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bioRxiv · 10.1101/2024.07.20.604398

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

Pandey, S.·Wohland, T.
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Abstract

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 EGFRs distribution on the PM using SRRFnTIRF, a spatiotemporal super-resolution microscopy technique that provides sub-100 nm resolution and ms-scale dynamics from the same dataset. 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: a) an ABD deletion mutant, EGFR{Delta}ABD-mEGFP serving as a negative control, and b) EGFR-mApple-F-tractin, where F-tractin is fused to the C-terminus of EGFR-mApple, serving as the positive control. We find that EGFR-mEGFP and EGFR{Delta}ABD-mEGFP show similar membrane dynamics, implying that EGFR-mEGFP dynamics and organization are independent of CA. EGFR dynamics show CA dependence when F-tractin is anchored to the cytoplasmic tail. Together, our results demonstrate that EGFR does not directly interact with the CA in its resting and activated state. SIGNIFICANCESRRFnTIRF is a spatiotemporal super-resolution microscopy technique that allows for the investigation of plasma membrane-cytoskeleton interactions. We investigate how cortical actin (CA) influences the dynamic behavior and structural organization of EGFR, employing specific probe targeting CA structure and dynamics. Our results suggest that EGFR, whether in its resting or activated state, does not directly bind to or interact with the CA. Any influence of CA on EGFR is indirect through membrane modulating activities of CA.

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Pandey, S., Wohland, T.. 2024-07-22. EGFR does not directly interact with cortical actin: A SRRF'n'TIRF Study. https://doi.org/10.1101/2024.07.20.604398

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