Probing protein interactions on a microtubule bench by fluorescence microscopy: Application to YB-1, a mRNA-binding protein
David Pastré (Université d’Évry)
A typical procedure adopted by biologists to analyze protein interactions is to take advantage of the high throughput capability of the two hybrid system, generally in yeast, or that of the combination of affinity purification with mass spectrometry to obtain an exhaustive list of potential partners for a protein of interest. However, these assays require cell lysis, antibodies and adsorption onto non physiological substrates leading to false positives and false negatives. There is therefore a need to control the relevance of the proposed interactions in a context closer to native conditions, such as in living mammalian cells. To that end, novel methods are currently developed to provide a better view on protein interactions. In line with this, we have proposed a novel technology to detect and quantify direct or indirect protein interactions by fluorescence microscopy in living mammalian cells using microtubules as platforms. Microtubules are micrometer-long rigid cylinders of about 25 nm in diameter that are present in the cytoplasm of all eukaryotic cells. Due to their geometry, they provide an ideal surface to probe molecular interactions by fluorescence microscopy. As a proof of concept, we used microtubules to probe the interactions between mRNA-binding proteins like YB-1 in the cytoplasm.