- Authors: Surve S. et al.
- Year: 2021
- Journal: J Cell Bio 220 e202107103
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: HeLa
Description: Human cervix epitheloid carcinoma cells
Knock-in: HeLa/mNG-NRAS cells were transfected with a PX459 plasmid expressing RAF1 gRNA 3 (Surve et al., 2019) and RAF1-mCh donor plasmid using jetOptimus transfection reagent at a ratio of 1:3. The cells were diluted for single-cell clonal selection and plated onto a 10-cm plate. The colonies were screened using fluorescence microscopic imaging and Western blotting with the RAF1 antibody.
The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS-containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.