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1.
Curr Biol ; 31(8): 1711-1725.e5, 2021 04 26.
Article in English | MEDLINE | ID: mdl-33651992

ABSTRACT

As a result of developmental synapse formation, the presynaptic neurotransmitter release machinery becomes accurately matched with postsynaptic neurotransmitter receptors. Trans-synaptic signaling is executed through cell adhesion proteins such as Neurexin::Neuroligin pairs but also through diffusible and cytoplasmic signals. How exactly pre-post coordination is ensured in vivo remains largely enigmatic. Here, we identified a "molecular choreography" coordinating pre- with postsynaptic assembly during the developmental formation of Drosophila neuromuscular synapses. Two presynaptic Neurexin-binding scaffold proteins, Syd-1 and Spinophilin (Spn), spatio-temporally coordinated pre-post assembly in conjunction with two postsynaptically operating, antagonistic Neuroligin species: Nlg1 and Nlg2. The Spn/Nlg2 module promoted active zone (AZ) maturation by driving the accumulation of AZ scaffold proteins critical for synaptic vesicle release. Simultaneously, these regulators restricted postsynaptic glutamate receptor incorporation. Both functions of the Spn/Nlg2 module were directly antagonized by Syd-1/Nlg1. Nlg1 and Nlg2 also had divergent effects on Nrx-1 in vivo motility. Concerning diffusible signals, Spn and Syd-1 antagonistically controlled the levels of Munc13-family protein Unc13B at nascent AZs, whose release function facilitated glutamate receptor incorporation at assembling postsynaptic specializations. As a result, we here provide direct in vivo evidence illustrating how a highly regulative and interleaved communication between cell adhesion protein signaling complexes and diffusible signals allows for a precise coordination of pre- with postsynaptic assembly. It will be interesting to analyze whether this logic also transfers to plasticity processes.


Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , Animals , Cell Adhesion Molecules , Drosophila , Drosophila Proteins/genetics , Receptors, Glutamate , Synapses
2.
Neuron ; 95(6): 1350-1364.e12, 2017 Sep 13.
Article in English | MEDLINE | ID: mdl-28867551

ABSTRACT

Neural information processing depends on precisely timed, Ca2+-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.


Subject(s)
Carrier Proteins/metabolism , Drosophila , Exocytosis/physiology , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Animals , Neuromuscular Junction/metabolism , Neuromuscular Junction/ultrastructure
3.
Nat Neurosci ; 19(10): 1311-20, 2016 10.
Article in English | MEDLINE | ID: mdl-27526206

ABSTRACT

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.


Subject(s)
Calcium Channels/metabolism , Carrier Proteins/metabolism , Drosophila melanogaster/metabolism , Presynaptic Terminals/metabolism , Synaptic Vesicles/metabolism , Animals , Carrier Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Female , GTPase-Activating Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Male , Models, Neurological , Mutation , Phosphoproteins/metabolism , Protein Isoforms , rab3 GTP-Binding Proteins/metabolism
4.
Elife ; 42015 Aug 14.
Article in English | MEDLINE | ID: mdl-26274777

ABSTRACT

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.


Subject(s)
Axonal Transport , Carrier Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/physiology , rab3 GTP-Binding Proteins/metabolism , Animals , Binding Sites , Carrier Proteins/genetics , DNA Mutational Analysis , Drosophila Proteins/genetics , Optical Imaging , Protein Binding , Protein Interaction Mapping , Protein Transport
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