Recombinant probes for visualizing endogenous synaptic proteins in living neurons

Area of Science:

• Neuroscience
• Molecular Biology
• Biotechnology

Background:

• Visualizing endogenous proteins in living neurons is crucial for understanding neuronal structure and function.
• Existing methods may have limitations in specificity or applicability to live imaging.

Purpose of the Study:

• To develop novel reagents for high-affinity binding and visualization of endogenous neuronal proteins PSD-95 and Gephyrin in living neurons.
• To create tools that report on the localization and quantity of synaptic proteins without altering neuronal function.

Main Methods:

• Generation of recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), targeting PSD-95 and Gephyrin.
• Fusion of FingRs to Green Fluorescent Protein (GFP) for visualization.
• Incorporation of a transcriptional regulation system to control FingR expression and reduce background fluorescence.
• Validation in dissociated neurons and brain slices.

Main Results:

• FingRs demonstrated high-affinity binding to endogenous PSD-95 and Gephyrin.
• Fusion proteins allowed visualization of excitatory and inhibitory synapses in living neurons.
• FingR expression did not affect target protein levels, synapse number, or synaptic strength.
• FingRs successfully reported localization and amount of endogenous synaptic proteins.

Conclusions:

• PSD-95 and Gephyrin FingRs are effective tools for visualizing synaptic proteins in living neurons.
• These FingRs can be used to study dynamic changes in synaptic strength in vivo.
• The developed FingR technology offers a promising approach for neuroscience research.