epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. Mutant cells that overexpress EGFR displayed a different kinetic profile to that of the wildtype MCF-10A cells that had a lower level of EGFR. Mutant MCF-10A cells had a higher rate for the initial disassembly of focal adhesion and a much lower rate for the alter reassembly of focal adhesions. These results suggest that the effects displayed by EGFR-overexpressing cells may contribute to the initiation and maintenance of a more favorable adhesion state for cell migration. Additionally, I report a complex de-adhesion process, which can be altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The IC₅₀ values attained from the dose dependencies are in strong agreement with the values reported in the literature. These results also demonstrated a quantitative relationship between the time-dependent [delta]D-response and the levels of focal adhesions. Furthermore, I report a novel application of QCM-D for the detection of EGCG-induced ROS formation. The results from this study showed that EGCG-induced [delta]D-response can be correlated with the change of EGCG-induced ROS formation. The QCM-D is a highly sensitive, non-invasive, and label-free technique that has a broad range of applications in the fundamental study of cellular processes, such as cell signaling and trafficking and mechanotransduction and holds promise to become an effective sensing platform for screening therapeutic agents and ROS formation.

Examining the ligand-induced change in cell adhesion using the quartz crystal microbalance with dissipation monitoring

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Examining the ligand-induced change in cell adhesion using the quartz crystal microbalance with dissipation monitoring

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