Abstract
Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.
Highlights
Membrane fusion is required for many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis
Since FRET is only possible between molecules that are in close proximity (1–10 nm), it is a reliable indicator of membrane fusion which permits the donor and acceptor to interact within a common continuous bilayer
In this paper we provide evidence that fusion initiation leading to nuclear envelope formation is GTP triggered, and requires PtdIns(4,5)P2 and PLC activity in the MV1 fraction
Summary
Membrane fusion is required for many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis. Recent work has integrated roles for both protein signalling and lipid modification in natural membrane fusion [1,2,3,4,5]. We have isolated a natural membrane vesicle fraction (MV1) from cytoplasm of fertilised oocytes. This membrane population consists of .50% phosphoinositides, is .100-fold enriched in a phosphatidylinositol-specific phospholipase C (PI-PLCc) and is essential for membrane fusion leading to nuclear envelope formation [3,6]. Using cell-free oocyte extracts to assemble nuclear envelopes from fusion of discrete membrane vesicle populations [7], we have shown that the early signalling events involve activation of a tyrosine kinase [8] which in turn activates PLCc in MV1 [3]. Subsequent formation of diacylglycerol (DAG) alters the lamellar structure of these precursor membranes, facilitating their fusion with the endoplasmic reticulum (ER)-
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