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Assembly of the Presynaptic Active Zone Available online at www.sciencedirect.com ScienceDirect Assembly of the presynaptic active zone Javier Emperador-Melero and Pascal S Kaeser In a presynaptic nerve terminal, the active zone is composed of In this review, we discuss recent progress and current sophisticated protein machinery that enables secretion on a models of the mechanisms of active zone assembly. The submillisecond time scale and precisely targets it toward focus of this review is on ‘primary’ active zone proteins, postsynaptic receptors. The past two decades have provided which we narrowly define as the proteins that are preferen- deep insight into the roles of active zone proteins in exocytosis, tially localized to the active zone membrane despite but we are only beginning to understand how a neuron the lack of transmembrane domains, and function together 2+ 2+ assembles active zone protein complexes into effective in coupling vesicle fusion to Ca influx through Ca molecular machines. In this review, we outline the fundamental channels (Box 1, Figure 1). These proteins include RIM, processes that are necessary for active zone assembly and Munc13, Bassoon/Piccolo, Liprin-a, ELKS and RIM-BP. discuss recent advances in understanding assembly Notably, this definition does not include SNARE proteins 2+ mechanisms that arise from genetic, morphological and orCa channels,butinsteadhighlightsthatoneactive zone biochemical studies. We further outline the challenges ahead function is to control the relative positioning of these for understanding this important problem. essential proteins [1,2]. We further define active zone assembly as the process that positions and maintains these Address proteins in a complex that supports exocytotic functions at Department of Neurobiology, Harvard Medical School, Boston, USA the presynaptic plasma membrane. Active zone assembly and function are closely linked. Any given component may Corresponding author: Kaeser, Pascal S ([email protected]) have assembly roles in recruiting other proteins to the active zone and direct roles in mediating exocytosis of Current Opinion in Neurobiology 2020, 63:95–103 vesicles. The following steps are essential for active zone This review comes from a themed issue on Cellular neuroscience assembly (Figure 2): Edited by Thomas Schwarz and Holly Cline the generation of active zone proteins, their sorting into axonal transport, and their capture in a nerve terminal the assembly of these active zone constituents into protein complexes https://doi.org/10.1016/j.conb.2020.03.008 the positioning and anchoring of these complexes at 0959-4388/ã 2020 Elsevier Ltd. All rights reserved. the presynaptic target membrane precisely opposed to postsynaptic receptors These steps are interconnected and do not necessarily occur in the same order for each protein. We highlight Introduction recent progress in dissecting each of these steps, first Transmission and processing of information in the brain focusing on the best understood step, assembly of mostly occur at synapses, sites of communication between the protein complex, and we explore key challenges that neurons at which a presynaptic nerve terminal contacts a lie ahead. postsynaptic cell. Synaptic transmission is initiated by the fusion of neurotransmitter-containing synaptic vesicles with The active zone protein complex and its the plasma membrane. This fusion step is mediated by a assembly set of conserved fusion proteins including SNAREs, their Superresolution microscopy has revolutionized the charac- 2+ regulators, and Ca sensors. While presynaptic fusion rates terization of active zone protein complexes and uncovered are low at rest, they dramatically increase upon opening of striking patterning of the active zone. In the vertebrate brain, 2+ voltage-gated Ca channels in response to action potential the large scaffold protein Bassoon is oriented such that its firing. Synaptic vesicle exocytosis is executed within less C-terminus is close to the presynaptic plasma membrane, than one millisecond upon presynaptic depolarization, is and its N-terminus reaches tens of nanometers into the precisely targeted toward postsynaptic receptors, is remark- presynaptic cytoplasm [3]. Other active zone proteins local- ably plastic, and is heterogeneous across synapse and neuro- ize between the Bassoon N-termini and C-termini [4 ], nal types. These important features of synaptic transmission indicating that they are part of the same complex. A similar are mediated by sophisticated protein machinery called the pattern occurs at the fly neuromuscular junction, where active zone (Box 1) [1,2]. Bruchpilot, a partial homologue of ELKS, is oriented with www.sciencedirect.com Current Opinion in Neurobiology 2020, 63:95–103 96 Cellular neuroscience Box 1 Active zone definitions Current mechanistic insight into assembly of active zone complexes strongly rely on gene knockout studies The most common active zone definitions have relied on function, morphology or protein biochemistry (also see Figure 1). (Table 1). The major message from these studies is that the active zone is a resilient structure. Specifically, Function. The active zone is the specialized area of the presynaptic plasma membrane where synaptic vesicles fuse [59]. Because the ablation of individual active zone proteins, or of active presynaptic plasma membrane contains several release sites zone protein families, typically results in at most mild [2,7 ,60], uncertainty in the terminology arises. A presynaptic assembly deficits, illustrated by impaired localization of release site could be considered an independent active zone, or an select interaction partners. At hippocampal synapses, for active zone could contain multiple independent release sites. example, removing RIMs leads to reduced levels of Morphology. The active zone has been defined in electron micro- 2+ Munc13-1 [12] and of CaV2 Ca channels [13]. For some scopic studies [61,62], where it appears as electron-dense material proteins, these functions differ strikingly across synapse attached to the presynaptic plasma membrane and opposed to the postsynaptic density. Often, the active zone membrane is associated types. Three examples are (1) knockout of RIM-BP2 that with electron dense cytoplasmic protrusions, or dense projections. leads to reduced Munc13-1 levels specifically at mossy These projections are variable in shape and prominent at some fiber synapses [14], (2) a recruitment function of ELKS1 synapses, for example ribbon synapses in the inner ear or retina, or for ubMunc13-2 that is restricted to a small subset of the fly neuromuscular junction [1]. forebrain synapses [15], and roles for RIM in Bassoon Biochemistry. The active zone is an insoluble protein complex clustering in dopamine axons but not at hippocampal anchored to the presynaptic plasma membrane [1]. Many proteins synapses [13,16]. Synapse-specific assembly roles may are present in this complex, including large scaffolding proteins that are preferentially localized to the active zone area (RIM, RIM-BP, also be revealed in functional phenotypes. Functions ELKS, Bassoon/Piccolo, Liprin-a and Munc13). Additional important of ELKS, for example, differ between excitatory and proteins that are present but often not restricted to the active zone inhibitory transmission, which may reflect distinct roles are SNARE proteins and their regulators, channels and receptors, in presynaptic assembly [17]. In contrast to these gener- cell-adhesion proteins, and cytoskeletal proteins. ally mild effects on active zone structure at typical small Combinations of criteria. Recently, superresolution microscopy is central synapses, there are morphologically unique used to describe the active zone combining various criteria. For synapses with prominent dense projections. There, single example, nano-scale localization of RIM or Munc13 and of exocy- totic events has led to the identification of multiple individual release proteins often account for the majority of the density, sites within an active zone [7 ,8 ]. Similarly, biochemical and mor- and genetic ablation of the main component leads to a loss phological parameters have been combined to localize protein of these densities [18–20]. Disrupting these electron complexes that contain multiple active zone members in an ordered densities, however, does not fully abolish exocytosis, fashion [4 ,5]. suggesting that machinery for secretion remains at least In this review, we use the term active zone to define the presynaptic partially intact. The overall resilience of active zone membrane specialization opposed to the postsynaptic density that structure indicates that redundant protein interactions contains multiple individual sites for vesicle docking and release. For simplicity, we focus on the primary components of the presynaptic within the active zone account for its stability. protein complexes that contain RIM, RIM-BP, Munc13, ELKS, Liprin- a, and Piccolo/Bassoon [1]. We further discuss the association of An important step forward in understanding redundancy these proteins with important components of the presynaptic plasma 2+ in active zone assembly came from the analysis of membrane, for example Ca channels and cell adhesion molecules, compound mutants [21 ,22 ,23 ]. Simultaneous knockout but do not include these proteins in the narrow active zone definition that we use here. of RIM and ELKS induced a strong disruption of active zone complexes with loss of Munc13-1, Bassoon, Piccolo, 2+ RIM-BP2, and CaV2.1 Ca channels, accompanied by a loss of
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