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Nuclear envelope (NE) reassembly during telophase and cytokinesis is an essential process for the completion of mitosis. NE reassembly is a proteolipid process that requires specific signalling and membrane compositions in order to conclude successfully. Previous research into NE reassembly has focused on the protein aspect, however the role of lipids is less well understood. There are currently two models for NE reassembly, one suggests that the endoplasmic reticulum (ER) is targeted to the chromosomes and consequently envelops them with the gaps between the tubules and sheets filled by nuclear pore complexes. The role of lipids in this model has not been investigated however it is suggested that membrane fusion is not required for NE reassembly. An alternative model based in a non-somatic system, has shown that diacylglycerol (DAG), a fusogenic lipid, is able and required to trigger localised membrane fusion in NE reassembly. DAG is a negative curvature lipid and its localised production can trigger fusion due to changes in the structure of the membrane. However, it is possible that these models are not mutually exclusive. While the fusion model has shown an important role for lipids and lipid-modifying enzymes in NE reassembly and membrane fusion, these discoveries have yet to be translated into mammalian systems. This study aims to determine the involvement of phospholipids and their derivatives in mammalian NE assembly, based on previous work from the fusion model, which has shown that DAG and PtdIns(4,5)P2 are involved in the membrane fusion which forms a complete and functional NE. The localisation of these lipids during mitosis has not previously been investigated in somatic cells. This study uses the C1 domains from protein kinase C epsilon (PKCε), and the pleckstrin homology (PH) domain from phospholipase C delta (PLCδ1), which bind directly to DAG and PtdIns(4,5)P2 respectively. These probes indicate the presence of DAG in the NE and PtdIns(4,5)P2 in the peri-nuclear region of interphase cells. For the first time we observe the localisation of the C1 domain to the newly forming NE during telophase suggesting a role for DAG in NE reassembly. The specific role of DAG has been investigated using the inducible dimerisation system, with which DAG kinase ε kinase domain (DGKεK) was inducibly targeted to the NE using the nuclear targeting sequence of lamin B receptor (LBR). Using this chemical biology tool we have been able to show the localised role of fusogenic lipids such as DAG in somatic cells. These unprecedented results have opened a new area of research in the field of the regulation of NE assembly and offer novel mechanisms in the previously described models. |
