Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced...

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Monitoring endogenous proteins, especially for dynamics and super-resolution imaging, remains challenging.
• Postsynaptic density-95 (PSD-95) is a key scaffold protein at excitatory synapses.
• Existing imaging methods often struggle with specificity and resolution for endogenous targets.

Purpose of the Study:

• To develop novel protein-based imaging probes for endogenous PSD-95.
• To validate these probes for super-resolution microscopy in neurons.
• To assess the utility of these probes for advanced imaging applications.

Main Methods:

• Protein engineering to evolve specific binders for PSD-95.
• Functional validation of binders to ensure no impact on PSD-95 activity.
• Application of engineered probes in super-resolution techniques (STED, PALM, DNA-PAINT).
• Testing probes in both live and fixed neurons.
• Utilizing binders to enhance genetically encoded calcium reporters at synapses.

Main Results:

• Successfully evolved protein binders that selectively recognize endogenous PSD-95.
• Confirmed that the binders do not interfere with PSD-95 function.
• Demonstrated the probes' efficacy in various super-resolution imaging modalities.
• Showcased the probes' utility in live and fixed neuronal imaging.
• Successfully enriched synaptic calcium reporters using the PSD-95 binders.

Conclusions:

• Evolved protein binders provide a robust platform for imaging endogenous PSD-95.
• These probes facilitate advanced fluorescence imaging, including super-resolution techniques.
• The developed platform offers new possibilities for studying synaptic structure and function.