title: Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging.
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.
authors.affiliation: Department of Cognitive Neuroscience, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands (AK)|Donders Institute for Brain, Cognition, and Behaviour, Centre for Neuroscience, 6525 AJ Nijmegen, The Netherlands (AK, KAW)
authors.forename: Aron
authors.initials: A
authors.lastname: Kos
authors.affiliation: Donders Institute for Brain, Cognition, and Behaviour, Centre for Neuroscience, 6525 AJ Nijmegen, The Netherlands (AK, KAW)|Language and Genetics Department, Max Planck Institute for Psycholinguistics, 6525 XD Nijmegen, The Netherlands (KAW)
authors.forename: Kai A
authors.initials: KA
authors.lastname: Wanke
authors.affiliation: Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland (AG, BBK, AA)
authors.forename: Anthony
authors.initials: A
authors.lastname: Gioio
authors.affiliation: Department of Cognitive Neuroscience, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands (AK)|Department of Molecular Animal Physiology, Radboud University, Nijmegen, The Netherlands (GJM)
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Martens
authors.affiliation: Department of Cognitive Neuroscience, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands (AK)|Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland (AG, BBK, AA)
authors.forename: Barry B
authors.initials: BB
authors.lastname: Kaplan
authors.affiliation: Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland (AG, BBK, AA)
authors.forename: Armaz
authors.initials: A
authors.lastname: Aschrafi
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000480:Alkynes / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001369:Axons / Q000502:physiology; D001386:Azides / Q000737:chemistry; D002540:Cerebral Cortex / Q000166:cytology; D057930:Click Chemistry; D003712:Dendrites / Q000502:physiology; D000076122:Disks Large Homolog 4 Protein; D004622:Embryo, Mammalian / Q000166:cytology; D005456:Fluorescent Dyes / Q000737:chemistry*; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000737:chemistry; D056656:Lab-On-A-Chip Devices; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry; D009474:Neurons / Q000378:metabolism*; D061251:Primary Cell Culture; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D017208:Rats, Wistar; D014230:Triazoles / Q000737:chemistry
chemical_list: D000480:Alkynes; D000596:Amino Acids; D001386:Azides; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D014230:Triazoles; C506098:azidohomoalanine; C551041:homopropargylglycine; D000409:Alanine; D005998:Glycine
keywords: FUNCAT; axon; click assay; local protein synthesis; microfluidic chambers; neurons; synapse