Live visualization of protein synthesis in axonal growth cones by microinjection of photoconvertible Kaede into Xenopus embryos.
Photoconvertible fluorescent proteins, such as Kaede, can be switched irreversibly from their native color to a new one. This property can be exploited to visualize de novo mRNA translation, because newly synthesized proteins can be distinguished from preexisting ones by their color. In this protocol, Kaede cDNA linked to the 3' untranslated region (UTR) of beta-actin is delivered into cells fated to become the retina by injection into Xenopus blastomeres. Brief exposure (6-10 s) to UV light (350-410 nm) of Kaede-positive retinal axons/growth cones efficiently converts Kaede from its native green fluorescence to red. The reappearance of the green signal reports the synthesis of new Kaede protein. This approach can be used to investigate the spatiotemporal control of translation of specific mRNAs in response to external stimuli and to test the efficiency of full-length versus mutant UTRs. The 3-d protocol can be adapted for broad use with other photoactivatable fluorescent proteins.
Leung
Kin-Mei
KM
Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.
Holt
Christine E
CE
D000199:Actins / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001757:Blastomeres / Q000378:metabolism* / Q000648:ultrastructure; D020439:Growth Cones / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000032:analysis* / Q000528:radiation effects; D008845:Microinjections / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012160:Retina / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D046509:Tissue Culture Techniques; D014466:Ultraviolet Rays; D014981:Xenopus
D000199:Actins; C513582:Kaede protein, Trachyphyllia geoffroyi; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins