Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release

European Neuroscience Institute Göttingen, Grisebachstrasse 5, 37077 Göttingen, Germany.
Science (Impact Factor: 33.61). 06/2006; 312(5776):1051-4. DOI: 10.1126/science.1126308
Source: PubMed


The molecular organization of presynaptic active zones during calcium influx–triggered neurotransmitter release is the focus
of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular
synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins
seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.

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    • "Ok6/+;UAS-Imac-RNAi. Stocks were obtained from: brp 69 (Kittel et al., 2006), Df(3R)S2.01 and rbp STOP1 (Liu et al., 2011a), aplip1 ex213 and aplip1 gen.rescue(ex213) gift from Catherine Collins (Klinedinst et al., 2013), srpk79D atc (Johnson et al., 2009), srpk79D vn (Nieratschker et al., 2009), UAS-Aplip1 GFP (Horiuchi et al., 2005), UAS-BRP-short straw (Schmid et al., 2008) and genomic BRP GFP (Matkovic et al., 2013). "
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    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.
    eLife Sciences 08/2015; 4. DOI:10.7554/eLife.06935 · 9.32 Impact Factor
    • "(M,M′) High-magnification imaging of Brp short ::GFP in the midline region of the main commissure. Both discrete small puncta in the size range of single active zones of NMJs (Kittel et al., 2006 "
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    ABSTRACT: Determining direct synaptic connections of specific neurons in the central nervous system (CNS) is a major technical challenge in neuroscience. As a corollary, molecular pathways controlling developmental synaptogenesis in vivo remain difficult to address. Here, we present genetic tools for efficient and versatile labeling of organelles, cytoskeletal components and proteins at single-neuron and single-synapse resolution in Drosophila mechanosensory (ms) neurons. We extended the imaging analysis to the ultrastructural level by developing a protocol for correlative light and 3D electron microscopy (3D CLEM). We show that in ms neurons, synaptic puncta revealed by genetically encoded markers serve as a reliable indicator of individual active zones. Block-face scanning electron microscopy analysis of ms axons revealed T-bar-shaped dense bodies and other characteristic ultrastructural features of CNS synapses. For a mechanistic analysis, we directly combined the single-neuron labeling approach with cell-specific gene disruption techniques. In proof-of-principle experiments we found evidence for a highly similar requirement for the scaffolding molecule Liprin-α and its interactors Lar and DSyd-1 (RhoGAP100F) in synaptic vesicle recruitment. This suggests that these important synapse regulators might serve a shared role at presynaptic sites within the CNS. In principle, our CLEM approach is broadly applicable to the developmental and ultrastructural analysis of any cell type that can be targeted with genetically encoded markers. © 2015. Published by The Company of Biologists Ltd.
    Development 01/2015; 142(2-2):394-405. DOI:10.1242/dev.115071 · 6.46 Impact Factor
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    • "DEVELOPMENT AZs in dysc and slo mutants facilitate enhanced spontaneous vesicle fusion. The BRP ring-like complex within the AZ surrounds a central core of VGCCs (Kittel et al., 2006). "

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