Molecular in situ topology of Aczonin/Piccolo and associated proteins at the mammalian neurotransmitter release site

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 31; pp. E392 - E401
• Main Authors: Limbach, Christoph, Laue, Michael M, Wang, Xiaolu, Hu, Bin, Thiede, Nadine, Hultqvist, Greta, Kilimann, Manfred W
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 02.08.2011; National Acad Sciences
• Series: PNAS Plus
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Animals; Binding Sites; Biological Sciences; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cytoskeletal Proteins - metabolism; electron microscopy; exocytosis; GTP-Binding Proteins - metabolism; Immunoblotting; Mammals; MEDICIN; MEDICINE; Membranes; Microscopy, Immunoelectron; Molecular structure; Multiprotein Complexes - metabolism; Multiprotein Complexes - ultrastructure; Nerve Tissue Proteins - metabolism; Neuropeptides - metabolism; Neurotransmitter Agents - metabolism; Neurotransmitters; ontogeny; plasma membrane; PNAS Plus; polypeptides; Protein Binding; protein structure; Proteins; Rats; Rats, Sprague-Dawley; scaffolding protein; space and time; Synapses - metabolism; Synapses - ultrastructure; synaptic transmission; topology
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1101707108

The protein machinery of neurotransmitter exocytosis requires efficient orchestration in space and time, for speed and precision of neurotransmission and also for synaptic ontogeny and plasticity. However, its spatial organization in situ is virtually unknown. Aczonin/Piccolo is a putative organizer protein of mammalian active zones. We determined by immunogold electron microscopy (EM) (i) the spatial arrangement (i.e., topology) of 11 segments of the Aczonin polypeptide in situ, and correlated it to (ii) the positioning of Aczonin-interacting domains of Bassoon, CAST/ELKS, Munc13, and RIM and (iii) the ultrastructurally defined presynaptic macromolecular aggregates known as dense projections and synaptic ribbons. At conventional synapses, Aczonin assumes a compact molecular topology within a layer 35 to 80 nm parallel to the plasma membrane (PM), with a "trunk" sitting on the dense projection top and a C-terminal "arm" extending down toward the PM and sideward to the dense projection periphery. At ribbon synapses, Aczonin occupies the whole ribbon area. Bassoon colocalizes with Aczonin at conventional synapses but not at ribbon synapses. At both conventional and ribbon synapses, CAST, Munc13, and RIM are segregated from Aczonin, closer to the PM, and Aczonin is positioned such that it may control the access of neurotransmitter vesicles to the fusion site.