Blobby: A Novel Active Zone Scaffold Protein Regulating Synaptic Organization, Sensory Processing, and Sleep in Drosophila

Abstract

Sensory processing and sleep are essential behaviors critical for survival from animal models to humans. The presynaptic active zone (AZ) plays a central role in both processes, yet how molecular diversity at the AZ contributes to functional variability and behavioral specificity remains unclear. In the Drosophila brain, distinct nanoscopic coupling distances between Ca²⁺ channels and the priming factors Unc13A and Unc13B underlie diverse modes of short-term plasticity (STP), which are essential for sensory decoding. Unc13A drives fast phasic transmission, while Unc13B mediates adaptive dynamics such as sensory prediction error coding. However, how this balance is maintained—and spatially controlled—at individual synapses had remained unresolved. Our recent study identified a previously unrecognized AZ protein, Blobby, encoded by the Drosophila gene CG42795. Blobby is a BRP-interacting factor homologous to human TBC1D30, a large Rab GTPase-activating protein (Rab-GAP). At the developing third-instar larval neuromuscular junction (NMJ), Blobby localizes adjacent to BRP scaffold and arrives at the newly forming AZ later than BRP. Loss of Blobby in blobbyNull mutant leads to ectopic accumulation of large BRP aggregates and a reduction in evoked synaptic transmission, which is likely due to a decrease in both release probability and number of release sites.Here, in my thesis, by combining biochemistry, genetics, diverse behavioral approaches, living imaging and super resolution microscopy, I provide a comprehensive characterization of Blobby in regulating nanoscopic localization of BRP and Unc13 proteins in the adult brain, and in its critical function in odor sensation and sleep regulation. The blobbyNull mutant is surprisingly homozygous adult-viable with severely reduced Mendelian ratio and the adult flies appeared healthy at young age. However, they suffered from a strong decrease of lifespan and hypersensitivity to oxidative stress. Furthermore, the blobbyNull mutant flies exhibit a marked loss of olfactory-driven behaviors in response to both appetitive and aversive odors. By mapping through the Drosophila olfactory circuit through a targeted knockout strategy, I show that Blobby is required in the excitatory projection neurons (PN) and the mushroom body (MB) Kenyon cells (KC), but not in the olfactory receptor neurons. Importantly, at the presynapse of PN in Calyx, loss of blobby either only in PN or throughout the whole brain causes nanoscopic redistribution of Unc13B toward AZ center and increases its overall levels, without affecting the localization of the tight-coupling Unc13A. Two-photon calciumimaging at PN::KC synapses reveal that PN-specific blobby knockout resulted in a drastic reduction in odor-evoked postsynaptic calcium response. Strikingly, knocking down Unc13B in PN was sufficient to rescue the olfactory sensitivity to both blobbyNull mutant and PN-specific blobby knockout flies. These data suggest Blobby is a bona fide regulator for sensory processing, most likely through the fine-tuning of synaptic transmission in the olfactory circuit via a redistribution of Unc13B. As sleep and sensory processing are closely coupled, I further show that loss of Blobby had very limited effects on the sleep patterns. In contrast, Unc13B mutant flies exhibited a strong increase in both daytime and nighttime sleep, with shorter sleep latency and more consolidated sleep. Remarkably, the sleep phenotypes of Unc13B mutant flies were fully rescued by loss of Blobby, indicating that the interaction between Blobby and Unc13B extends from the coding of sensory information to the maintenance of sleep. In conclusion, Blobby is an essential AZ protein in tuning synaptic transmission via nanoscopic localization and balancing of Unc13 isoforms. The interactive regulatory relationship between Blobby and Unc13B in sleep and odor sensation highlights the role of synapse and synaptic plasticity in gating, filtering and integrating behavioral relevant information. By identifying novel regulators such as Blobby in regulating core AZ scaffolds and release factors, we will provide better molecular understandings in bridging nanometer-scale vesicle priming to organism-scale control of sleep and sensory behavior.