Coupling the Structural and Functional Assembly of Synaptic Release Sites

Abstract

Information processing in our brains depends on the exact timing of calcium (Ca²⁺)-activated exocytosis of synaptic vesicles (SVs) from unique release sites embedded within the presynaptic active zones (AZs). While AZ scaffolding proteins obviously provide an efficient environment for release site function, the molecular design creating such release sites had remained unknown for a long time. Recent advances in visualizing the ultrastructure and topology of presynaptic protein architectures have started to elucidate how scaffold proteins establish "nanodomains" that connect voltage-gated Ca²⁺ channels (VGCCs) physically and functionally with release-ready SVs. Scaffold proteins here seem to operate as "molecular rulers or spacers," regulating SV-VGCC physical distances within tens of nanometers and, thus, influence the probability and plasticity of SV release. A number of recent studies at Drosophila and mammalian synapses show that the stable positioning of discrete clusters of obligate release factor (M)Unc13 defines the position of SV release sites, and the differential expression of (M)Unc13 isoforms at synapses can regulate SV-VGCC coupling. We here review the organization of matured AZ scaffolds concerning their intrinsic organization and role for release site formation. Moreover, we also discuss insights into the developmental sequence of AZ assembly, which often entails a tightening between VGCCs and SV release sites. The findings discussed here are retrieved from vertebrate and invertebrate preparations and include a spectrum of methods ranging from cell biology, super-resolution light and electron microscopy to biophysical and electrophysiological analysis. Our understanding of how the structural and functional organization of presynaptic AZs are coupled has matured, as these processes are crucial for the understanding of synapse maturation and plasticity, and, thus, accurate information transfer and storage at chemical synapses.

Keywords: AZ scaffold protein superfamilies; active zone assembly; calcium channel positioning; coupling distances; release sites.

FIGURE 1

AZ protein superfamilies in invertebrates and vertebrates. Molecular structure of AZ proteins grouped into superfamilies. (A) RIM superfamily is composed of RIM/Unc10, Bassoon, Piccolo, Fife, and Clarinet. These proteins possess an N-terminal zinc-finger domain (ZF), a PDZ domain and two C2 domains. Mammalian Bassoon and Piccolo proteins additionally possess three coiled-coil domains (CC). (B) ELKS/CAST/BRP superfamily of proteins contains multiple CC regions and an additional C-terminal IWA motif in the mammalian isoforms (I). (C) M(U)nc13 superfamily consists of Unc13L (C. elegans), Unc13 (A,B isoforms in Drosophila) and Munc13-1—4 in mammals. These proteins have two/three C2 domains, a calmodulin binding site (Cbs) (with the exception of Unc13B), a C1 domain, and a MUN domain. (D) dRIM-BP (Drosophila) and RIM-BP form a superfamily of AZ proteins that possess an interruption of three contiguous FN3 domains between their first and last SH3 domains (E) Syd-2 /Liprinα family contain five CC regions and three C-terminally located SAM domains. (F) Syd-1 family possess a PDZ domain, C2 domain and a unique Rho-GAP domain.

FIGURE 2

Stable and specific positioning of Unc13A and B at Drosophila NMJ synapses. (A,D) Two-color STED microscopy images of synaptic boutons or individual planar AZs (B,C,E,F) from third instar larvae of the displayed genotypes labeled with antibodies to indicated proteins. (G) Mean intensity profile of Unc13A and Unc13B immunoreactivity plotted from the center of the AZ (the reference center being that of the BRP signal). The intensity maximum of the average fluorescence profile was found 70 nm from the AZ center for Unc13A and at 120 nm for Unc13B. Scale bars: (A,D): 1.5 μm; (B,C,E,F):50 nm; (H) 250 nm (Modified from Böhme et al., 2016). (H) Long-term FRAP of motoneuronally overexpressed Unc13A-GFP at muscle 26/27 depicts a stable integration of Unc13A that requires hours to recover completely. Dashed box shows bleached bouton before and directly after the fluorescence bleaching. Fluorescence recovery was measured 0.5; 1; 2; 4; 6; or 8 h after bleaching. Different time points were measured in different animals. (I) Quantification of percentage recovery over time. Bleached Unc13A-GFP showed a slow fluorescence recovery, exhibiting a tau of 6.88 ± 0.55 h, single exponential recovery fit. Data are mean ± SEM. Scale bar:5 μm (Modified from Reddy-Alla et al., 2017). Reproduced with permission, from Böhme et al., 2016 (A—G) and from Reddy-Alla et al., 2017 (H,I).

FIGURE 3

A schematic depicting AZ assembly at Drosophila NMJs. (A) Depicts a schematic of the early AZ scaffold recruitment and assembly. Here, Spn (Spinophilin) acts antagonistically to dSyd-1 (Drosophila Syd-1). Immobile dNrx-1 (Drosophila Neurexin-1) molecules get stabilized at the presynaptic membrane by an interaction with the PDZ domain of dSyd-1. Once dNrx-1 is immobilized at the presynaptic membrane, it forms a bridge with its postsynaptic partner dNlg1 (Drosophila Neuroligin1). In this way, trans-synaptic contact can also initiate pre- and postsynaptic scaffold assembly. Spn competes with dSyd-1 to bind dNrx-1, with its PDZ domain, thus reducing the amount of dNrx-1 available for dSyd-1 binding. This mechanism helps control the seeding and ultimately the number of AZs at a presynaptic bouton. In addition, dLiprinα also binds to dSyd-1 and together these proteins are known to recruit Unc13B to nascent synaptic sites. Unc13B mediated loose coupling of SVs, facilitates SV-fusion and thus NT (neurotransmitter) release into the synaptic cleft. (B) Illustrates a schematic of the late AZ scaffold assembly process. Once a nascent site is established, incorporation of postsynaptic GluRIIA (Glutamate receptor type IIA) on the postsynaptic scaffold is observed, while BRP (Bruchpilot); dRIM-BP (Drosophila RIM-binding protein), and Unc13A proteins are recruited in a second wave of AZ scaffold assembly and center themselves at VGCC localizations. (C) Illustrates the AZ scaffold maturation process at a developing AZ. A stable AZ scaffold forms with BRP and dRIM-BP centered on the VGCCs; here represented by Cac (Drosophila Cacophony). Pre-existing dLiprinα and Unc13B are pushed towards the extremities of the AZ scaffold. The N-terminus of dSyd-1 localizes close to the C-terminal region of BRP. In addition, dSyd-1 still maintains its binding partners the dNrx-1 and dLiprinα, thus tethering the early scaffold to the late AZ scaffold structure. BRP mediated VGCC clustering occurs at the presynaptic terminus, while at the postsynapse the incorporation of GluRIIB (Glutamate receptor type IIB) receptors, predominantly occurs outside the GluRIIA receptor field. Unc13A localizes SVs for tight coupling at approximate distances around 70 nm from the center of the scaffold.

FIGURE 4

Loose and tight coupling at Drosophila NMJ AZs. A schematic of loose and tight coupling occurring at single NMJ AZs. Loose coupling of SVs, depicted on the left-hand side of the AZ, occurs at low Ca²⁺ concentrations, at a rough distance of 140 nm from the AZ center and localize at Unc13B signals that are positioned 120 nm from the center. Tight coupling, depicted on the right-hand side of the AZ, occurs near high Ca²⁺ concentrations, that is typically positioned at a 70 nm distance from the AZ center or at Unc13A localizations, which are roughly positioned 50 nm from the AZ center.