Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis

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.