RAS Image-Based Screens

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Membrane localization of mutant KRAS. Upper left, cell nuclei. Lower left, cell membranes. Upper right, mutant KRAS. Lower right, merged.

Credit: Alla Brafman, NCI RAS Initiative

Oncogenic RAS signaling from the cell membrane is a major driver of the cancer phenotype, and published evidence suggests that RAS signaling may be mediated by and dependent on dimerization and higher order clustering of RAS molecules. Our group develops and applies new optical microscopy techniques to understand how RAS molecules move and signal in living cells.  We use that understanding to develop assays to screen for disruption and mislocalization of RAS proteins and complexes.

Our Progress

We have developed image-based assays for localization of KRAS suitable for drug screens.

We have developed BRET (bioluminescence-based) assays for monitoring interactions of RAS molecules with RAF and RALGDS in living cells.

We have developed cell lines with a biosensor that reports on ERK activity to monitor the RAS/RAF/MEK/ERK pathway in live cells.

We have modeled the mobility and clustering of single Halo-tagged KRAS molecules in the membranes of living cells using single-molecule microscopy.

Our Projects

  • Measure and characterize the dynamics of KRAS molecules in living cells using single molecule microscopy
  • Monitor interactions of KRAS with other proteins in the membranes of living cells
  • Develop high-content screening assays to identify molecules that disrupt the ability of KRAS to localize to the cell membrane and interact with effectors

Tools We Use

  • Single-molecule tracking
  • Step photobleaching
  • Photo-activated localization microscopy and stochastic optical reconstruction microscopy (PALM/Storm) imaging
  • Labeling proteins with dyes (Halo and Snap tags) and fluorescent fusions
  • BRET (bioluminescence resonance energy transfer)
  • Confocal microscopy and live cell imaging

Collaborations

The RAS Image-Based Screens Group has collaborated with:

Larry Botticelli
Pharma Arava
 
Krishna Kota and Rajini Mudhasani 

United State Army Medical Research Institute for Infectious Diseases
 
Andreas Gosberg
Eli Lilly and Company
 
Kirk Gustafson
National Cancer Institute
 
Emil Lou
University of Minnesota

Contact

Dr. Tommy Turbyville

Dr. Tommy Turbyville, RAS Image-Based Screens Group Lead

For more information, contact the RAS Image-Based Screens Group team lead:

Dr. Tommy Turbyville
301-846-6892
Thomas.Turbyville@nih.gov

  • Updated: September 15, 2016