Kittel, R.J. et al. Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312, 1051-1054

European Neuroscience Institute Göttingen, Grisebachstrasse 5, 37077 Göttingen, Germany.
Science (Impact Factor: 31.48). 06/2006; 312(5776):1051-4. DOI: 10.1126/science.1126308
Source: PubMed

ABSTRACT 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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Available from: Katrin I Willig, Aug 19, 2015
    • "(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 · 6.27 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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    • "The AZ membrane is associated with an electron-dense protein matrix, termed the cytomatrix of the active zone (CAZ), that is partially resistant to detergent extraction (Phillips et al., 2001). CAZ components (Gundelfinger and Fejtova, 2012) include giant multidomain proteins such as piccolo, bassoon, ELKS (also termed CAST), relatives of Drosophila bruchpilot (BRP) (Kittel et al., 2006) and Fife (Bruckner et al., 2012), Rab3 interacting molecules (RIMs) (Kaeser et al., 2011), RIM binding proteins (RBPs) (Liu et al., 2011), Munc13, liprins, and G protein coupled receptor kinase 2 interacting protein (GIT) (Kim et al., 2003). Endocytic retrieval of exocytosed SV proteins involves adaptor proteins (Dittman and Ryan, 2009) such as AP-2, stonins (Fergestad and Broadie, 2001; Mullen et al., 2012), and AP180 (Koo et al., 2011). "
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    ABSTRACT: Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.
    Cell Reports 05/2014; 7(5). DOI:10.1016/j.celrep.2014.04.051 · 8.36 Impact Factor
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