Piccolo (also known as Aczonin) is the largest CAZ protein in the scaffold, with a molecular weight of ~550 kDa, and it shares with Bassoon a high degree of sequence homology (50 – 80 % common sequence identity), at two N-terminal zinc-fingers and three coil-coil domains together known as the Piccolo-Bassoon homology domains. These domains enable Bassoon-Piccolo interactions at the central CC2 and allow Bassoon and Piccolo complexes to compete for binding with other synaptic and scaffold proteins26. For example the N-terminal zinc finger domains of Piccolo and Bassoon bind to Siah125, though it is not yet understood whether the proteins collaborate or compete to inhibit Siah1 activity, while the
The N-terminal zinc-finger domains of Piccolo additionally have 40 % and 39 % homology to the zinc- finger domains of rabphilin-3A and RIM respectively, and contain a PRA1 binding site that has been shown to mediate Piccolo interactions with rab3A and VAMP2 receptors, thus linking Piccolo and synaptic vesicles pools at the AZ49. Piccolo possesses a few proline-rich sequences over its structure. A proline patch in the N-terminus of piccolo interacts with actin binding protein 1(Abp1), which links Piccolo by interacting with actin, dynamin50, and a GTPase that mediates the fission of SV vesicles51.
The central region of Piccolo also possess another proline patch that interacts with profilin, an actin-binding protein, that influences the actin-dynamics within presynaptic terminal52. This enables Piccolo to link the CAZ scaffold of proteins to cytoskeleton of the presynaptic terminus as well as incorporate itself into the process of endocytic fission at the AZ site.
Piccolo possesses two C2 domains, namely C2A and C2B domains, within its C-terminus that are not present in Bassoon. The C2A domain has been shown to undergo a conformation switch, upon binding Ca2+, that promotes dimerization and Ca2+-dependent phospholipid binding implicating a role for Piccolo in short-term plasticity53. Overall, by virtue of its large size and its CAZ interaction partners, Piccolo incorporates itself into the CAZ scaffold and links the CAZ scaffold the action cytoskeleton and SV recycling events at the presynaptic terminus.
Introduction Munc-13s, RIMs and CAST/ELKs
1.2.2. Munc-13s, RIMs and CAST/ELKs
Munc13, the vertebrate specific isoform of unc-13 found in C. elegans, is a small yet integral CAZ protein that interacts and neuronal Rab3 isoforms (RIMs) to regulate presynaptic neurotransmitter transmission54,55,56. CAST/ELKs are quintessential CAZ as a scaffold proteins that despite having different roles in different synapses are always involved in organizing the CAZ structure18. All three proteins bind to specific C-terminal subdomains of Bassoon and Piccolo and are therefore integral members of the CAZ scaffold31,34,26,31,48,57.A schematic depiction of these interactions can be referred to in Figure 2.
Three Munc13 genes exist in the brain Munc13-1, Munc13-2, and Munc13-3, are roughly ~222 kDa in weight and all three isoforms share three evolutionary conserved C2 domains present in the N- terminal, central, and C-terminal regions of the protein56,58.
Additionally, the MUN domain, when expressed in hippocampal neurons lacking Munc13s, rescues the SV priming deficits59. SV priming is a maturation step that occurs between the vesicle docking and SNARE-mediated SV fusion steps.
The presence of SV priming is attributed to the combination of opposing electrophysiological and ultrastructural observations noted in Munc13-1 knockout and Munc13-1 and Munc13-2 double knockout mice cultures. Glutamatergic hippocampal neurons of Munc13s, upon inspection for ultrastructural deficits, showed that docked SV vesicles numbers remained unchanged, despite the complete loss of evoked EPSCs in synapses deficient for Munc13s60,61. These results suggest Munc13s use their conserved MUN domain to play a crucial role for synaptic vesicle maturation.
The role of Munc13s in SV priming is compounded by the interaction of Munc13-1’s C2A domain with N-terminal zinc-finger domain of RIM. Disruption of this interaction in RIM deficient synapses prevents synaptic vesicles from reaching and fusing with the presynaptic membrane, as the lack of RIM forces Munc13-1 C2A domains to homo-dimerize thereby inhibiting Munc13’s SV priming and fusion competence55,62. This observation suggests that the N-terminus of RIM is essential for activating Munc13-1 molecules to mediate SV priming.
The RIM protein family consists of neuronal Rab3 isoforms that regulate neurotransmitter release at the presynaptic terminus. RIMs were first isolated and identified in a yeast two-hybrid screen of Rab3C against a rat brain cDNA library,
Introduction Munc-13s, RIMs and CAST/ELKs and found isolated on the synaptic vesicle fraction. The protein is 1554 amino acids long and consists of an N-terminal zinc finger domain comprised of alpha helices, alanine and proline-rich patches, a postsynaptic density domain, and C-terminal C2A and C2B domains. The RIM protein sequence is well conserved in invertebrates, but in vertebrates at least four RIM genes exist, RIM1—RIM4, with two isoforms each for RIM1 and RIM254.
The N terminal alpha helical domain of RIM binds to synaptic vesicle component rab3, forming a tight link between RIMs and SVs55. In addition the central PDZ domain of RIM proteins binds to N- and P-/Q-type Ca2+ channels. The interaction RIMs and Ca2+ channels is necessary for recruiting and organizing Ca2+ channels at the AZ. Deletion of the PDZ domain of RIMs not only affects the clustering of Ca2+ channel at the presynaptic membrane, but causes a decrease in Ca2+influx, which in turn reduces the priming of SVs and thereby fails to interact Ca2+
channels to synaptic vesicles63.
CAST or CAZ-associated structural protein, and ELKS (named after its highest a.a. content glutamate (E), leucine (L), lysine (K), and serine (S)) are CAZ proteins that are 957 a.a. and 948 a.a.-long, respectively, and share a 71 % a.a.
identity64. Two isoforms for ELKS: ELKS𝜀 and ELKS𝛼 exist. ELKS𝛼 is the neuron specific isoform that is involved in CAZ organization, while ELKS𝜀 are ubiquitously expressed, do not localize to the CAZ, and have been implicated in GTP-dependent Rab6 interaction that mediates its secretory traffic to membranes64,65.
CAST and ELKS𝛼 colocalizes with Bassoon in neuronal cultures and possess four coil-coil domains and a C-terminal IWA conserved domains33,64.The first two coil-coil domains (spanning 680 a.a. of their N-terminus), are essential for CAST and ELKS𝛼 targeting to the CAZ, while the CC2 domain of CAST competitively binds to the CC3 domains of CAZ scaffold proteins; Bassoon and Piccolo48. Although knocking out CAST does not yield an effect on synaptic transmission in their central excitatory synapses, it however crucially impairs retinal ribbon synapse transmission. Knockout of the CAST protein produces smaller AZs and diminished transmission in excitatory neurons66, and enlarged resting SV pools in inhibitory neuronal terminals, suggesting that CAST influences SV priming67. Together these five AZPs build a network of proteins localized in close proximity to the AZ site, link the site to SVs and prime them, arrange Ca2+ channel to the AZ site, promote the structural stability of scaffold by inhibiting local degradation mechanisms and link the scaffold to the dynamically changing actin cytoskeleton in the presynaptic terminus (Figure 2).
Introduction CAZ Assembly
Figure 2: Ultrastructural and schematic organization of AZPs and SV pools of central synapses.
Modified from Gundlefinger et al, 2016. (A) Cryo-electron micrograph rat central excitatory synapse (originally published in Rostaing et al., 2006). (B) Schematic organization of SVs pools within presynaptic boutons, the reserve SV pool tethered via synapsin and the docked SV pool are implanted in the CAZ (red tethers). (C) Schematic of CAZ proteins, interaction partners guiding the SV clustering, translocation, docking, priming and fusion at the presynaptic terminus.
Scale bar A, 200nm.