Combined targeted Omic and Functional Assays Identify Phospholipases A₂ that Regulate Docking/Priming in Calcium-Triggered Exocytosis.

Deepti Dabral,Jens R Coorssen

doi:10.3390/cells8040303

Deepti Dabral, Jens R Coorssen

Open Access

https://doi.org/10.3390/cells8040303

Copy DOI

Abstract

The fundamental molecular mechanism underlying the membrane merger steps of regulated exocytosis is highly conserved across cell types. Although involvement of Phospholipase A2 (PLA2) in regulated exocytosis has long been suggested, its function or that of its metabolites—a lyso-phospholipid and a free fatty acid—remain somewhat speculative. Here, using a combined bioinformatics and top-down discovery proteomics approach, coupled with lipidomic analyses, PLA2 were found to be associated with release-ready cortical secretory vesicles (CV) that possess the minimal molecular machinery for docking, Ca2+ sensing and membrane fusion. Tightly coupling the molecular analyses with well-established quantitative fusion assays, we show for the first time that inhibition of a CV surface calcium independent intracellular PLA2 and a luminal secretory PLA2 significantly reduce docking/priming in the late steps of regulated exocytosis, indicating key regulatory roles in the critical step(s) preceding membrane merger.

Highlights

Fusion is a ubiquitous fundamental cellular process enabling merger of biological membranes; this includes the defining release step of exocytosis following merger of secretory vesicles with the plasma membrane (PM)
As CSC consist of cortical secretory vesicles (CV) endogenously docked on the PM and undergo Ca2+ -triggered exocytosis in vitro, the potential presence and influence(s) of Phospholipase A2 (PLA2) activities was first assessed using CSC and selective
As the lipid detection approach used is somewhat selective for unsaturated lipid species [56], in order to ensure a full assessment of potential PLA2 activities, CSC were labelled with NBD-PC; as previously documented, such exogenous substrates are readily incorporated into CSC and CV membranes [49]

Summary

Introduction

Fusion is a ubiquitous fundamental cellular process enabling merger of biological membranes; this includes the defining release step of exocytosis following merger of secretory vesicles with the plasma membrane (PM). In a cell free system, fusion of secretory vesicles with target membranes increased in the presence of PLA2 suggesting that the enzyme or its metabolites affected membrane merger or the steps immediately preceding it [5,6,9]. Studies identified endogenous PLA2 activity in purified synaptic vesicles (SV) [5,6,19,20]. While some proteomic analyses have failed to identify SV associated PLA2 [21,22], suggesting very low abundance and/or activity, at least one study identified iPLA2γ in synaptosomes [23]. When synaptosomes were fractionated into free and docked SV pools prior to proteomic analysis, a patatin-like phospholipase domain containing 8 (Gene ID 157819923; known as iPLA2 γ) was found to be more abundant in the docked SV pool [24]

Methods

Results

Discussion

Conclusion