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

Abstract

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.scaffolding protein | protein structure N eurotransmitter release is confined to a specialized area of the presynaptic plasma membrane (PM) known as the active zone. Calcium-dependent exocytosis at the synapse is an elaborate, multistep process that is fast and precise, yet highly restrained and subject to modulation (i.e., presynaptic plasticity) (1). A solubilization-resistant lattice of proteins associated with the inner face of the presynaptic PM, the cytomatrix at the active zone (CAZ), is apparent on electron microscopy (EM) imaging and seems to integrate and organize many proteins of the presynaptic machinery (2).When mammalian conventional synapses are stained with heavy metal ions, EM reveals regularly spaced, cone-shaped structures of a size of approximately 50 nm attached to the presynaptic PM, which are known as dense projections (DPs) or, collectively, the "presynaptic particle web" or "presynaptic grid." It has been possible to isolate DP-like particles by subcellular fractionation, and even to reconstitute similar aggregates after previous solubilization, and many proteins of the presynaptic molecular machinery have been detected in them (3). These findings suggest that the presynaptic machinery may be organized into modular units of homogeneous size and shape, and possibly also of defined stoichiometry and spatial organization of its constituent proteins. An organized positioning of individual proteins and protein domains could allow these supramolecular assemblies to function as "molecular machines" and contribute to the high speed, precision, and fine-tuning that is characteristic for synaptic exocytosis. Morphological organization of active z...

Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses

“…2). Two studies (tom Dieck et al, 2005;Limbach et al, 2011) reported Munc13-1 expression at photoreceptor ribbon synapses (but see Schmitz et al, 2001). An explanation for these findings is that the antibodies used there detected both Munc13-1 and ubMunc13-2, since Figure 6.…”

“…1B,C, illustrating the crossreactivity of the polyclonal anti-Munc13 antibody with Munc13-1, Munc13-2, and Munc13-3), or against the N terminus of Munc13-1 (Limbach et al, 2011), which is highly homologous to ubMunc13-2. Indeed, the latter study acknowledges significant cross-reactivity of the antibody used with ubMunc13-2 (Limbach et al, 2011).…”

“…High-resolution imaging at the lightand electron-microscopic levels showed that ubMunc13-2 is not directly associated with ribbons, but discretely localized at the CAZ (Limbach et al, 2011) and along the plasma membrane of the invagination (Fig. 5).…”

Excitable behavior can explain the “ping‐pong” mode of communication between cells using the same chemoattractant

“…Interestingly, sequence alignment shows a very weak homology of SO to the proteins of aczonin/piccolo family. In mammalian synapses, these proteins are thought to regulate the transition of neurotransmitter-loaded vesicles from the reserve to the release-ready pool and thus are involved in the coordination of rapid fusion of multiple vesicles [56].…”

Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences

“…The CAZ is an electron-dense region in electron micrographs and integrates many proteins that regulate neurotransmitter secretion in presynaptic terminals. Piccolo and its interacting proteins are positioned to support synaptic vesicle supply to the site of release and support synaptic reliability during intense activity (Limbach et al, 2011). The Piccolo protein consists of two Zinc-finger, one PDZ, three-coiled coil, and two C2 domains (C2A and C2B).…”