Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
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.
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Author details
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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