EGFR Membrane Dynamics and Organization Investigated by Camera-Based Multi-Parameter Fluorescence Imaging with High Spatiotemporal Resolution

Abstract

The epidermal growth factor receptor is a prototypical receptor tyrosine kinase that has been investigated for four decades. However, the exact oligomerization state of the receptor in the cellular plasma membrane, its dynamics, and its relation to membrane domains and cytoskeletal structures is still under investigation. One of the hurdles is that the optimization of fluorescence methods to investigate structure, dynamics and oligomerization require different optimization strategies and thus their measurements have been performed in mutually exclusive ways. Here we overcome this problem by recording data in a total internal reflection microscope (TIRFM) at 1000 frames per second with optical resolution. We then use computational approaches to achieve super-resolution images (&lt100 nm) and extract quantitative parameters on fluorophore dynamics and oligomerization. For this purpose we combine imaging fluorescence correlation spectroscopy (imaging FCS) with super-resolution radial fluctuation (SRRF) analysis and number and brightness (N&B) analysis to extract spatial and temporal super-resolution data from the exact same sample simultaneously. Measurements were conducted on CHO cells that were co-transfected with a filamentous actin marker (LifeAct GFP) and mApple labeled epidermal growth factor receptor (EGFR-mApple). We achieve a spatial resolution of about 60 nm on actin filaments using SRRF, demonstrate diffusion with transient trapping for EGFR, and show that the majority of EGFR are dimerized in the absence of ligand. Interestingly, since the imaging FCS, N&B, and SRRF data were obtained simultaneously, we can show that the diffusion coefficient and the oligomerization state of EGFR are negatively correlated and that the diffusion coefficient shows no correlation with cytoskeletal actin structures.