Understanding synaptic complexity through recoding of endogenous neuronal receptor complexes using genetic code expansion and genome editing

• Applicant: Professor Dr. Markus Sauer
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells ; Biophysics
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545292095

The accurate visualization of synaptic proteins requires a careful balance between high-resolution imaging and maintenance of the physiological integrity of these proteins. Our project aims to determine the precise subcellular and subsynaptic localization of the various AMPAR complexes, as well as their dynamics. Therefore, we will develop refined labeling methods based on genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click labeling with tetrazine dyes for site-specific labeling of AMPAR and auxiliary subunits with minimal linkage error to unlock the full potential of latest single-molecule localization microscopy methods such as three-dimensional (3D) lattice light-sheet dSTORM and DNA-PAINT of hippocampal neurons and brain slices. Using these methods, we aim to investigate the dynamic of the interactions between AMPARs and auxiliary subunits such as TARPs, CNIHs, and GSG1l because this is crucial for the understanding of their role in synaptic physiology and represents a significant challenge in the field. In addition, the methods we plan to develop will enable us to investigate how different configurations of AMPAR complexes, such as GluA1/GluA2/GluA3 tri-heteromeric and GluA1/GluA2 di-heteromeric assemblies, contribute to the diversity of synaptic responses in different brain regions and cell types. By combining the expertise of two leading teams in the fields of synaptic protein biology (Team Choquet) and advanced super-resolution imaging coupled with innovative protein labeling techniques (Team Sauer) with a proven track record of collaboration, our project aims to fundamentally transform the methods used to study synaptic proteins and deepen our understanding of their functions and interactions.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Professor Dr. Daniel Choquet