Spatio-Temporal Dynamics and Metabolic Alterations of P53 Upon DNA Damage

Abstract

For biological reaction-diffusion systems, live single cell spatio-temporal analysis of protein dynamics provides a mean to observe stochastic biochemical signaling which may lead to better understanding of cancer cell invasion, stem cell differentiation and other fundamental biological processes. We used the Number and Molecular Brightness (N&B) method to map aggregation processes of the p53 protein in the live cells upon DNA damage with cisplatin (a chemotherapy agent); thus revealing the spatial distribution of events, the site of tetramer formation and the time sequence of the aggregation events with quantitative information about the distribution and size of any intermediate aggregates that are formed. p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migration and repair. Upon cell stress, p53 will form tetramers in specific chromatin sites and activate genes that trigger cell cycle arrest and/or apoptosis. To gain information regarding fast dynamic processes we use line scanning on a convention confocal microscope to reveal the transient binding dynamics across the nucleus. Using fluctuation analysis we were able to calculate binding rates of p53 to DNA sites. To gain information about changes in cell metabolism we use the phasor FLIM approach that we have established in our lab. We found that there is higher concentration of free NADH in the nucleus after cisplatin damage. This work is supported in part by NIH grants P50 GM076516 and P41 GM103540.