Robert J. Kittel

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) 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.

Maturation of active zone assembly by Drosophila Bruchpilot

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila.

Three ionotropic glutamate receptor subunits, designated GluRIIA, GluRIIB, and GluRIII, have been identified at neuromuscular junctions of Drosophila. Whereas GluRIIA and GluRIIB are redundant for viability, it was shown recently that GluRIII is essential for both the synaptic localization of GluRIIA and GluRIIB and the viability of Drosophila. Here we identify a fourth and a fifth subunit expressed in the neuromuscular system, which we name GluRIID and GluRIIE. Both new subunits we show to be necessary for survival. Moreover, both GluRIID and GluRIIE are required for the synaptic expression of all other glutamate receptor subunits. All five subunits are interdependent for receptor function, synaptic receptor expression, and viability. This indicates that synaptic glutamate receptors incorporate the GluRIII, GluRIID, and GluRIIE subunit together with either GluRIIA or GluRIIB at the Drosophila neuromuscular junction. At this widely used model synapse, the assembly of four different subunits to form an individual glutamate receptor channel may thus be obligatory. This study opens the way for a further characterization of in vivo glutamate receptor assembly and trafficking using the efficient genetics of Drosophila.

Glutamate receptor dynamics organizing synapse formation in vivo

Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Here, we imaged postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIAGFP) at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, formed independently of existing synapses and grew continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments showed that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially entered growing PSDs. After entering PSDs, receptors were largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchanged faster and with no apparent preference for growing synapses. We show here that new glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs.

Activity-dependent site-specific changes of glutamate receptor composition in vivo

The subunit composition of postsynaptic non–NMDA-type glutamate receptors (GluRs) determines the function and trafficking of the receptor. Changes in GluR composition have been implicated in the homeostasis of neuronal excitability and synaptic plasticity underlying learning. Here, we imaged GluRs in vivo during the formation of new postsynaptic densities (PSDs) at Drosophila neuromuscular junctions coexpressing GluRIIA and GluRIIB subunits. GluR composition was independently regulated at directly neighboring PSDs on a submicron scale. Immature PSDs typically had large amounts of GluRIIA and small amounts of GluRIIB. During subsequent PSD maturation, however, the GluRIIA/GluRIIB composition changed and became more balanced. Reducing presynaptic glutamate release increased GluRIIA, but decreased GluRIIB incorporation. Moreover, the maturation of GluR composition correlated in a site-specific manner with the level of Bruchpilot, an active zone protein that is essential for mature glutamate release. Thus, we show that an activity-dependent, site-specific control of GluR composition can contribute to match pre- and postsynaptic assembly.

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

Naked dense bodies provoke depression.

At presynaptic active zones (AZs), the frequently observed tethering of synaptic vesicles to an electron-dense cytomatrix represents a process of largely unknown functional significance. Here, we identified a hypomorphic allele, brpnude, lacking merely the last 1% of the C-terminal amino acids (17 of 1740) of the active zone protein Bruchpilot. In brpnude, electron-dense bodies were properly shaped, though entirely bare of synaptic vesicles. While basal glutamate release was unchanged, paired-pulse and sustained stimulation provoked depression. Furthermore, rapid recovery following sustained release was slowed. Our results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression.

Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications

Significance Controlling neuronal activity in live tissue is a long sought-after goal in the neurosciences. Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin that can be genetically expressed to depolarize neurons with light. Thereby, this “optogenetic tool” delivers cellular specificity and elegant options for studying the neuronal basis of behavior in intact organisms. Unfortunately, low-light transmission through pigmented tissue greatly complicates light delivery to target cells and curtails experiments in freely moving animals. This study introduces a ChR mutant, ChR2-XXL, that gives rise to the largest photocurrents of all ChRs published so far and increases light sensitivity more than 10,000-fold over wild-type ChR2 in Drosophila larvae. As a result, behavioral photostimulation is evoked in freely moving flies using diffuse, ambient light. Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. Here, we describe the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. We validated the benefits of the variant in intact flies by eliciting simple and complex behaviors. We demonstrate efficient and prolonged photostimulation of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.

The Adhesion GPCR Latrophilin/CIRL Shapes Mechanosensation

G-protein-coupled receptors (GPCRs) are typically regarded as chemosensors that control cellular states in response to soluble extracellular cues. However, the modality of stimuli recognized through adhesion GPCR (aGPCR), the second largest class of the GPCR superfamily, is unresolved. Our study characterizes the Drosophila aGPCR Latrophilin/dCirl, a prototype member of this enigmatic receptor class. We show that dCirl shapes the perception of tactile, proprioceptive, and auditory stimuli through chordotonal neurons, the principal mechanosensors of Drosophila. dCirl sensitizes these neurons for the detection of mechanical stimulation by amplifying their input-output function. Our results indicate that aGPCR may generally process and modulate the perception of mechanical signals, linking these important stimuli to the sensory canon of the GPCR superfamily.

The Ig cell adhesion molecule Basigin controls compartmentalization and vesicle release at Drosophila melanogaster synapses

Synapses can undergo rapid changes in size as well as in their vesicle release function during both plasticity processes and development. This fundamental property of neuronal cells requires the coordinated rearrangement of synaptic membranes and their associated cytoskeleton, yet remarkably little is known of how this coupling is achieved. In a GFP exon-trap screen, we identified Drosophila melanogaster Basigin (Bsg) as an immunoglobulin domain-containing transmembrane protein accumulating at periactive zones of neuromuscular junctions. Bsg is required pre- and postsynaptically to restrict synaptic bouton size, its juxtamembrane cytoplasmic residues being important for that function. Bsg controls different aspects of synaptic structure, including distribution of synaptic vesicles and organization of the presynaptic cortical actin cytoskeleton. Strikingly, bsg function is also required specifically within the presynaptic terminal to inhibit nonsynchronized evoked vesicle release. We thus propose that Bsg is part of a transsynaptic complex regulating synaptic compartmentalization and strength, and coordinating plasma membrane and cortical organization.