Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides

Membrane fusion plays a central role in many cell processes from vesiculartransport to nuclear envelope reconstitution at mitosis but the mechanisms thatunderlie fusion of natural membranes are not well understood. Studies withsynthetic membranes and theoretical considerations indicate that accumulation oflipids characterised by negative curvature such as diacylglycerol (DAG)facilitate fusion. However, the specific role of lipids in membrane fusion ofnatural membranes is not well established. Nuclear envelope (NE) assembly wasused as a model for membrane fusion. A natural membrane population highlyenriched in the enzyme and substrate needed to produce DAG has been isolated andis required for fusions leading to nuclear envelope formation, although itcontributes only a small amount of the membrane eventually incorporated into theNE. It was postulated to initiate and regulate membrane fusion. Here we use amultidisciplinary approach including subcellular membrane purification,fluorescence spectroscopy and Förster resonance energy transfer(FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstratethat initiation of vesicle fusion arises from two unique sites where thesevesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmicreticulum-derived membranes that make up the bulk of the NE to ultimatelyenclose the chromatin. We show how initiation of multiple vesicle fusions can becontrolled by localised production of DAG and propagated bidirectionally.Phospholipase C (PLCγ), GTP hydrolysis and(phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P₂) arerequired for the latter process. We discuss the general implications of membranefusion regulation and spatial control utilising such a mechanism.