Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila

Abstract

Neurotransmitters are released at presynaptic active zones (AZs). In the fly Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. We employ nc82 to identify Bruchpilot protein (BRP) as a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC, which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of BRP displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of the bruchpilot locus (brp) coincides with neuronal differentiation. Panneural reduction of BRP expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, we observe loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence "bruchpilot"-crash pilot). We propose that BRP is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila.

Engrailed Alters the Specificity of Synaptic Connections ofDrosophilaAuditory Neurons with the Giant Fiber

“…2G-J ) and also to another interneuron projecting into the JO-A region (Fig. 2G,K ), tentatively identified as AMMC-A1 Tootoonian et al, 2012). It is clear that the JO axons are filled directly via the GF and not via the AMMC-A1 neuron, because the contralateral homolog of the latter also fills strongly yet on that side no JO axons are coupled to it (data not shown).…”

“…After removal, brains or pupal antennae were processed for antibody labeling, cleared, and mounted as described previously (Blagburn, 2008;Pézier and Blagburn, 2013). The nc82 [anti-Bruchpilot (Brp); Wagh et al, 2006] and the 4D9 [anti-En and Invected (Inv); Patel et al, 1989] antibodies were obtained from the Developmental Studies Hybridoma Bank and used at a dilution of 1:20. Anti-En rabbit polyclonal antibody (D-300; Santa Cruz Biotechnology) was used at 1:200.…”

“…6A) is the first neuronal circuit in Drosophila that was found to be amenable for electrophysiology (Levine and Tracey, 1973;Tanouye and Wyman, 1980;Thomas and Wyman, 1984;Allen et al, 1999;Allen and Godenschwege, 2010). In addition to the mechanosensory inputs from the JO that we are investigating here, the GF is known to receive synaptic inputs from the visual system onto its lateral dendrites (Bassemir and Strausfeld, 1983;Mu et al, 2012).…”

“…Recently, these electrical synaptic inputs have been recorded directly from the GF cell body in response to sound and air movements (Tootoonian et al, 2012;Lehnert et al, 2013;Mu et al, 2014), and it appears that a dendrite of GF is inserted directly in the region of JO-A afferent projections (Lehnert et al, 2013). In this study, we find that Enexpressing JONs are the ones that form these synapses and show anatomically and electrophysiologically that perturbation of En expression in the JONs alters their synaptic connections with the GF.…”

Swiss Cheese, Drosophila Ortholog of Hereditary Spastic Paraplegia Gene NTE, Maintains Neuromuscular Junction Development and Microtubule Network

“…We analyzed presynaptic marker Bruchpilot and the postsynaptic marker Dlg in sws mutants to check whether the satellite bouton change is associated with alterations in the subcellular localization of synaptic proteins. For active zone analysis, we performed immunohistochemical staining with nс82 antibodies against a D. melanogaster-active zone protein-Bruchpilot [18]. Active zone number in SWS-RNAi and sws 1 was not diferent from the control but was reduced in sws 76−15 ( Figure 5), which is, in our view, with a decrease in the number of satellite boutons.…”