Research/key-initiatives/ras/screens-assays/imaging
Contents
RAS Image-Based Screens
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
For more information, contact the RAS Image-Based Screens Group team lead:
Dr. Tommy Turbyville
301-846-6892
Thomas.Turbyville@nih.gov
