Recombinant probes for visualizing endogenous synaptic proteins in living neurons

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Carrier Proteins / analysis*
  • Carrier Proteins / genetics
  • Chlorocebus aethiops
  • Disks Large Homolog 4 Protein
  • Gene Expression Profiling / methods*
  • Intracellular Signaling Peptides and Proteins / analysis*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Membrane Proteins / analysis*
  • Membrane Proteins / genetics
  • Nerve Tissue Proteins / analysis
  • Nerve Tissue Proteins / genetics
  • Neurons / chemistry*
  • Neurons / physiology
  • Recombinant Proteins / analysis*
  • Recombinant Proteins / genetics
  • Synapses / chemistry
  • Synapses / physiology

Substances

  • Carrier Proteins
  • DLG4 protein, human
  • Disks Large Homolog 4 Protein
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Recombinant Proteins
  • gephyrin