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 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.

Phosphatidylinositol 4,5-bisphosphate drives Ca2+-independent membrane penetration by the tandem C2 domain proteins synaptotagmin-1 and Doc2β

“…6, C-F). In both lipid and content-mixing assays, elevation of [PIP 2 ] enhanced fusion of v-and t-SNARE vesicles prior to the addition of Ca 2ϩ , consistent with the capacity of PIP 2 to drive Ca 2ϩ -independent activation of syt1 (36). Increasing PIP 2 likewise enhanced membrane fusion after the addition of Ca 2ϩ , consistent with published findings using in vitro fusion assays (43) and PIP 2 uncaging in chromaffin cells (36).…”

“…Furthermore, because [PIP 2 ] can reach Ͼ5 mol % at release sites (29) and the plasma membrane contains ϳ10 -15 mol % PS (46), it is likely that the docked and/or primed configurations of syt1 and Doc2␤ involve some degree of insertion into the plasma membrane. However, although the PIP 2 uncaging technique of Walter et al (36) provides useful mechanistic insights, we note that we are not aware of studies showing such rapid up-regulation of PIP 2 at exocytotic sites in endogenous systems.…”

“…Thus, a rapid increase in [PIP 2 ], e.g. via optical uncaging of caged PIP 2 as performed by Walter et al (36), might trigger syt1-dependent release via two nonexclusive mechanisms: recruitment of additional Ca 2ϩ sensors that penetrate the plasma membrane or driving deeper penetration by Ca 2ϩ sensors that are already present at release sites ( Figs. 6 and 7).…”

“…According to current models of tandem C2 domain protein function, Ca 2ϩ is required for membrane penetration and thus the triggering of exocytosis. However, recent findings have challenged this model by demonstrating that rapid uncaging of PIP 2 can trigger syt1-dependent exocytosis without a measurable change in [Ca 2ϩ ] i (36). Given the apparent requirement of Ca 2ϩ for membrane penetration by syt1, how could a stepwise increase in available PIP 2 evoke exocytosis?…”

Regulation of synaptic release‐site Ca2+ channel coupling as a mechanism to control release probability and short‐term plasticity

“…different Munc13 isoforms shifted synapses from highto-low release probability and from STD to STF [54]. (M)Unc13 proteins are well-known targets of second messenger cascades such as DAG or phosphatidylinositol bisphosphate (PIP 2 ) [31,32,[125][126][127][128][129][130]. Therefore, the isoform-specific differences of pV r and STP might partly be explained by differing action of these second messengers on specific isoforms.…”