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: 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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    • "(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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    • "We also examined the abundance of Bruchpilot (Brp), an active zone-organizing protein [45], in an effort to determine if Activin/TGF-β signaling influences the accumulation of presynaptic molecules. In contrast to the postsynaptic components, deposition of Brp at the synapses was not decreased in the babo and smox mutants (Fig. S4A and A′). "
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    ABSTRACT: Members of the TGF-β superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. In this study, we analyzed whether the Activin branch of the TGF-β superfamily also contributes to NMJ development and function. We find that elimination of the Activin/TGF-β type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. Our results demonstrate that anterograde Activin/TGF-β signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling, our findings indicate that the two branches of the TGF-β superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function.
    PLoS ONE 09/2014; 9(9):e107443. DOI:10.1371/journal.pone.0107443 · 3.23 Impact Factor
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