Oral Presentation 2014 International Biophysics Congress

A role for PLD in the late steps of exocytosis (#166)

T. P. Rogasevskaia 1 , Jens Coorssen 2
  1. Biology, Mount Royal University, Calgary, AB, Canada
  2. Molecular Physiology, and the Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Sydney, NSW, Australia

Molecular mechanisms underlying the docking, priming, Ca2+ sensing, and fusion steps of regulated exocytosis remain the subject of much research and speculation. While immediate roles for phospholipase-generated fusogens have largely been excluded from fusion per se, upstream/modulatory effects are likely (i.e. ‘tuning’ of local membrane composition). Phospholipase D (PLD) and its product phosphatidic acid (PA) have been of particular interest. Using the cortical vesicle (CV) model system enabled coupling of quantitative functional-molecular assays with selective pharmacological manipulations to test for the role of PLD-derived PA in late steps of exocytosis. There was selective inhibition of the rate of CV fusion following treatments with different PLD inhibitors but a consistent inhibitory effect on the extent, Ca2+ sensitivity and initial rate of fusion when the inhibitors were used to treat intact eggs prior to CV isolation. Assays of inter-membrane attachment indicated that PLD-derived PA acts in CV docking. To localize the PLD activity, the fluorescent PLD substrate, NBD-phosphatidylcholine, was used to label isolated CV and sheets of plasma membrane (PM) with docked CV (i.e. cell surface complexes; CSC). High resolution lipid analysis identified pronounced NBD-PA generation in CSC rather than in CV, localizing PLD activity to the inner monolayer of the PM, presumably associated with CV docking sites. An alternate activity assay also showed highest PLD activity on CSC relative to the intact egg, cytosol or CV membrane. Using siramesine to selectively block PA in the CV membrane also inhibited docking and fusion kinetics. As a whole, the data indicate upstream roles for PA in docking and/or late priming steps rather than directly in fusion, and the likely localization of PLD with vesicle docking sites. Alterations in optimal local PA concentration may thus modulate the fusion process, particularly kinetics, through effects on docking.