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

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Title: Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.
Authors: Xiaolin Nan1,2 nan@ohsu.edu, Collisson, Eric A.2,3, Lewis, Sophia2, Jing Huang2, Tamgüney, Tanja M.3, Liphardt, Jan T.1, McCormick, Frank3, Gray, Joe W.2,3 grayjo@ohsu.edu, Chu, Steven4 schu@stanford.edu
Source: Proceedings of the National Academy of Sciences of the United States of America. 11/12/2013, Vol. 110 Issue 46, p18519-18524. 6p.
Subjects: Serine/threonine kinases, High resolution imaging, Growth factors, Cell membranes, Dimers
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 sufficient step for RAF activation. [ABSTRACT FROM AUTHOR]
Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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DbLabel: Engineering Source
An: 92018843
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  Data: Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.
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  Data: <searchLink fieldCode="AR" term="%22Xiaolin+Nan%22">Xiaolin Nan</searchLink><relatesTo>1,2</relatesTo><i> nan@ohsu.edu</i><br /><searchLink fieldCode="AR" term="%22Collisson%2C+Eric+A%2E%22">Collisson, Eric A.</searchLink><relatesTo>2,3</relatesTo><br /><searchLink fieldCode="AR" term="%22Lewis%2C+Sophia%22">Lewis, Sophia</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Jing+Huang%22">Jing Huang</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Tamgüney%2C+Tanja+M%2E%22">Tamgüney, Tanja M.</searchLink><relatesTo>3</relatesTo><br /><searchLink fieldCode="AR" term="%22Liphardt%2C+Jan+T%2E%22">Liphardt, Jan T.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22McCormick%2C+Frank%22">McCormick, Frank</searchLink><relatesTo>3</relatesTo><br /><searchLink fieldCode="AR" term="%22Gray%2C+Joe+W%2E%22">Gray, Joe W.</searchLink><relatesTo>2,3</relatesTo><i> grayjo@ohsu.edu</i><br /><searchLink fieldCode="AR" term="%22Chu%2C+Steven%22">Chu, Steven</searchLink><relatesTo>4</relatesTo><i> schu@stanford.edu</i>
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  Data: <searchLink fieldCode="DE" term="%22Serine%2Fthreonine+kinases%22">Serine/threonine kinases</searchLink><br /><searchLink fieldCode="DE" term="%22High+resolution+imaging%22">High resolution imaging</searchLink><br /><searchLink fieldCode="DE" term="%22Growth+factors%22">Growth factors</searchLink><br /><searchLink fieldCode="DE" term="%22Cell+membranes%22">Cell membranes</searchLink><br /><searchLink fieldCode="DE" term="%22Dimers%22">Dimers</searchLink>
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  Label: Abstract
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  Data: 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. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1073/pnas.1318188110
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        Text: English
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      – SubjectFull: High resolution imaging
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      – SubjectFull: Growth factors
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      – SubjectFull: Cell membranes
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      – SubjectFull: Dimers
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      – TitleFull: Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.
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              Text: 11/12/2013
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              Y: 2013
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