Molecular scaffold reorganization at the transmitter release site with vesicle exocytosis or botulinum toxin C1.
Neurotransmitter release sites at the freeze-fractured frog neuromuscular junction are composed of inner and outer paired rows of large membrane particles, the putative calcium channels, anchored by the ribs of an underlying protein scaffold. We analysed the locations of the release site particles as a reflection of the scaffold structure, comparing particle distributions in secreting terminals with those where secretion was blocked with botulinum toxin A, which cleaves a small segment off SNAP-25, or botulinum toxin C1, which cleaves the cytoplasmic domain of syntaxin. In the idle terminal the inner and outer paired rows were located approximately 25 and approximately 44 nm, respectively, from the release site midline. However, adjacent to vesicular fusion sites both particle rows were displaced towards the midline by approximately 25%. The intervals between the particles along each row were examined by a nearest-neighbour approach. In control terminals the peak interval along the inner row was approximately 17 nm, consistent with previous reports and the spacing of the scaffold ribs. While the average distance between particles in the outer row was also approximately 17 nm, a detailed analysis revealed short 'linear clusters' with a approximately 14 nm interval. These clusters were enriched at vesicle fusion sites, suggesting an association with the docking sites, and were eliminated by botulinum C1, but not A. Our findings suggest, first, that the release site scaffold ribs undergo a predictable, and possibly active, shortening during exocytosis and, second, that at the vesicle docking site syntaxin plays a role in the cross-linking of the rib tips to form the vesicle docking sites.
Stanley
Elise F
EF
Toronto Western Research Institute, University Health Network, MP14-320, 399 Bathurst Street, Toronto ON M5T 2S8, Canada. estanley@uhnres.utoronto.ca
Reese
Tom S
TS
Wang
Gary Z
GZ
D000818:Animals; D001001:Anura; D001905:Botulinum Toxins / Q000494:pharmacology*; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D005614:Freeze Fracturing / Q000379:methods; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D018377:Neurotransmitter Agents / Q000378:metabolism*; D016011:Normal Distribution; D050765:Qa-SNARE Proteins; D013572:Synaptic Vesicles; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D022161:Transport Vesicles / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050765:Qa-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096324:botulinum toxin type C