Protein composition of axonal dopamine release sites in the striatum
Abstract
Dopamine is an important modulator of cognition and movement. We recently found that evoked dopamine secretion is fast and relies on active zone-like release sites. Here, we used in vivo biotin-identification (iBioID) proximity proteomics in mouse striatum to assess which proteins are present at these sites. Using three release site baits, we identified proteins that are enriched over the general dopamine axonal protein content, and they fell into categories including active zone, Ca2+ regulatory and synaptic vesicle proteins. We also detected many proteins not previously associated with vesicular exocytosis. Knockout of the presynaptic organizer protein RIM strongly decreased the hit number obtained with iBioID, while Synaptotagmin-1 knockout did not. α-Synuclein, a protein linked to Parkinson's disease, was enriched at release sites, and its enrichment was lost in both tested mutants. We conclude that RIM organizes scaffolded dopamine release sites and provide a proteomic assessment of the composition of these sites.
Data availability
All data generated or analyzed in this study are included in the figures and the source data tables. Source data files are provided for Figs. 1 to 3, Fig. 1 - figure supplement 2, Fig. 2 - figure supplements 1 and 2, and Fig. 3 - figure supplements 1 and 2.
Article and author information
Author details
Funding
National Institute of Neurological Disorders and Stroke (R01NS103484)
- Pascal S Kaeser
National Institute of Neurological Disorders and Stroke (R01NS083898)
- Pascal S Kaeser
National Institute of Neurological Disorders and Stroke (F31NS105159)
- Lauren Kershberg
Harvard Medical School (Lefler Foundation)
- Pascal S Kaeser
Harvard Medical School (Brooks Fellowship)
- Aditi Banerjee
Harvard Medical School (Quan Fellowship)
- Lauren Kershberg
Harvard Medical School (Dean's Innovation Grant)
- Pascal S Kaeser
Harvard Medical School
- Pascal S Kaeser
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were performed according to institutional guidelines of Harvard University, and were in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by the Harvard University Animal Care and Use Committee (protocol number IS00000049).
Copyright
© 2022, Kershberg 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
-
- 2,017
- views
-
- 381
- downloads
-
- 9
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
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
Cell survival in metazoans depends on cell attachment to the extracellular matrix (ECM) or to neighboring cells. Loss of such attachment triggers a type of programmed cell death known as anoikis, the acquisition of resistance to which is a key step in cancer development. The mechanisms underlying anoikis resistance remain unclear, however. The intracellular F-actin cytoskeleton plays a key role in sensing the loss of cell–ECM attachment, but how its disruption affects cell fate during such stress is not well understood. Here, we reveal a cell survival strategy characterized by the formation of a giant unilocular vacuole (GUVac) in the cytoplasm of the cells whose actin cytoskeleton is disrupted during loss of matrix attachment. Time-lapse imaging and electron microscopy showed that large vacuoles with a diameter of >500 nm accumulated early after inhibition of actin polymerization in cells in suspension culture, and that these vacuoles subsequently coalesced to form a GUVac. GUVac formation was found to result from a variation of a macropinocytosis-like process, characterized by the presence of inwardly curved membrane invaginations. This phenomenon relies on both F-actin depolymerization and the recruitment of septin proteins for micron-sized plasma membrane invagination. The vacuole fusion step during GUVac formation requires PI(3)P produced by VPS34 and PI3K-C2α on the surface of vacuoles. Furthermore, its induction after loss of matrix attachment conferred anoikis resistance. Our results thus show that the formation of a previously unrecognized organelle promotes cell survival in the face of altered actin and matrix environments.
-
- Cell Biology
- Plant Biology
Plants distribute many nutrients to chloroplasts during leaf development and maturation. When leaves senesce or experience sugar starvation, the autophagy machinery degrades chloroplast proteins to facilitate efficient nutrient reuse. Here, we report on the intracellular dynamics of an autophagy pathway responsible for piecemeal degradation of chloroplast components. Through live-cell monitoring of chloroplast morphology, we observed the formation of chloroplast budding structures in sugar-starved leaves. These buds were then released and incorporated into the vacuolar lumen as an autophagic cargo termed a Rubisco-containing body. The budding structures did not accumulate in mutants of core autophagy machinery, suggesting that autophagosome creation is required for forming chloroplast buds. Simultaneous tracking of chloroplast morphology and autophagosome development revealed that the isolation membranes of autophagosomes interact closely with part of the chloroplast surface before forming chloroplast buds. Chloroplasts then protrude at the site associated with the isolation membranes, which divide synchronously with autophagosome maturation. This autophagy-related division does not require DYNAMIN-RELATED PROTEIN 5B, which constitutes the division ring for chloroplast proliferation in growing leaves. An unidentified division machinery may thus fragment chloroplasts for degradation in coordination with the development of the chloroplast-associated isolation membrane.