EGFR does not directly interact with cortical actin: A SRRF'n'TIRF study.

The epidermal growth factor receptor (EGFR) governs pivotal signaling pathways in cell proliferation and survival, with mutations implicated in numerous cancers. The organization of EGFR on the plasma membrane (PM) is influenced by the lipids and the cortical actin (CA) cytoskeleton. Despite the presence of a putative actin-binding domain (ABD) spanning 13 residues, a direct interaction between EGFR and CA has not been definitively established. While disrupting the cytoskeleton can impact EGFR behavior, suggesting a connection, the influence of the static actin cytoskeleton has been found to be indirect. Here, we investigate the potential interaction between EGFR and CA, as well as the extent to which CA regulates EGFR's distribution on the PM using SRRF'n'TIRF, a spatiotemporal super-resolution microscopy technique that provides sub-100 nm resolution and ms-scale dynamics from the same data set. To label CA, we constructed PMT-mEGFP-F-tractin, which combines an inner leaflet targeting domain PMT, fluorescent probe mEGFP, and the actin-binding protein F-tractin. In addition to EGFR-mEGFP, we included two control constructs: 1) an ABD deletion mutant, EGFRΔ