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

Xiaolin Nan, Eric A. Collisson, Sophia Lewis, Jing Huang, Tanja M. Tamgüney, Jan T. Liphardt, Frank McCormick, Joe Gray, Steven Chu

Research output: Contribution to journalArticle

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Abstract

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 suf ficient step for RAF activation.

Original languageEnglish (US)
Pages (from-to)18519-18524
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number46
DOIs
StatePublished - Nov 12 2013

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Microscopy
Spatial Analysis
Membrane Fusion
Protein-Serine-Threonine Kinases
Neoplasms
Cytoplasm
Proteins
Cell Membrane
Technology
Growth
Single Molecule Imaging

Keywords

  • Cancer signaling
  • Single molecule imaging
  • Superresolution optical microscopy

ASJC Scopus subject areas

  • General

Cite this

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling. / Nan, Xiaolin; Collisson, Eric A.; Lewis, Sophia; Huang, Jing; Tamgüney, Tanja M.; Liphardt, Jan T.; McCormick, Frank; Gray, Joe; Chu, Steven.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, No. 46, 12.11.2013, p. 18519-18524.

Research output: Contribution to journalArticle

Nan, Xiaolin ; Collisson, Eric A. ; Lewis, Sophia ; Huang, Jing ; Tamgüney, Tanja M. ; Liphardt, Jan T. ; McCormick, Frank ; Gray, Joe ; Chu, Steven. / Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling. In: Proceedings of the National Academy of Sciences of the United States of America. 2013 ; Vol. 110, No. 46. pp. 18519-18524.
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