Quantifying protein interactions in vivo

Research Areas:

Cell biology, Biochemistry, Protein-protein interactions in vivo, S. cerevisiae

Imaging Needs:

Accurate fluorescence quantitation in living cells over time.

Imaging System:
  • Olympus IX81 microscope
  • Olympus Biosystems ScanR software (modified in-house) for automated image acquisition
  • 100x/NA 1.45 objective lens
  • Hamamatsu ORCA-ER camera
  • ImageJ software for image analysis (custom macros for automated PICT)
Measuring protein interactions in cells, whole organisms

Measuring protein interactions in living cells is one thing, but what if you want to measure interactions within an intact, living organism? Find out how Takeaki Ozawa and colleagues screen for GPCR ligands in mice in Illuminating Activity—in vitro and in vivo. Read now.

THE QUESTION

How do you measure protein-protein interactions in their native environment—that is, inside a living cell?

THE BARRIERS

In vitro methods for measuring protein-protein interactions are well established, but may not faithfully reproduce events in the complex and crowded environment inside the cell. In vivo methods like FRET exist, but are technically challenging.

THE SOLUTION

Detection and Characterization of Protein Interactions In Vivo by a Simple Live-Cell Imaging Method
Oriol Gallego, Tanja Specht, Thorsten Brach, Arun Kumar, Anne-Claude Gavin, Marko Kaksonen
PLoS One. 2013; 8(5): e62195. PMCID: PMC3641059.

Gallego, et al,1 have developed a new method—dubbed PICT for Protein Interactions from Complexes After Translocation—that uses a simple fluorescent readout and a translocation-based reporter system to measure protein-protien interactions in vivo.

Using the bait-prey approach familiar to many molecular biologists as a two-hybrid system, Gallego, et al,1 add a few extra bells and whistles to convert this system into one that reports protein-protien interactions without relying on a transcriptional readout. They start with the rapamycin-inducible binding pair of FRB and FKBP. They fuse FKBP-RFP to an anchor protein on the cell surface and FRB to a bait protein. The bait protein binds to its GFP-fused partner, and then rapamycin is added to bind FRB-bait to FKBP-RFP-anchor. If the bait is bound to its partner, the GFP and RFP signals will co-localize.

Gallego, et al,1 demonstrated their approach with the Ste5-Ste11-Ste50 complex of S. cerevisiae, and were able to achieve accurate measurement of protein interactions using a Hamamatsu ORCA-ER camera.

THE POSSIBILITIES

Measuring the dynamics of protein complexes is critical for understanding the mechanisms behind many cellular processes. Gallego, et al,1 used the ORCA-ER camera, but what if you want to use the luminescence instead of fluorescence? Hamamatsu’s may be a better choice. Find out how Takeaki Ozawa and colleagues use the ImagEM to detect GPCR binding—read Illuminating Activity—in vitro and in vivo.

References

  1. Oriol Gallego, Tanja Specht, Thorsten Brach, Arun Kumar, Anne-Claude Gavin, Marko Kaksonen. Detection and Characterization of Protein Interactions In Vivo by a Simple Live-Cell Imaging Method. PLoS One. 2013; 8(5): e62195. PMCID: PMC3641059
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