Ultrastructural identification of storage compartments and localization of activity-dependent secretion of neurotrophin 6 in hippocampal neurons.
A modulatory role of neurotrophins (NTs) in activity-dependent neuronal plasticity by pre- and postsynaptic mechanisms is now well established. In this context, it is important to identify the storage compartments and to localize the precise site(s) and mechanism of NT secretion in order to deduce the spatial and temporal availability of NTs. We approached these questions at the ultrastructural level, exploiting the unique property of NT6 to bind tightly to heparan sulfate proteoglycans at the neuronal surface (R. Götz et al., 1994, Nature 372, 266-269), permitting the localization of secretion sites excluding diffusion artifacts. The myc tagging of NT6 permitted glutaraldehyde fixation and hence good preservation of the membrane structure, permitting immunogold labeling of NT6myc at the neuronal surface. NT6myc is preferentially secreted from neurites compared to neuronal cell bodies. In agreement with light-microscopic observations, the ultrastructural localization of NT6myc by postembedding procedures showed a predominant localization in ER-like membrane-confined compartments, partially associated with microtubules.
Gärtner
A
A
Department of Neurobiochemistry, Max-Planck-Institute of Neurobiology, Am Klopferspitz 18a, Martinsried, D-82152, Germany.
Shostak
Y
Y
Hackel
N
N
Ethell
I M
IM
Thoenen
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000648:ultrastructure; D005404:Fixatives; D005976:Glutaral; D006031:Glycosylation; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism; D007150:Immunohistochemistry; D008870:Microtubules / Q000648:ultrastructure; D008958:Models, Molecular; D009414:Nerve Growth Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009473:Neuronal Plasticity; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry; D016707:Tissue Fixation / Q000379:methods; D014162:Transfection
D005404:Fixatives; D019812:Heparan Sulfate Proteoglycans; D009414:Nerve Growth Factors; D011498:Protein Precursors; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C075936:neurotrophin 6; D006493:Heparin; D005976:Glutaral