# Dissecting the assembly of neurotransmitter release sites

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2024 · $666,601

## Abstract

Project Summary
Neurotransmitter release at synapses critically depends on the precise assembly of the secretory machine.
Within a presynaptic nerve terminal, synaptic vesicles fuse at the active zone, a protein scaffold that forms
release sites apposed to postsynaptic receptors. This protein complex contains RIM, ELKS, Munc13, RIM-BP,
Liprin-α and Bassoon/Piccolo as central components. Recent work provides ground for new models of
how these proteins assemble into functional release sites. First, the active zone is remarkably resilient and
ablation of individual genes has at most modest effects on its assembly. Instead, combined deletions of RIM,
ELKS, or RIM-BP strongly disrupt active zone assembly, establishing scaffolding redundancy. Second, current
studies have led to a working model of assembly through liquid-liquid phase separation, with robust contributions
of multivalent low-affinity interactions to assembly. Regardless of exact mechanisms, an overarching model
that arises from these and other studies is that the active zone is a dynamic protein network that is held together
by redundant, low-affinity protein binding. This is different from conventional models in which master organizers
mediate assembly through rigid complexes with well-defined stoichiometries.
Here, we build on our and other’s recent progress with the goal to identify what mechanisms mediate assembly
of the initial active zone scaffold, and how opposing surfaces of these active zone protein networks interact with
the target plasma membrane and with the synaptic vesicle cluster, respectively. We will use a three-pronged
approach to answer these questions. Aim 1 defines roles and mechanisms of RIM in active zone assembly.
We build on our finding that RIM drives recruitment of interacting proteins after removing scaffolding redundancy
through RIM+ELKS knockout. We test the model that RIM organizes active zones through a two-step process
that mechanistically separates RIM-targeting to active zones from RIM’s activity in recruiting other active zone
proteins. Aim 2 dissects how synaptic vesicle clusters and active zones, two presynaptic sub-
compartments, interact with one another. We rely on a new, “in-synapse” reconstitution approach and test
parallel models to define which binding activities are sufficient to mediate vesicle docking. Aim 3 determines
active zone anchoring mechanisms at the target plasma membrane. This aim makes use of our unique
collection of conditional and compound mutants to solve the long-standing question of how the active zone
scaffolds are physically attached to the right place at the target membrane. We use state-of-the-art methodology
including conditional gene knockout, stimulated emission depletion (STED) microscopy, fluorescence recovery
after photobleaching (FRAP), high pressure freezing- and correlative light-electron microscopy (CLEM), and
electrophysiology to answer these questions.
Our work will establish mechanistic models on how the targ...

## Key facts

- **NIH application ID:** 10867447
- **Project number:** 5R01MH113349-08
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Pascal Simon Kaeser
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $666,601
- **Award type:** 5
- **Project period:** 2017-03-13 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10867447

## Citation

> US National Institutes of Health, RePORTER application 10867447, Dissecting the assembly of neurotransmitter release sites (5R01MH113349-08). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10867447. Licensed CC0.

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