Conference Agenda

Here we discover a mechanism for presynaptic pre-assembly that positions cytosolic active zone proteins temporally upstream of the cell-adhesion molecules they bind do. Based on molecular dynamics simulations followed by in vivo structure/function analyses, we propose that the cytosolic active zone scaffold SYD-1 interacts with membrane phospholipids to promote active zone protein clustering. SYD-1 subsequently recruits the cell adhesion molecule neurexin to stabilize those clusters. We find that PIP2-interacting residues in SYD-1’s C2 and PDZ domains are redundantly necessary for proper active zone assembly. Moreover, depleting PIP2 from the membrane by temporal degradation of the PIP2 enzyme ppk-1 leads to a loss of SYD-1 and degradation of the presynaptic density as assessed by EM. Finally, we propose that the short yet evolutionarily conserved gamma isoform of neurexin represents a minimal neurexin sequence that can stabilize previously assembled presynaptic clusters, potentially a core function of this critical protein.

Complex protein architectures often require specific assembly-promoting proteins. At presynaptic active zones (AZs), scaffold proteins play a crucial role in orchestrating the release of synaptic vesicles (SVs) by establishing a detailed nanoscale architecture that connects voltage-gated Ca2+ channels with the SV release machinery. Although recent theories suggest that liquid condensate formation may drive developmental AZ assembly, the exact mechanisms governing this process remain largely unknown.

We identified "Blobby" as a novel AZ scaffold-localizing and assembly promoting protein. Blobby is characterized by intrinsically unstructured and coiled-coil domains, and resides in the AZ scaffold through extensive contacts with the ELKS/BRP scaffold protein. Loss of Blobby on the one hand resulted in an ectopic accumulation of AZ scaffold proteins, forming ectopic "blobs" within synaptic boutons. Moreover, the molecular nanoscale architecture was disrupted entailing a decreased AZ density of voltage-gated Ca2+ channels, resulting in inefficient SV release as observed through electrophysiological measurements. Developmental reduction of Blobby within olfactory sensory neuron drastically impaired smell function.

Our findings suggest that the unique constraints of the AZ assembly process have driven the evolution of a specific assembly factor. This factor likely promotes the liquid-like characteristics of scaffold proteins, enabling assembly to take place properly.

The liberation of chemical neurotransmitters from presynaptic release sites is fundamental to neural communication and its plastic adaptation enables homeostasis. The signaling lipid diacylglycerol (DAG) potentiates transmitter release by binding the C1 domain of Unc13 proteins, major release site constituents and essentials factors to localize and fuse transmitter-containing vesicles with the presynaptic plasma membrane. We used the Drosophila melanogaster neuromuscular junction as a model synapse to investigate signaling pathways converging on DAG/Unc13. We found that presynaptic homeostatic potentiation (PHP) – induced by the pharmacological inhibition of postsynaptic neurotransmitter receptors – coincided with the elevation of local DAG levels and the subsynaptic enrichment of Unc13. Similar effects were seen upon pharmacological treatment with phorbol-esters which mimic DAG and occlude PHP. Because the transsynaptic signals inducing PHP are poorly understood we further investigated upstream processes. We identified monoamines as putative targets whose acute application similarly affected local DAG- and Unc13 levels while potentiating neurotransmitter release. Moreover, presynaptic manipulation of their G-protein coupled receptors occluded PHP. Our collective data uncovers an innate signaling pathway that acutely modulates neurotransmitter release and may control homeostasis via DAG-induced subsynaptic Unc13 concentration.