Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis.

Alexander M Walter,Iwona Ziomkiewicz,Anthony W Mccarthy,Rainer Müller,Jens Rettig,Paulo S Pinheiro,André Nadler,Gregor Reither,Carsten Schultz,Bertil Hille,Martin Kruse,Volker Haucke,Jakob Balslev Sørensen,Martin Lehmann,Keimpe Db Wierda,Bassam Tawfik

doi:10.7554/elife.30203

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 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.

Highlights

Signal transduction between cells depends on the regulated exocytosis of vesicles to liberate neurotransmitters, neuropeptides and hormones
The assembly of the thermodynamically stable neuronal SNARE complex formed by the vesicular SNARE protein VAMP2/synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1 and SNAP-25 provides energy needed for vesicle fusion (Jahn and Fasshauer, 2012)
Capacitance measurements in chromaffin cells showed that following PI(4,5)P2 uncaging exocytosis is enhanced, and the Readily Releasable Pool (RRP) increased, which we demonstrate is specific to PI(4,5) P2 by contrasting the effects of DAG-uncaging

Summary

Introduction

Signal transduction between cells depends on the regulated exocytosis of vesicles to liberate neurotransmitters, neuropeptides and hormones. Neuronal exocytosis is driven by an evolutionarily conserved machinery that targets vesicles to the plasma membrane, attaches them to the membrane (sometimes referred to as vesicle ‘docking’), and molecularly matures (‘primes’) them to a fusioncompetent state. Primed vesicles reside in the so-called Readily Releasable Pool (RRP) whose fusion triggering leads to transmitter/hormone release. The assembly of the thermodynamically stable neuronal SNARE complex formed by the vesicular SNARE protein VAMP2/synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1 and SNAP-25 provides energy needed for vesicle fusion (Jahn and Fasshauer, 2012). SNARE proteins are already required for membrane attachment and priming (de Wit et al, 2009; Imig et al, 2014; Walter et al, 2010), and during fusion they continue to influence fusion pore properties through their transmembrane domains

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