Synaptic Protein Dynamics Measured by Fluorescence Correlation Spectroscopy

Abstract

The activity dependent adjustment of synaptic strength (synaptic plasticity) involves the reorganization of post-synaptic proteins. The fast diffusion of synaptic proteins has been shown to play an important role in such molecular rearrangements, but the dynamic events that lead to the formation of molecular complexes are still poorly understood. Taking advantages of Single Particle Tracking (SPT) and FRAP approaches, our group has recently demonstrated that during inhibitory long term potentiation stimuli (iLTP) Gephyrin and GABAA receptors are accumulated at post-synaptic inhibitory sites. In addition we have shown that the synaptic clustering of the scaffold protein gephyrin do not precede the synaptic inclusion of GABA receptors. However, the coordinated dynamics of gephyrin and GABAA receptors outside and inside the synapse remain to be elucidated. To investigate the diffusion dynamics of the main synaptic proteins we exploited here Fluorescence Correlation Spectroscopy (FCS), an established technique to study the mobility of molecules and to measure the protein diffusion parameters with high statistics in unperturbed live cell, in both the plasma membrane and the cytoplasmic space. We calibrated an FCS method (FCS) to study the coordinated multilevel organization of receptors and adhesion molecules that laterally diffuse on the plasma membrane and the scaffold proteins that diffuse in the cytoplasmic space. Our data suggest that, the scaffold protein freely diffuse in the extrasynaptic space and is suddenly immobilized at both synaptic site and extrasynaptic clusters. This approach may clarify the coordinated diffusion of membrane synaptic proteins and cytoplasmic scaffold proteins thus clarifying the mechanisms of synaptic clustering in basal conditions and during synaptic plasticity.