AbstractSynaptic function and plasticity requires a delicate balance between overall structural stability and the continuous rearrangement of the components that make up the presynaptic active zone and the postsynaptic density (PSD). Photoactivated localization microscopy (PALM) has provided a detailed view of the nanoscopic structure and organization of some of these synaptic elements. Still lacking, are tools to address the morphing and stability of such complexes at super-resolution. We describe an approach to quantify morphological changes and energetic states of multimolecular assemblies over time. With this method, we studied the scaffold protein gephyrin, which forms postsynaptic clusters that play a key role in the stabilization of receptors at inhibitory synapses. Postsynaptic gephyrin clusters exhibit an internal microstructure composed of nanodomains. We found, that within the PSD, gephyrin molecules continuously undergo spatial reorganization. This dynamic behavior depends on neuronal activity and cytoskeleton integrity. The proposed approach also allowed access to the effective energy responsible for the tenacity of the PSD despite molecular instability.Significant statementSuper-resolution microscopy has become an important tool for the study of biological systems, allowing detailed, nano-scale structural reconstruction, single molecule tracking, particle counting, and interaction studies. However, quantification tools that take full advantage of the information provided by this technology are still lacking. We describe a novel quantification method to obtain information related to the size, directionality, dynamics, and stability of clustered structures from super-resolution microscopy. With this method, we studied the stability of gephyrin clusters, the main inhibitory scaffold protein. We found that gephyrin molecules continuously undergo reorganization based on neuronal activity and changes in the cytoskeleton.
Sylites: Multipurpose markers for the visualization of inhibitory synapses
