INVESTIGATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR ORGANIZATION AND DYNAMICS ON THE PLASMA MEMBRANE OF LIVE CELLS USING FLUORESCENCE TECHNIQUES

Abstract

The epidermal growth factor receptor (EGFR) is a plasma membrane protein that is a crucial regulator of cell proliferation and survival, and its mutations and dysregulated signalling have been implicated in many cancers. However, a clear understanding of its oligomerization state, dynamics, and interactions with the actin cytoskeleton and other plasma membrane components has been elusive. This thesis is an investigation of these questions in live CHO-K1 cells using total internal reflection fluorescence (TIRF) microscopy-based fluorescence spectroscopy techniques. First, brightness-calibrated camera-based number and brightness (N&B) analysis was established for estimating EGFR oligomerization in resting cells, after drug treatments, and using EGFR mutants. Next, a strategy was developed to combine multiple fluorescence techniques and obtain mutually consistent structure and dynamics information of EGFR behaviour and interactions simultaneously. Cross-correlation analysis between any two parameters provided new insights into EGFR biology that could not be obtained from separate sequential measurements.