A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency
Abstract
Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between Src homology 3 and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of SV organization and spontaneous release.
Data availability
Raw measures and intermediate data processing of images and electrophysiology traces are submited in source files appended to this submission.Source images and electrophysiology traces reported in this study are fully available upon request to the corresponding author (Etienne Herzog - https://orcid.org/0000-0002-0058-6959).
Article and author information
Author details
Funding
Agence Nationale de la Recherche (ANR-12-JSV4-0005-01 VGLUT-IQ)
- Etienne Herzog
Agence Nationale de la Recherche (ANR-10-LABX-43 BRAIN)
- Yann Humeau
- Etienne Herzog
Agence Nationale de la Recherche (ANR-10-IDEX-03-02 PEPS SV-PIT)
- Etienne Herzog
Agence Nationale de la Recherche (PSYVGLUT 09-MNPS-033)
- Salah El Mestikawy
Agence Nationale de la Recherche (ANR-10-INBS-04)
- Fabrice P Cordelières
European Commission (Erasmus-Mundus European Neuroscience Campus Program)
- Xiao Min Zhang
- Etienne Herzog
Fondation pour la Recherche Médicale (FDT20120925288)
- Kätlin Silm
Fondation pour la Recherche Médicale (ING20150532192)
- Maria Florencia Angelo
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: The experimental design and all procedures were in accordance with the European guide for the care and use of laboratory animals, approved by the ethics committee of Bordeaux Universities (CE50), and registered with the french ministry for research under the APAFIS n{degree sign}1692. Every effort was made to minimize the number of animals used and their suffering.
Reviewing Editor
- Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Germany
Version history
- Received: July 22, 2019
- Accepted: October 27, 2019
- Accepted Manuscript published: October 30, 2019 (version 1)
- Version of Record published: November 18, 2019 (version 2)
Copyright
© 2019, Zhang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,245
- Page views
-
- 383
- Downloads
-
- 9
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
- Microbiology and Infectious Disease
Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the P. falciparum exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria causing parasite, PfEMP1. We generated independent TurboID fusions of 2 proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.
-
- Cell Biology
The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.