Analyses of p73 Protein Oligomerization and p73-MDM2 Interaction in Single Living Cells Using In Situ Single Molecule Spectroscopy.

Protein oligomerization and protein-protein interaction are crucial to regulate protein functions and biological processes. p73 protein is a very important transcriptional factor and can promote apoptosis and cell cycle arrest, and its transcriptional activity is regulated by p73 oligomerization and p73-MDM2 interaction. Although extracellular studies on p73 oligomerization and p73-MDM2 interaction have been carried out, it is unclear how p73 oligomerization and p73-MDM2 interaction occur in living cells. In our study, we described an in situ method for studying p73 oligomerization and p73-MDM2 interaction in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. Lentiviral transfection was used to transfect cells with a plasmid for either p73 or MDM2, each fused to a different fluorescent protein. p73 oligomerization was evaluated using brightness per particle, and the p73-MDM2 interaction was quantified using the cross-correlation value. We constructed a series of p73 mutants in three domains (transactivation domain, DNA binding domain, and oligomerization domain) and MDM2 mutants. We systematically studied p73 oligomerization and the effects of p73 oligomerization and the p73 and MDM2 structures on the p73-MDM2 interaction in single living cells. We have found that the p73 protein can form oligomers and that the p73 structure changes in the oligomerization domain significantly influence its oligomerization. p73 oligomerization and the structure changes significantly affect the p73-MDM2 interaction. Furthermore, the effects of inhibitors on p73 oligomerization and p73-MDM2 interaction were studied.