Application of Fluorescence Correlation Spectroscopy to Study Dynamics of Proteins Involved in Neuronal Synapse-to-Nucleus Signaling

Abstract

Activity-dependent gene transcription is required for the long-term stabilization of synaptic plasticity, a process that is key to learning and memory. We are studying the transcription factor, NFAT, which has been shown to be actuated by postsynaptic signaling. Ca2+ influx through voltage-gated L-type calcium channels plays an exclusive role in initiating NFAT signaling via the action of the Ca2+/calmodulin- phosphatase calcineurin (CaN). CaN dephosphorylates multiple residues on NFAT, resulting in the exposure of its nuclear localization sequence and subsequent translocation to the nucleus. A required component of this cascade is the A-kinase anchoring protein (AKAP) 79/150, which serves to recruit CaN to the channel. New evidence from our group indicates that NFAT is also tethered to this complex via a transient association with AKAP.This scenario suggests a model wherein NFAT signaling is instigated by local synaptic activity and the actuated transcription factor is then conveyed to the nucleus over long intracellular distances via diffusion or active transport. However, the molecular mechanisms of this process have yet to be elucidated. A potentially powerful approach to investigate the dynamics of the signaling molecules in this pathway is fluorescence correlation spectroscopy (FCS). This technique yields quantitative information on protein mobility in living cells by measuring intensity fluctuations produced by fluorescent (or fluorescently tagged) molecules diffusing through a confined detection volume.Here we present FCS measurements characterizing NFAT dynamics in response to induced synaptic activity in cultured hippocampal neurons, utilizing models that account for dendritic morphology. Additionally, we have found that there is a prominent component in the autocorrelation curve due to GFP photophysics (triplet state transitions and dynamic quenching) in FCS data from these cells, which requires further modifications to the standard fitting models.