title: Snap-25 is polarized to axons and abundant along the axolemma: an immunogold study of intact neurons.
abstract: SNAP-25, synaptosomal associated protein of 25 kDa, is reported to be a t-SNARE (target receptor associated with the presynaptic plasma membrane) involved in the docking and fusion of synaptic vesicles. We present here the first ultrastructural localization of SNAP-25 in intact neurons by pre-embedding EM immunocytochemistry in rat brains, hippocampal slice cultures, and PC12 cells. In differentiated neurons, SNAP-25 labeling was clearly membrane-associated. The labeling was most prominent in the plasma membrane of axons and excluded from the plasma membranes of soma and dendrites. Furthermore, SNAP-25 did not appear to be restricted to the synaptic junctions. SNAP-25 labeling was seen in the cytoplasm of the soma and large dendrites, mostly associated with the Golgi complexes. There were also some SNAP-25 labeled tubulo-vesicular structures in the cytoplasm of the soma and the axons, but rarely in the smaller dendrites. In PC12 cells, after 5-10 minutes of high potassium (75 mM) stimulation in the presence of HRP, SNAP-25 labeling appeared, additionally, on HRP-filled early endosomes. After a longer (20-30 minutes) HRP incubation, most of the later stage endosomes and lysosomes were loaded with HRP but they were negative for SNAP-25. These results suggest that SNAP-25 is sorted out of these late endosomal compartments, and that the bulk of the SNAP-25 protein is probably recycled back to the axolemma from the early endosomes. In contrast, in those samples which were incubated with HRP for longer periods, there were still some SNAP-25-positive vesicular structures which were HRP-negative. These structures most likely represent anterograde vesicles that carry newly synthesized SNAP-25 from the soma to the axolemma by axonal transport. SNAP-25 appears to be sorted at the Golgi complex to reach the axolemma specifically. Its widespread distribution all along the axolemma does not support the view of SNAP-25 as a t-SNARE limited for synaptic exocytosis.
authors.affiliation: NINDS EM Facility, NICHD, NIH, Bethesda, MD 20892, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Tao-Cheng
authors.forename: J
authors.initials: J
authors.lastname: Du
authors.forename: C J
authors.initials: CJ
authors.lastname: McBain
mesh_terms: D000818:Animals; D001369:Axons / Q000737:chemistry* / Q000648:ultrastructure; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D003593:Cytoplasm / Q000737:chemistry / Q000648:ultrastructure; D003712:Dendrites / Q000737:chemistry / Q000648:ultrastructure; D011992:Endosomes / Q000737:chemistry / Q000648:ultrastructure; D006624:Hippocampus / Q000166:cytology; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000032:analysis; D016253:Microscopy, Immunoelectron; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure*; D019581:Neuropil / Q000737:chemistry / Q000648:ultrastructure; D016716:PC12 Cells; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins; D013572:Synaptic Vesicles / Q000737:chemistry / Q000648:ultrastructure; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins
chemical_list: D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D011188:Potassium