Oral Presentation 2014 International Biophysics Congress

Imaging the diffusive route of transcription factor activity in live cells by fluctuation analysis (#14)

Elizabeth Hinde 1 , Elvis Pandzic 1 , Enrico Gratton 2 , Katharina Gaus 1
  1. Medicine, University of New South Wales, Sydney, NSW, Australia
  2. Biomedical Engineering, University of California, Irvine, CA, USA

Dynamic changes in higher order chromatin structure modulate the accessibility of DNA toward transcription, DNA repair and replication. The spatiotemporal scale upon which these dynamics occur however, render them ‘invisible’ to live cell imaging. Here we present a novel method to measure the real time accessibility of chromatin that is based on pair correlation analysis of EGFP (an inert tracer) molecular flow throughout the chromatin network1. From detected changes in the arrival time of EGFP molecules into and out of the chromatin, this method detects millisecond changes in protein access to condensed chromatin with sub-micron resolution. Using this method we have probed the naturally occurring changes in chromatin compaction during the cell cycle and in combination with laser micro-irradiation, monitored for the first time the in vivo structural rearrangements of chromatin during repair2,3. The pair correlation method thus offers a unique opportunity to probe the directionality of intra-nuclear traffic, by measuring the accessibility of the nuclear landscape and the role it plays in determining the diffusive routes adopted by molecules. Given that pair correlation analysis can be combined with other optical information4, more recently we have employed this method with a brightness analysis to look at how the diffusive route of the STAT3 transcription factor is modulated by oligomeric state. Given that homo-oligomerisation is critical to the function of many transcription factors we envisage this multiplexed method of analysis will enable advancements in our understanding of how gene expression is regulated at the single molecule level.