Probing protein interactions in living mammalian cells on a microtubule bench.

Mirela Boca,Alix Mephon-Gaspard,David Pastré,Patrick A Curmi,Dmitry A Kretov,Bénédicte Desforges

doi:10.1038/srep17304

Mirela Boca, Alix Mephon-Gaspard + Show 4 more

Open Access

https://doi.org/10.1038/srep17304

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Abstract

Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases.

Highlights

We present a new method which uses the microtubule network as an intracellular platform to detect protein interactions in living cells
To explore the validity of this method, we first considered whether Tau, a microtubule-associated protein, can help to bring a bait protein onto microtubules, which is a prerequisite for the present detection scheme
Microtubule-associated proteins (MAPs) like Tau can be used for this purpose (Fig. 1)

Summary

Results and Discussion

Tau brings bait proteins onto microtubules and preserves the accessibility of baits to molecular partners. Other RNA-binding proteins which do not colocalize with the bait protein under physiological conditions may be brought to microtubules artificially To quantify such bias and discard false positive colocalizations, the spearman coefficient was measured in cells expressing both bait and prey proteins at varying levels (Fig. 4A,B). For both YB-1-RFP-Tau, Lin28-RFP-Tau and G3BP-RFP-Tau, the results indicate that the spearman coefficient increases gradually with the bait expression levels. The value of the spearman coefficients in the virtual absence of bait protein were extrapolated from the plots of the spearman coefficient versus bait expression level (Fig. 4B) This analysis shows that, in unperturbed cells, YB-1-RFP-Tau significantly interacts with YB-1-GFP, moderately with Lin28-GFP and poorly with G3BP1-GFP. High throughput data can be possibly collected using this method but requires optimal lateral resolution with oil-immersed lenses to clearly distinguish microtubules in both fixed and living cells

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