# Synaptic transmission: Munc13 assembles onto PI(4,5)P2-rich domains into trimers that cooperate to capture vesicles

## TL;DR

The paper shows how Munc13 proteins form trimers on specific membrane regions to capture synaptic vesicles, a key step in preparing them for rapid release.

## Contribution

The study reveals that Munc13 trimers assemble on PI(4,5)P2-rich domains and capture vesicles, initiating synaptic vesicle priming.

## Key findings

- Munc13-1 cycles between upright trimeric and lateral hexameric conformations.
- Munc13 trimers capture vesicles via C2C domains on PI(4,5)P2-rich domains.
- Trimer interface mutations disrupt vesicle priming and trimer formation.

## Abstract

Munc13-1 primes synaptic vesicles for rapid release at the presynaptic membrane. Reconstitution on supported bilayers and Cryo-EM reveal that Munc13-1 cycles between upright (trimeric) and lateral (hexameric) conformations. Munc13 binds PI(4,5)P2, a key lipid for vesicle docking and fusion. We show that Munc13 assembles into trimer clusters on PI(4,5)P2 domains. These clusters efficiently capture vesicles via C2C domains. Trimer interface mutations disrupt vesicle priming. These observations prompted us to propose that priming begins with vesicle capture by Munc13 trimers, which transition into lateral hexamers upon vesicle binding, a process promoted by diacylglycerol production from PI(4,5)P2 hydrolysis.

Munc13-1 is a key protein involved in priming synaptic vesicles for rapid release at the presynaptic plasma membrane. It was previously revealed that Munc13-1 cycles between at least two alternate conformations, an upright (open) molecular conformation organized as a trimer and a lateral (closed) conformation organized as a hexagon. Munc13 binds PI(4,5)P2, a plasma membrane phospholipid essential for vesicle docking and fusion. We report that Munc13 is recruited to PI(4,5)P2 domains induced by a Syntaxin-1A juxta-membrane peptide in supported bilayers. Statistical analysis of the copy numbers of Munc13 within the domains suggests that Munc13 exists in clusters of three molecules, i.e., it assembles into trimers. The trimeric clusters disappear with engineered interface mutations disrupting the upright trimers in cryo-EM as well as reconstituted vesicle priming suggesting the trimers observed on bilayers are identical to the upright trimer structures. These upright trimers can also be identified by cryo-electron tomography on vesicles containing PI(4,5)P2 and Syntaxin-1A. Clusters of 3 or more Munc13 trimers forming on PI(4,5)P2 domains efficiently capture phosphatidylserine-containing small unilamellar vesicles via their C2C domains, as shown by the effect of mutations that disrupt synaptic vesicle binding in synapses. We propose a two-step model for vesicle priming: i) synaptic vesicles are captured by clusters of upright trimers of Munc13 that self-assemble within PI(4,5)P2 enriched domains; ii) these trimers transition into lateral hexamers when vesicles have bound and the transition would be promoted when the closed conformation of Munc13 is stabilized by diacylglycerol binding, resulting from Ca2+-dependent hydrolysis of PI(4,5)P2.