摘要：SummaryNRas is a key mediator of the mitogenic pathway in normal cells and in cancer cells. Its dynamics and nanoscale organization at the plasma membrane (PM) facilitate its signaling. Here, we used two-color photoactivated localization microscopy to resolve the organization of individual NRas and associated signaling proteins in live melanoma cells, with resolution down to ∼20 nm. Upon EGF activation, a fraction of NRas and BRAF (dis)assembled synchronously at the PM in co-clusters. NRas and BRAF clusters associated with GPI-enriched domains, serving as possible nucleation sites for these clusters. NRas and BRAF association in mutual clusters was reduced by the NRas farnesylation inhibitor lonafarnib, yet enhanced by the BRAF inhibitor vemurafenib. Surprisingly, dispersed NRas molecules associated with the periphery of self-clusters of either Grb2 or NF1. Thus, NRas-mediated signaling, which is critical in health and disease, is regulated by dynamic interactions with functional clusters of BRAF or other related proteins at the PM.Graphical abstractDisplay OmittedHighlights•Two-color PALM resolved the dynamic interactions of NRas in live melanoma cells•Upon EGF activation, NRas and BRAF (dis)assembled synchronously in co-clusters•NRas and BRAF co-clustering was reduced by lonafarnib, yet enhanced by vemurafenib•NRas signaling is regulated by interactions with BRAF, Grb2, NF1, and GPI clustersBiological sciences; Cell biology; Biophysics; Molecular biology
