[PDF][PDF] Recombinant probes for visualizing endogenous synaptic proteins in living neurons

GG Gross, JA Junge, RJ Mora, HB Kwon, CA Olson… - Neuron, 2013 - cell.com
GG Gross, JA Junge, RJ Mora, HB Kwon, CA Olson, TT Takahashi, ER Liman…
Neuron, 2013cell.com
The ability to visualize endogenous proteins in living neurons provides a powerful means to
interrogate neuronal structure and function. Here we generate recombinant antibody-like
proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind
endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused
to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of
the FingR incorporates a transcriptional regulation system that ties FingR expression to the …
Summary
The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices, FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.
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