Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling

Significance This paper describes a quantitative imaging approach based on photoactivated localization microscopy for precise protein localization and stoichiometric analysis inside an intact cell. With this approach, we directly resolved individual protein monomers, dimers, and multimers in fixed mammalian cells. We used this technique to study the dimerization and multimerization of the protein kinase RAF, a putative mechanism for mutant Rat sarcoma (RAS)-mediated RAF activation as well as for the paradoxical activation of RAF/MAPK in RAF WT cells and tumors upon treatment with RAF kinase inhibitors. We presented direct evidence that RAF indeed forms dimers and occasionally higher-order multimers under various activating conditions. These observations provide critical insight into the biological regulation of RAF signaling and oncogenesis. The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF–CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.