Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
- University of Copenhagen, Denmark
- European Molecular Biology Laboratory (EMBL), Germany
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Germany
- University of Washington School of Medicine, United States
- Saarland University, Germany
Abstract
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging bypasses CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.
Article and author information
Author details
Jakob Balslev Sorensen
Funding
European Union 7th Framework programme (HEALTH-F2-2009-242167)
- Jakob Balslev Sorensen
The independent Research Fond Denmark
- Jakob Balslev Sorensen
The Novo Nordisk Foundation
- Jakob Balslev Sorensen
The Lundbeck Foundation
- Paulo S Pinheiro
- Jakob Balslev Sorensen
European Molecular Biology Laboratory
- Carsten Schultz
Deutsche Forschungsgemeinschaft (Transregio83)
- Alexander M Walter
- Carsten Schultz
Deutsche Forschungsgemeinschaft (Emmy Noether Programme)
- Alexander M Walter
National Institutes of Health (R37NS008174)
- Bertil Hille
Alexander von Humboldt-Stiftung
- Martin Kruse
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Permission to keep and breed knockout mice for this study was obtained from The Danish Animal Experiments Inspectorate (2006/562−43, 2012−15−2935−00001). The animals were maintained in an AAALAC-accredited stable in accordance with institutional guidelines as overseen by the Institutional Animal Care and Use Committee (IACUC).
Copyright
© 2017, Walter 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.
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Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
eLife 6:e30203.
https://doi.org/10.7554/eLife.30203
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