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

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Protein oligomerization and protein-protein interactions regulate protein function.
• The p73 protein, a transcriptional factor, is involved in apoptosis and cell cycle arrest.
• Its activity is modulated by p73 oligomerization and p73-MDM2 interaction, but these processes in living cells remain unclear.

Purpose of the Study:

• To develop and apply an in situ method for studying p73 oligomerization and p73-MDM2 interaction in living cells.
• To investigate the structural determinants of p73 oligomerization and their impact on p73-MDM2 interaction.
• To examine the effects of inhibitors on these cellular processes.

Main Methods:

• Utilized fluorescence cross-correlation spectroscopy (FCCS) combined with fluorescent protein labeling.
• Employed lentiviral transfection to express p73 and MDM2 fused to distinct fluorescent proteins.
• Assessed p73 oligomerization via brightness per particle and p73-MDM2 interaction using cross-correlation values.
• Constructed and analyzed various p73 and MDM2 mutants.

Main Results:

• Confirmed that p73 protein forms oligomers in living cells.
• Demonstrated that structural changes in the p73 oligomerization domain significantly influence p73 oligomerization.
• Showed that p73 oligomerization and structural alterations critically affect the p73-MDM2 interaction.
• Investigated the impact of inhibitors on p73 oligomerization and p73-MDM2 interaction.

Conclusions:

• The study successfully established an in situ method to observe p73 oligomerization and p73-MDM2 interactions in living cells.
• Structural dynamics of p73, particularly in its oligomerization domain, are key regulators of its oligomeric state and interaction with MDM2.
• These findings provide novel insights into the regulation of p73 function within a cellular context and the potential for therapeutic intervention.