Evidence That Ion-Based Signaling Initiating at the Cell Surface Can Potentially Influence Chromatin Dynamics and Chromatin-Bound Proteins in the Nucleus.

We have developed tools and performed pilot experiments to test the hypothesis that an intracellular ion-based signaling pathway, provoked by an extracellular stimulus acting at the cell surface, can influence interphase chromosome dynamics and chromatin-bound proteins in the nucleus. The experimental system employs chromosome-specific fluorescent tags and the genome-encoded fluorescent pH sensor SEpHluorinA227D, which has been targeted to various intracellular membranes and soluble compartments in root cells of Arabidopsis thaliana. We are using this system and three-dimensional live cell imaging to visualize whether fluorescent-tagged interphase chromosome sites undergo changes in constrained motion concurrently with reductions in membrane-associated pH elicited by extracellular ATP, which is known to trigger a cascade of events in plant cells including changes in calcium ion concentrations, pH, and membrane potential. To examine possible effects of the proposed ion-based signaling pathway directly at the chromatin level, we generated a pH-sensitive fluorescent DNA-binding protein that allows pH changes to be monitored at specific genomic sites. Results obtained using these tools support the existence of a rapid, ion-based signaling pathway that initiates at the cell surface and reaches the nucleus to induce alterations in interphase chromatin mobility and the surrounding pH of chromatin-bound proteins. Such a pathway could conceivably act under natural circumstances to allow external stimuli to swiftly influence gene expression by affecting interphase chromosome movement and the structures and/or activities of chromatin-associated proteins.