In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons.
Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click chemistry'. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous.
Dieterich
Daniela C
DC
Division of Biology, California Institute of Technology, Pasadena, California, USA.
Hodas
Jennifer J L
JJ
Gouzer
Géraldine
G
Shadrin
Ilya Y
IY
Ngo
John T
JT
Triller
Antoine
A
Tirrell
David A
DA
Schuman
Erin M
EM
D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000480:Alkynes / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D017551:Microdialysis; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D009474:Neurons / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology*; D040901:Proteomics / Q000379:methods*; D045663:Quantum Dots; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods*
D000480:Alkynes; D000596:Amino Acids; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; C506098:azidohomoalanine; C551041:homopropargylglycine; D000409:Alanine; D005998:Glycine