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Spatial-temporal dynamics and metabolic alterations of p53 upon cellular stress

  • Author(s): Bagilthaya, Swathi
  • Advisor(s): Digman, Michelle
  • et al.
Abstract

p53 is a tumor suppressor protein that plays a very important role in determining the fate of damaged cells. Depending on the extent of damage, p53 being a transcription factor, induces target genes that are involved in cell repair mechanisms and apoptosis. In doing so, it prevents proliferation of abnormal cells that could lead to tumorigenesis. Primarily existing in its monomeric or dimeric form, when activated, it binds to DNA as a tetramer. The localization of these tetramers in cells has never been mapped. Since p53 is mutated in 50% of human cancers, its ability to tetramerize efficiently and hence bind to the DNA is disrupted leading to tumor progression. Here we use the Number and Brightness (N&B) analysis, a powerful method to measure protein oligomerization pixel by pixel from raster scanned images thereby providing spatial maps of p53 aggregates. The research described here shows, for the first time, the oligomerization maps of the p53 protein and its mutant counterparts to establish the crucial role of p53 tetramers in tumor suppression. In addition, p53 also regulates the metabolism of the cell by modulating important metabolic pathways upon cellular stress. To determine whether this switch is indicative of the balance between apoptosis and DNA repair, the phasor approach to lifetime imaging microscopy (FLIM) was employed to detect the free and bound lifetime of reduced nicotinamide adenine dinucleotide (NADH). The shifts in lifetime are informative of the level of stress and give an indication whether the cell is undergoing cell cycle arrest or apoptosis. The ratios of free and bound NADH obtained from this data may be used as a marker of transcriptional activity. The N&B and FLIM results together provide an insight to p53 activation and this information can be further exploited to improve the field of cancer research.

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