Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging

J Histochem Cytochem. 2016 May;64(5):323-33. doi: 10.1369/0022155416641604. Epub 2016 Mar 29.

Abstract

A steady accumulation of experimental data argues that protein synthesis in neurons is not merely restricted to the somatic compartment, but also occurs in several discrete cellular micro-domains. Local protein synthesis is critical for the establishment of synaptic plasticity in mature dendrites and in directing the growth cones of immature axons, and has been associated with cognitive impairment in mice and humans. Although in recent years a number of important mechanisms governing this process have been described, it remains technically challenging to precisely monitor local protein synthesis in individual neuronal cell parts independent from the soma. This report presents the utility of employing microfluidic chambers for the isolation and treatment of single neuronal cellular compartments. Furthermore, it is demonstrated that a protein synthesis assay, based on fluorescent non-canonical amino acid tagging (FUNCAT), can be combined with this cell culture system to label nascent proteins within a discrete structural and functional domain of the neuron. Together, these techniques could be employed for the detection of protein synthesis within developing and mature neurites, offering an effective approach to elucidate novel mechanisms controlling synaptic maintenance and plasticity.

Keywords: FUNCAT; axon; click assay; local protein synthesis; microfluidic chambers; neurons; synapse.

Publication types

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

MeSH terms

  • Alanine / analogs & derivatives
  • Alanine / chemistry
  • Alkynes / chemistry
  • Amino Acids / chemistry*
  • Animals
  • Axons / physiology
  • Azides / chemistry
  • Cerebral Cortex / cytology
  • Click Chemistry
  • Dendrites / physiology
  • Disks Large Homolog 4 Protein
  • Embryo, Mammalian / cytology
  • Fluorescent Dyes / chemistry*
  • Glycine / analogs & derivatives
  • Glycine / chemistry
  • Intracellular Signaling Peptides and Proteins / biosynthesis
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Lab-On-A-Chip Devices
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / chemistry
  • Neurons / metabolism*
  • Primary Cell Culture
  • Protein Biosynthesis*
  • RNA, Messenger / metabolism*
  • Rats, Wistar
  • Triazoles / chemistry

Substances

  • Alkynes
  • Amino Acids
  • Azides
  • Disks Large Homolog 4 Protein
  • Dlg4 protein, rat
  • Fluorescent Dyes
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • RNA, Messenger
  • Triazoles
  • azidohomoalanine
  • homopropargylglycine
  • Alanine
  • Glycine