Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

Neural information processing depends on precisely timed, Ca-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.

Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) we show that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation.

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.DOI: http://dx.doi.org/10.7554/eLife.17523.001

Chemical synaptic transmission relies on the Ca2+-induced fusion of transmitter-laden vesicles whose coupling distance to Ca2+ channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Here, using electron- and super-resolution microscopy at the Drosophila neuromuscular junction we quantitatively map vesicle:Ca2+ channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. We show that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca2+-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. We propose activity-dependent inhibition of vesicle un-priming or release site activation as novel facilitation mechanisms.

1 Synaptic transmission is mediated by neurotransmitter release at presynaptic active zones (AZs) 2 followed by postsynaptic neurotransmitter detection. Plastic changes in transmission maintain 3 functionality during perturbations and enable memory formation. Postsynaptic plasticity targets 4 neurotransmitter receptors, but presynaptic plasticity mechanisms directly regulating the 5 neurotransmitter release apparatus remain largely enigmatic. Here we describe that AZs consist 6 of nano-modular release site units and identify a molecular sequence adding more modules 7 within minutes of plasticity induction. This requires cognate transport machinery and a discrete 8 subset of AZ scaffold proteins. Structural remodeling is not required for the immediate 9 potentiation of neurotransmitter release, but rather necessary to sustain this potentiation over 10 longer timescales. Finally, mutations in Unc13 that disrupt homeostatic plasticity at the 11 neuromuscular junction also impair shot-term memory when central neurons are targeted, 12 suggesting that both forms of plasticity operate via Unc13. Together, while immediate synaptic 13 potentiation capitalizes on available material, it triggers the coincident incorporation of modular 14 release sites to consolidate stable synapse function. 16Neurotransmitter-laden synaptic vesicles (SVs) release their content at presynaptic active zones 17 (AZs) in response to Ca 2+ influx through voltage gated channels that respond to action-potential 18 (AP) depolarization. Neurotransmitter binding to postsynaptic receptors subsequently leads to 19 their activation for synaptic transmission. Modulation of transmission strength is called synaptic 20 plasticity. Long-term forms of synaptic plasticity are major cellular substrates for learning, 21 memory, and behavioral adaptation 1, 2 . Mechanisms of long-term synaptic plasticity modify the 22 structure and function of the presynaptic terminal and/or the postsynaptic apparatus. AZs are 23 covered by complex scaffolds composed of a conserved set of extended structural proteins. 24 ELKS/Bruchpilot (BRP), RIM, and RIM-binding protein (RBP) functionally organize the 25 coupling between Ca 2+ -channels and release machinery by immobilizing the critical (M)Unc13 26 release factors in clusters close to presynaptic Ca 2+ -channels and thus generate SV release sites, 27 at both mammalian and Drosophila synapses 3-12 . Whether and how discrete AZ release sites and 28 the associated release machinery are reorganized during plastic changes remains unknown. 29 One crucial form of presynaptic plasticity is the homeostatic control of neurotransmitter 30 release. This process, referred to as presynaptic homeostatic potentiation (PHP), is observed in 31 organisms ranging from invertebrates to humans, but is perhaps best illustrated at the larval 32 neuromuscular junction (NMJ) of Drosophila melanogaster 13, 14 . Here, PHP requires the core 33 AZ-scaffolding proteins RIM, RBP and Fife 15-17 and physiologically coincides with the 34 upregulat...

Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol–protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.

Synaptic transmission relies on the rapid fusion of neurotransmitter-containing synaptic vesicles (SVs), which happens in response to action potential (AP)-induced Ca influx at active zones (AZs). A highly conserved molecular machinery cooperates at SV-release sites to mediate SV plasma membrane attachment and maturation, Ca sensing, and membrane fusion. Despite this high degree of conservation, synapses - even within the same organism, organ or neuron - are highly diverse regarding the probability of APs to trigger SV fusion. Additionally, repetitive activation can lead to either strengthening or weakening of transmission. In this review, we discuss mechanisms controlling release probability and this short-term plasticity. We argue that an important layer of control is exerted by evolutionarily conserved AZ scaffolding proteins, which determine the coupling distance between SV fusion sites and voltage-gated Ca channels (VGCC) and, thereby, shape synapse-specific input/output behaviors. We propose that AZ-scaffold modifications may occur to adapt the coupling distance during synapse maturation and plastic regulation of synapse strength.