Myelinated axons contain beta-actin mRNA and ZBP-1 in periaxoplasmic ribosomal plaques and depend on cyclic AMP and F-actin integrity for in vitro translation.
Periaxoplasmic ribosomal plaques (PARPs) are periodic structural formations containing ribosomes, which are likely cortical sites of translation along myelinated fibers. beta-actin mRNA, and its trans-acting binding factor, zipcode-binding protein-1, were co-distributed within PARP domains of axoplasmic whole-mounts isolated from goldfish Mauthner, rabbit and rat nerve fibers. The distribution of co-localization signals of fluorophore pixels, however, was asymmetric in PARP domains, possibly indicative of endpoint trafficking of RNPs. beta-actin mRNA in RNA extracted from axoplasm of single Mauthner fibers was confirmed by RT-PCR. A metabolically active isolated Mauthner fiber system, which required cAMP to activate translation, was developed in order to probe cycloheximide-sensitivity, and the importance of the actin cytoskeleton. cAMP greatly stimulated protein synthesis in axoplasm after a period of pre-incubation, while being inhibited strongly by cycloheximide, or by cytochalasin D. Cytochalasin D reduced incorporation only modestly in the associated myelin sheath. We conclude that mechanisms for targeting and localizing beta-actin mRNA to discrete PARP domains are probably similar to those described for dendritic synaptic domains. Moreover, optimal translation in axoplasm depends on the integrity of the actin cytoskeleton, and can be modulated by cAMP as well.
Sotelo-Silveira
José
J
Department of Protein and Nucleic Acids, Instituto de Investigaciones Biologicas Clemente Estable, Montevideo, Uruguay.
Crispino
Marianna
M
Puppo
Agostina
A
Sotelo
José R
JR
Koenig
Edward
E
D000199:Actins / Q000235:genetics*; D000818:Animals; D001369:Axons / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D006054:Goldfish; D017403:In Situ Hybridization / Q000379:methods; D066298:In Vitro Techniques; D009413:Nerve Fibers, Myelinated / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012270:Ribosomes / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology
D000199:Actins; C498634:Igf2bp1 protein, rat; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000242:Cyclic AMP