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Neuron. 2013 Jun 19;78(6):971-85. doi: 10.1016/j.neuron.2013.04.017.

Recombinant probes for visualizing endogenous synaptic proteins in living neurons.

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1
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.

Abstract

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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PMID:
23791193
PMCID:
PMC3779638
DOI:
10.1016/j.neuron.2013.04.017
[Indexed for MEDLINE]
Free PMC Article

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