Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
Paulo S Pinheiro
Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
André Nadler
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
Iwona Ziomkiewicz
Neurosecretion group, Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Discovery Sciences, AstraZeneca, Cambridge, United Kingdom
Martin Kruse
Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, United States
Gregor Reither
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
Jens Rettig
Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, Homburg, Germany
Martin Lehmann
Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
Volker Haucke
Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
Bertil Hille
Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, United States
Carsten Schultz
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its 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 may bypass 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.
