Abstract RAS GTPases are central drivers of proliferation, differentiation, morphology, and apoptosis, yet oncogenic RAS remains largely untreatable: only two KRAS G12C–specific therapies are approved, benefit a minority of patients, and face clinical resistance. A key unresolved problem is how RAS activates its main effector, RAF, inside cells. Bulk affinity measurements show strong RAS–RAF binding but obscure the lifetimes of individual encounters and their spatial context. Here we integrate single-molecule with both in vitro and live-cell approaches to quantify the spatiotemporal dynamics of RAS–RAF binding. To directly visualize one-to-one RAS–RAF and RAF–RAF interactions in a TIRF microscope, we conducted dynamic single-molecule pulldowns: we built artificial 2- or 8-lipid membranes on coverslips (supported lipid biolayers) with tethered recombinant human RAS proteins. Then, BRAF or CRAF proteins from crude human cell lysates were captured by the membrane-bound RAS. This system allowed us to quantitate the biophysical parameters of RAS-RAF complexes diffusing on a lipid bilayer in real time. Strikingly, most individual RAS-RAF encounters only lasted a few tens of milliseconds. These short-lived interactions are consistent with constant interaction turnover detected in conventional co-immunoprecipitation assays. In live cells, we built upon a human HEK cell line devoid of endogenous H/N/KRAS and A/B/CRAF genes (6 KOs), to stably express halo-KRAS4b and snap-CRAF at stoichiometric, low levels. Then, we imaged single-molecule halo-KRAS4b and snap-CRAF using simultaneous two-color TIRF video acquisition, and analyzed the dynamics of the RAS/RAF interaction. Similar to our in vitro results, we observed that CRAF exhibits very short membrane dwell times in live cells, which increased upon MAPK pathway activation with soluble EGF. By bridging millisecond-scale binding kinetics with MAP kinase pathway activation, our work outlines how transient RAS–RAF contacts are regulated in space and time. This could reveal potential vulnerabilities for therapeutic intervention beyond current RAS inhibitors. Citation Format: Rodrigo E. Cáceres-Gutiérrez, Rebika Shreshta, Jean Castillo-Badillo, Vanessa Wall, Scott Eury, Katie Powell, William Burgan, Min Hong, Peter Frank, Dwight V. Nissley, Frank McCormick, Thomas Turbyville. Spatiotemporal control of RAS-RAF signaling at the single-molecule level abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5976.
Cáceres-Gutiérrez et al. (Fri,) studied this question.