Transport of a synaptotagmin-YFP fusion protein in sympathetic neurons during early neurite outgrowth in vitro after transfection in vivo.
Developing neurons are engaged in neurite outgrowth as well as the synthesis and transport of proteins involved in synaptic transmission. Very little is known about when transport is established in these rudimentary neurites. We used a novel technique to visualize protein transport during the early hours of neurite outgrowth in culture. Recombinant adenoviruses were used to express a synaptotagmin-YFP fusion protein in the superior cervical ganglia of neonatal rats in vivo and protein transport was examined in neuronal cultures established from the superior cervical ganglions (SCGs). We find that, as early as 4h in culture, synaptotagmin-YFP was present in the cytoplasm, lamellipodia, filopodia and growth cones. Protein expression appeared punctate in neurites at 8h in vitro and is consistent with a vesicular localization. These results indicate that the machinery to transport synapse-specific proteins is functional in rudimentary neurites at this time and indicates that this technique can be used to study early neuronal development.
Narayan
Sujatha
S
Department of Biology, Bryn Mawr College, 101 North Merion Avenue, Bryn Mawr, PA 19010, USA.
Greif
Karen F
KF
D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism / Q000821:virology; D005260:Female; D020439:Growth Cones / Q000378:metabolism / Q000821:virology; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016501:Neurites / Q000502:physiology*; D009474:Neurons / Q000378:metabolism* / Q000821:virology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D017783:Superior Cervical Ganglion / Q000166:cytology* / Q000821:virology; D050857:Synaptotagmins; D013997:Time Factors; D014162:Transfection; D014760:Viral Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D014760:Viral Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D050857:Synaptotagmins