A new method to probe protein interactions in living mammalian cells using microtubules as platforms
In the SABNP laboratory, we have developed a novel and unconventional technology to probe direct or indirect protein interactions in living mammalian cells using microtubules (MT) as platforms
A protein of interest, the bait, is brought onto microtubules and the presence of putative molecular partners (RNA or protein), attracted by the bait protein, is then detected on microtubules by fluorescence microscopy. To bring the bait protein on microtubules, we used a fusion to a microtubule-binding domain, Tau. An additional linker domain was used to separate the bait protein from the microtubule surface.
As a proof of concept, we used this method to probe interactions between mRNA-binding proteins like YB-1 and G3BP1 which are known to interact with mRNA in the cytoplasm. We demonstrated that mRNA molecules were brought on microtubules when both YB-1 and G3BP were used as bait proteins. We then probed the interactions between different mRNA-binding proteins on microtubules and, for two of them, detected their cooperative binding to mRNA. These results were in agreement with conventional gel mobility shift assays and pull down assays. In addition, we show that two point mutations in the cold-shock domain of YB-1 disrupt its cooperative binding to mRNA in cells.
Finally, we also tested whether the domain of application of the present method can be extended to proteins other than RNA-binding proteins and found that this method can also be used with membrane proteins like Cx43.
These first results indicated that microtubules can be used as platforms to detect protein interactions in mammalian cells. Such an approach should allow to investigate the molecular partners of pathogenic protein involved in human diseases.