Chung, HC; (2015) The role of fusogenic vesicles in the regulation of nuclear envelope assembly. Doctoral thesis , University College London (UCL).

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Abstract

Earlier studies of the male pronucleus (MPN) formation in echinoderms have isolated fusogenic vesicles, known as MV1, essential for nuclear envelope (NE) assembly. MV1 is a membrane compartment with elevated levels of phosphoinositides, a lipid-modifying enzyme PLCγ and its upstream regulator SFK1. These are responsible for production of localised diacylglycerol (DAG) which facilitates membrane fusion. The fusion mechanism in mammalian NE assembly is uncharacterised. We hypothesise MV1-like vesicles are responsible for completing the NE in mammalian cells by sealing the NE gaps at telophase. To study the fusion mechanism in mammalian NE assembly, subcellular fractions analogous to MV1 were isolated from CHO and HeLa cells. However, these membrane fractions were rarely separated from the ER, Golgi and endosomes subfractions. Electron tomography confirmed the presence of membrane vesicles (MVs) in the vicinity of the NE gaps at telophase. Moreover, the quantity of these MVs was significantly higher in the regions of large gap versus regions without gap, suggesting a putative consumption of these MVs for gap closure. Furthermore, PtdIns(3,4,5)P3 and class I PI3 kinase were detected in endomembranes at the vicinity of the mammalian NE implying the presence of MV1-like vesicles. DAG-enriched MVs were detected in interphase cells using a miniSOG-conjugated C1aC1b domain; they may be involved in house-keeping fusion events. Using a chemically-induced dimerisation system and correlative light and electron microscopy, localised DAG production was demonstrated to be essential for mammalian NE assembly. Acute DAG depletion in the NE resulted in NE fragmentation. Fragmented NE was rescued by addition of exogenous DAG. Also, we show for the first time that Golgi-DAG depletion led to fragmented NE, signifying that the Golgi may produce fusogenic vesicles. Our findings show that the regulatory mechanisms involved in the MPN formation in fertilised echinoderm eggs are also conserved in mammalian NE assembly during mitosis.

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