InteractiveFly: GeneBrief

bruchpilot: Biological Overview | References

Neurotransmitters are released at presynaptic active zones (AZs). In Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. Nc82 was used to identify Bruchpilot protein (Brp), a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC (Note: CAST stands for cytomatrix at the active zone associated structural protein), 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 bruchpilot 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, loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence 'bruchpilot' - German for crash pilot), were observed. It is proposed that Brp is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila (Wagh, 2007). Further studies show that in brp mutants Ca2+ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. Brp-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006)

Neurotransmitter release during chemical communication between nerve cells takes place at synaptic active zones, specific sites characterized ultrastructurally by presynaptic membrane thickenings decorated with synaptic vesicles and surrounded by additional synaptic vesicle accumulations. Often, electron-dense projections of various shapes (plaques, pyramids, T-shaped structures, ribbons) extend from the active zone into the presynaptic cytoplasm. Considerable efforts have been undertaken in recent years to identify and functionally characterize the protein components of these projections and the cytoskeletal matrix associated with the active zone (CAZ). This complex meshwork of proteins most likely constitutes an essential part of the molecular machinery mediating neurotransmitter release. The fine regulation of this process is believed to be central to nervous system operation including higher functions such as learning, memory, and cognition (Wagh, 2007)

In vertebrates, several components associated with the presynaptic active zone have been characterized. In addition to the general cytoskeletal proteins actin and spectrin, the large protein Bassoon (420 kDa) (tom Dieck, 1998; Shapira, 2003) is specifically found at the CAZ. This protein has been shown to be required for structural active zone formation and/or maintenance. Piccolo (530 kDa) (Fenster, 2000) contains several putative protein-protein interaction domains and together with Bassoon is assumed to organize components of the active zone, including Rab3-interacting molecule (RIM1), Munc-13, and the CAZ-associated structural protein (ELKS/CAST/ERC) (Wagh, 2007)

Vertebrate ELKS/CAST/ERC proteins were identified as AZ components by purifying synaptic densities from rat brain followed by electrophoresis and mass spectrometry (Ohtsuka, 2002) and, independently, in a yeast-two-hybrid screen of a rat-brain cDNA library as proteins interacting with the RIM1α PDZ domain (Wang, 2002). Several isoforms have been reported to be transcribed from two genes (Wang, 2002; Deguchi-Tawarada, 2004). Two isoforms (CAST1/ERC2 and CAST2α/ERC1b) are brain-specific and contain several coiled-coil domains as well as a C-terminal IWA motif essential for binding the PDZ domain of RIM1 (Ohtsuka, 2002). ELKS/CAST/ERCs form large oligomeric protein complexes with the other known proteins of the CAZ (Munc-13, RIM1, Piccolo, Bassoon) and are believed to be involved in the molecular organization of presynaptic active zones (Ko, 2003) where they regulate the release of neurotransmitter (Takao-Rikitsu, 2004; Wagh, 2007 and references therein).

Although most proteins shown to be relevant for structure and/or function of the vertebrate nervous system are conserved in invertebrates, apparently no Bassoon or Piccolo homologs are encoded in the Drosophila genome. This study identified the Drosophila bruchpilot gene (brp) coding for a protein (Brp) that contains an N-terminal domain with significant sequence homology to vertebrate ELKS/CAST/ERC and a large C-terminal domain rich in coiled-coil structures similar to several cytoskeletal proteins. The structure of this gene was analyzed; the Drosophila Brp protein localizes at the presynaptic active zone. By analyzing various transgenic RNAi lines, the effects were characterized of reduction of Brp expression on synaptic ultrastructure, as well as neurotransmitter release and observe behavioral defects, including unstable flight (hence bruchpilot, crash pilot, named after an old German movie about a pilot who always crashes his planes but survives). It is speculated that Brp might combine functions of ELKS/CAST/ERC and a cytoskeletal structural protein in a single polypeptide that is highly conserved among insects (Wagh, 2007).

Over the last years, some insight into assembly and molecular composition of vertebrate presynaptic active zones has been gained. Cytoskeletal elements like actin and spectrin, as well as large active-zone-specific proteins like Piccolo and Bassoon, seem to form a structural meshwork organizing various components of the active zone. The third coiled-coil domain of Bassoon contains a motif that is highly homologous to the corresponding region of Piccolo and binds in competition to Piccolo the fifth predicted coiled-coil domain of ELKSα/CAST1. This binding of ELKSα/CAST1 to Bassoon seems to be involved in neurotransmitter release (Takao-Rikitsu, 2004). ELKSα/CAST1 itself has been shown to bind RIM1 via the C-terminal PDZ binding motif IWA (Ohtsuka, 2002). RIM1 is a target of the Rab3A small G protein (Wang, 1997) and interacts with Munc13-1. Together with vesicular proteins, this complex might control the recruitment of vesicles and regulate their subsequent fusion with the presynaptic membrane. Recently, ELKS has been shown to function in insulin exocytosis of pancreatic β cells (Ohara-Imaizumi, 2005). Deletion of the elks gene in C. elegans neither relocates RIM nor produces an obvious structural or functional phenotype, although C. elegans ELKS (Deken, 2005) like its mammalian homologs binds to the PDZ domain of RIM (Wagh, 2007)

Although there is a wealth of information on the ultrastructure of insect synapses, the molecular composition of their synaptic active zones is almost completely unknown. This work shows that in Drosophila, a protein with homology to the ELKS/CAST/ERC protein family of vertebrates localizes at the presynaptic active zones of neuronal terminals. The rather uniform distribution of tiny nc82 stained spots in all adult and larval neuropil regions suggests that Brp is present at active zones of most if not all synapses of Drosophila. Thus, the Brp protein provides an entry point to study general active-zone formation and function in this species (Wagh, 2007)

The open-reading frame of the cDNA identified by RT-PCR corresponds in size to the protein recognized by MAB nc82. The fact that the prominent Northern blot signal is about 5.5 kb larger than the known cDNA could indicate that the brp mRNA abundantly expressed in heads likely contains a long 3'UTR. Long 3'UTRs are found in several brain mRNAs (e.g., elav, fne). Possibly, the gene contains alternative polyadenylation sites giving rise to an abundant 11 kb mRNA and a less-abundant mRNA that contains the 3'end of cDNA AT09405 but is not detected in the Northern blot. This hypothesis is supported by two RT-PCR experiments with independent primer pairs downstream of the 3'end of cDNA AT09405. The signal at 2.0 kb cannot be interpreted with present cDNA information. Both signals were identically reproduced in two independent head mRNA blots hybridized with probes specific for either CG12933 and CG30336 or CG30337, or containing the entire cDNA sequence. No difference was observed with these three probes. Transcripts containing ORF CG12932, on the other hand, apparently are not abundantly expressed in adult heads and may or may not belong to the brp transcription unit (Wagh, 2007)

The combined evidence from MS analysis, bacterial cDNA expression, ectopic expression of GFP-labeled Brp, and the RNAi experiments proves that Brp represents the active-zone protein recognized by MAB nc82. Analysis of the amino acid sequence of Drosophila Brp predicts two leucine zipper domains and a glutamine-rich C terminus but no transmembrane domains. High homologies among human ELKSα, C. elegans CAST, and Brp are found in three regions of the proteins. However, no PDZ interaction motif (IWA) for RIM interaction as seen in several mammalian ELKS/CAST isoforms and, interestingly, in the C. elegans homolog seems to be present in insect Brp. No Drosophila protein is detected by BLAST analysis with conserved C-terminal sequences of worm or human. However, for the large C terminus of Brp (1260 aa) significant sequence similarities to cytoskeletal proteins such as plectin, myosin heavy chain, and restin are observed, suggesting a possible cytoskeletal role or interaction of the C terminus of Brp (Wagh, 2007)

The expression of Brp is not restricted to the glutamatergic type I boutons of the NMJ, but Brp is present in active zones of presumably all synapses. Consistently, those layers in the visual neuropil that contain high levels of either choline acetyltransferase or GABA/glutamic acid decarboxylase do not show reduced nc82 staining, and nc82 staining is found in both glutamatergic and nonglutamatergic synaptic boutons of larval NMJs. It was furthermore demonstrated that normal Brp levels are required for normal synaptic transmission at histaminergic synapses (Wagh, 2007)

This study directly address the structural, physiological, and systemic function of Brp, a member of the ELKS/CASTl/ERC family. The ultrastructure of synaptic active zones in terminals of larval motorneurons and adult photoreceptors is impaired when Brp protein levels are severely reduced. The loss of T bars in the lamina is compatible with the hypothesis that Brp may be required for anchoring the T bars to the presynaptic membrane. The fact that similar frequencies of T bars is seen in wild-type photoreceptor terminals (16.8%) and of conspicuous membrane densities in brp-RNAi^C8-expressing terminals (19.5%) supports the proposition that these membrane thickenings may in fact represent presynaptic sites without typical T bars. However, it is unlikely that Brp is restricted to T bars because about 13%-37% of the active zones of type Ib boutons at NMJs of larval muscles 6/7 have no T bar, whereas in light microscopical preparations, only 3% of the active zones identified by their postsynaptic receptor fields contained no Brp label. Unfortunately, in these experiments the epitope recognized by MAB nc82 did not tolerate tissue fixation conditions required for immunoelectron microscopical analysis of T bars (Wagh, 2007)

Regarding a role of Brp in synaptic function, two RNAi lines with panneural expression were tested. Both showed a reduction in Brp protein levels and a decrease in evoked transmitter release as reflected by reduced EJC amplitude at larval NMJs, whereas miniature EJC amplitude and frequency were indistinguishable from wild-type controls (Wagh, 2007)

The genotype brp-RNAi^B12/elav-Gal4 caused embryonic lethality. When Brp was suppressed only in the eye, adult brp-RNAi^B12/gmr-Gal4 flies had a strong effect on eye development ('rough eye') and in some cases, in addition to the lack of ON/OFF transients, showed a much smaller ERG receptor potential than the other three lines. However, because this lethal and rough eye phenotype is observed only for a single RNAi line, it cannot be excluded that unspecific side effects might play a role. The description of the true loss-of-function phenotype will therefore have to await the generation of a genuine null mutant. A special advantage in this context is that Brp presumably is present at all synapses. Therefore, it is possible not only to asses the functionality of transgenic constructs at the electrophysiological level but also to score their effects at the behavioral level by genetically controlled, spatially, and/or temporally selective expression or suppression (Wagh, 2007)

In mammals ELKS/CAST/ERC isoforms apparently have both neuronal and nonneuronal roles. Drosophila Brp seems to correspond to the neuronal CAST isoforms, whereas the nonneuronal functions might be specific to vertebrates. Vesides the protein described here, the only published molecule localized specifically at Drosophila presynaptic active zones is the Ca²⁺ channel encoded by the cacophony gene. This channel seems to be responsible for providing the calcium trigger for evoked neurotransmitter release. The findings indicate, however, that the molecular structure of the presynaptic active zone might be more conserved between vertebrate and insect synapses than thought previously because of the lack of Piccolo and Bassoon homologs in insects. In the future, studying active-zone formation and function in Drosophila will be a valuable addition to similar studies in vertebrates, especially considering the powerful genetic tools available for Drosophila (Wagh, 2007)

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

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) are entirely lost, Ca²⁺ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. BRP-like proteins seem to establish proximity between Ca²⁺ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006).

Synaptic communication is mediated by the fusion of neurotransmitter-filled vesicles with the presynaptic membrane at the active zone, a process triggered by Ca²⁺ influx through clusters of voltage-gated channels. The spacing between Ca²⁺ channels and vesicles at active zones is particularly thought to influence the dynamic properties of synaptic transmission (Kittel, 2006).

The larval Drosophila neuromuscular junction (NMJ) is frequently used as a model of glutamatergic synapses. The monoclonal antibody Nc82 specifically stains individual active zones by recognizing a coiled-coil domain protein of roughly 200 kD named Bruchpilot (Brp). Brp shows homologies to the mammalian active zone components CAST [cytoskeletal matrix associated with the active zone (CAZ)-associated structural protein], also called ERC (ELKS, Rab6-interacting protein 2, and CAST). Whereas confocal microscopy recognized diffraction limited spots, the subdiffraction resolution of stimulated emission depletion (STED) fluorescence microscopy revealed donut-shaped Brp structures at active zones. Viewed perpendicular to the plane of synapses, both single and multiple 'rings' were uncovered, of similar size to freeze-fracture-derived estimates of fly active zones. The donuts were up to 0.16 µm high, as judged by images taken parallel to the synaptic plane (Kittel, 2006).

Brp seems to demark individual active zones associated with clusters of Ca²⁺ channels. Transposon-mediated mutagenesis allowed isolation of a mutant chromosome (brp⁶⁹) in which nearly the entire open reading frame of Brp was deleted. brp mutants [brp⁶⁹/df(2R)BSC29] develop into mature larvae but do not form pupae. The Nc82 label is completely lost from the active zones of brp mutant NMJs but can be restored by re-expressing the brp cDNA in the brp mutant background with use of the neuron-specific driver lines ok6-GAL4. This also rescued larval lethality. Mutants had slightly smaller NMJs and somewhat fewer individual synapses. However, individual receptor fields, identified by the glutamate receptor subunit GluRIID, were enlarged in brp mutants. Thus, principal synapse formation occurred in brp mutants, with individual postsynaptic receptor fields increased in size but moderately decreased in number (Kittel, 2006).

In electron micrographs of brp mutant NMJs, synapses with pre- and postsynaptic membranes in close apposition were present at regular density, and consistent with the enlarged glutamate receptor fields postsynaptic densities appeared larger while otherwise normal. However, intermittent rufflings of the presynaptic membrane were noted, and brp mutants completely lacked presynaptic dense projections (T-bars). Occasionally, very little residual electron-dense material attached to the presynaptic active zone membrane was identified. After re-expressing the Brp protein in the mutant background, T-bar formation could be partially restored, although these structures were occasionally somewhat aberrant in shape. Thus, Brp assists in the ultrastructural assembly of the active zone and is essential for T-bar formation (Kittel, 2006).

In brp mutant larvae a drastic decrease was noted in evoked excitatory junctional current (eEJC) amplitudes by using two-electrode voltage clamp recordings of postsynaptic currents at low stimulation frequencies. This drop in current amplitude could be partially rescued through brp re-expression within the presynaptic motoneurons by using either elav-GAL4 or ok6-GAL4. In contrast, the amplitude of miniature excitatory junctional currents (mEJCs) in response to single, spontaneous vesicle fusion events was increased over control levels. This is consistent with the enlarged individual glutamate receptor fields of brp mutants and excludes a lack of postsynaptic sensitivity as the cause of the reduced eEJC amplitudes (Kittel, 2006).

It follows that the number of vesicles released per presynaptic action potential (AP) (quantal content) was severely compromised at brp mutant NMJs and could not be attributed solely to the moderate decrease in synapse number. The ultrastructural defects of brp mutant synapses may interfere with the proper targeting of vesicles to the active zone membrane and thereby impair exocytosis. The number of vesicles directly docked to active zone membranes was slightly decreased in brp mutants. However, the amplitude distribution and sustained frequency of mEJCs showed that brp mutant synapses did not appear to suffer from extrasynaptic release, as would be caused by a misalignment of vesicle fusion sites with postsynaptic receptors. Consistent with the appropriate deposition of exo- and endocytotic proteins, an apparently normal distribution of Syntaxin, Dap160, and Dynamin was observed at brp mutant synapses (Kittel, 2006).

The exact amplitude and time course of AP-triggered Ca²⁺ influx in the nerve terminal governs the amplitude and time course of vesicle . Nerve-evoked responses of brp mutants were delayed when compared with controls, whereas in contrast mEJC rise times were unchanged. Thus, evoked vesicle fusion events were less synchronized with the invasion of the presynaptic terminal by an AP. Spatiotemporal changes in Ca²⁺ influx have a profound effect on short-term plasticity. Whereas at 10 Hz controls exhibited substantial short-term depression of eEJC amplitudes, brp mutants showed strong initial facilitation before stabilizing at a slightly lower but frequency-dependent steady-state current. As judged by the initial facilitation at 10 Hz, neither a reduction in the number of releasable vesicles nor available release sites could fully account for the low quantal content of brp mutants at moderate stimulation frequencies. Furthermore, the altered short-term plasticity of brp mutant synapses suggested a change in the highly Ca²⁺-dependent vesicle release probability. Paired-pulse protocols were applied to the NMJ. Closely spaced stimuli lead to a buildup of residual Ca²⁺ in the vicinity of presynaptic Ca²⁺ channels, enhancing the probability of a vesicle within this local Ca²⁺ domain to undergo fusion after the next pulse. The absence of marked facilitation at control synapses could be explained by a depletion of release-ready vesicles. At brp mutant NMJs, however, the prominent facilitation at short interpulse intervals showed that the enhancement of release probability strongly outweighed the depletion of releasable vesicles. Thus, initial vesicle release probability was low, and release at brp synapses particularly benefited from the accumulation of intracellular Ca²⁺ (Kittel, 2006).

Vesicle fusion is highly sensitive to the spacing between Ca²⁺ channels and vesicles at release sites. It has been calculated that doubling this distance from 25 to 50 nm decreases the release probability threefold, and the larger this distance, the more effective the slow synthetic Ca²⁺ buffer EGTA should become in suppressing release. Indeed, after bath application of membrane permeable EGTA-AM, the reduction of evoked vesicle release was more pronounced at brp mutant than at control NMJs (Kittel, 2006).

The Ca²⁺-channel subunit Cacophony governs release at Drosophila NMJs. By using a fully functional, GFP (green fluorescent protein)-labeled variant (Cac^GFP), Ca²⁺ channels were visualized in vivo. Consistently, Ca²⁺ channel expression was severely reduced over the entire NMJ and at synapses lacking Brp (Kittel, 2006).

Thus, it is concluded that brp mutants suffer from a diminished vesicle release probability due to a decrease in the density of presynaptic Ca²⁺ channel clusters. It is conceivable that Brp tightly surrounds but is not part of the T-bar structure, contained within the unlabeled center of donuts. Brp may establish a matrix, required for both T-bar assembly as well as the appropriate localization of active zone components including Ca²⁺ channels, possibly by mediating their integration into a restricted number of active zone slots. Related mechanisms might underlie functional impairments of mammalian central synapses lacking active zone components (Altrock, 2003) and natural physiological differences between synapse types. Electron microscopy has identified regular arrangements at active zones of mammalian CNS synapses ('particle web') and frog NMJs ('ribs'), where these structures have also been proposed to organize Ca²⁺ channel clustering. At calyx of Held synapses (an axosomatic synapse in the auditory brainstem), both a fast and a slow component of exocytosis have been described. The fast component recovers slowly and is believed to owe its properties to vesicles attached to a matrix tightly associated with Ca²⁺ channels, whereas the slow component recovers faster and is thought to be important for sustaining vesicle release during tetanic stimulation. In agreement with this concept, the absence or impairment of such a matrix at brp synapses has a profound effect on vesicle release at low stimulation frequencies, but this effect subsides as the frequency increases. The sustained frequency of mEJCs at brp synapses could be explained if spontaneous fusion events arise from the slow release component or a pathway independent of evoked vesicle fusion (Kittel, 2006).

Synapses lacking Brp and T-bars exhibited a defective coupling of Ca²⁺ influx with vesicle fusion, whereas the vesicle availability did not appear rate-limiting under low frequency stimulation. The activity-induced addition of presynaptic dense bodies has been proposed to elevate vesicle release probability. This work supports this hypothesis and suggests an involvement of Brp or related factors in synaptic plasticity by promoting Ca²⁺ channel clustering at the active zone membrane (Kittel, 2006).

Search PubMed for articles about Drosophila Bruchpilot

Altrock, W. D., et al. (2003). Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon. Neuron 37(5): 787-800. Medline abstract: 12628169

Deguchi-Tawarada, M., et al. (2004). CAST2: identification and characterization of a protein structurally related to the presynaptic cytomatrix protein CAST. Genes Cells 9(1): 15-23. Medline abstract: 14723704

Deken, S. L., et al. (2005). Redundant localization mechanisms of RIM and ELKS in Caenorhabditis elegans. J. Neurosci. 25(25): 5975-83. Medline abstract: 15976086

tom Dieck, S., et al. (1998). Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J. Cell Biol. 142(2): 499-509. Medline abstract: 9679147

Fenster, S. D. et al. (2000). Piccolo, a presynaptic zinc finger protein structurally related to bassoon. Neuron 25: 203-214. Medline abstract: 10707984

Kittel, R. J., et al. (2006). Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312(5776): 1051-4. Medline abstract: 16614170

Ko, J., Na, M., Kim, S., Lee, J. R. and Kim, E. (2003). Interaction of the ERC family of RIM-binding proteins with the liprin-alpha family of multidomain proteins. J. Biol. Chem. 278: 42377-42385. Medline abstract: 12923177

Ohara-Imaizumi, M., et al. (2005). ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic {beta} cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy. Mol. Biol. Cell 16: 3289-3300. Medline abstract: 15888548

Ohtsuka, T. et al. (2002). Cast: a novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and munc13-1. J. Cell Biol. 158(3): 577-90. Medline abstract: 12163476

Shapira, M., et al. (2003). Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles. Neuron 38: 237-252. Medline abstract: 12718858

Takao-Rikitsu, E., et al. (2004). Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release. J. Cell Biol. 164(2): 301-11. Medline abstract: 14734538

Wagh, D. A., (2006). Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron 49(6): 833-44. Medline abstract: 16543132

Wang, Y., et al. (2002). A family of RIM-binding proteins regulated by alternative splicing: implications for the genesis of synaptic active zones. Proc. Natl. Acad. Sci. 99: 14464-14469. Medline abstract: 12391317

Wang, Y., et al. (1997). Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature 388: 593-598. Medline abstract: 9252191

date revised: 10 February 2008

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