A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Abstract

Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca 2+ -triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca 2+ -triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.

Negatively Charged Lipids as a Potential Target for New Amphiphilic Aminoglycoside Antibiotics

“…Another striking characteristic of cardiolipin is the comparatively small cross-section of its headgroup relative to the cross-section of its four large tail groups. This discrepancy results in a molecule with a large intrinsic negative curvature (16), also described previously for phosphatidylethanolamine (PE) (17), which facilitates the insertion of membrane proteins. The cross-sectional size difference further explains the location of cardiolipin-enriched regions at the pole and/or the division septum, which can in turn relate to the polar localization of many proteins, including * This work was supported by the Belgian Funds for Scientific Research those involved in cell division and osmosensing (6).…”

The Role of Phospholipase D in Regulated Exocytosis

“…0.2-m MCE filter) in 50 ml Falcon tubes. Incubation of eggs with inhibitors was as previously described (33), with continuous slow and gentle mixing for either 20 min or 20 h at 7°C. Butanol was delivered as 1-4% aqueous baseline intracellular media (BIM) solutions; SRM, FIPI, HLP, QRC, and VU compounds were delivered from dimethylformamide stock solutions to a final dimethylformamide concentration of Յ1% for QRC and Յ0.05% for other inhibitors.…”

“…CV "settle" fusion assays to assess the capacity of endogenous CV membrane components to independently establish intermembrane attachment (i.e. docking) were also carried out as described (30,32,33,39,42,45); in short, suspended CV (A 405 0.4) were pipetted into microplates and left to settle into contact for 1 h followed by standard challenge with Ca 2ϩ to trigger fusion responses. The Wallac Victor2 V Microplate Reader (PerkinElmer Life Sciences) was used for all light scattering measurements to assess fusion responses.…”

“…Thus, although an essential role for PLD in the fusion step of regulated exocytosis was excluded some time ago using human platelets (31), in terms of the more recent studies noted it was deemed important to further evaluate the function(s) of this critical enzyme in the late steps of the exocytotic pathway. Selective small molecules provide an effective tool to study the biological effects of critical enzymes and lipids on the fusion process, particularly using the CV model system (19,32,33). This is also noteworthy considering the range of different inhibitors available to target PLD activity and thus inhibit PA generation (e.g.…”

“…an ex vivo "cell culture" system; Ref. 33) and acute CV treatments (30,32,33,39,40) with a variety of selective PLD1 and PLD2 inhibitors to quantitatively address functional and molecular aspects of the involvement of PLD-derived PA in regulated exocytosis. Fluorescently labeled PC was used as a substrate, to localize PLD activities.…”