Nuclear Envelope Formation In Vitro: A Sea Urchin Egg Cell-Free System

Cell Biophysics Laboratory, Cancer Research UK (CR-UK), London Research Institute (LRI), London, UK.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 02/2009; 464:207-23. DOI: 10.1007/978-1-60327-461-6_12
Source: PubMed


The formation of the nuclear envelope (NE) typically occurs once during every mitotic cycle in somatic cells, and also around the sperm nucleus following fertilization. Much of our understanding of NE assembly has been derived from systems modeling the latter event in vitro. In these systems, demembranated sperm nuclei are combined with fertilized egg cytoplasmic extracts and an ATP-regenerating system and in a multistep process they form the functional double bilayer of the NE. Using a system that we developed from sea urchin gametes, we have demonstrated that NE assembly is regulated by membrane vesicles in a spatial and temporal fashion, emphasizing the roles of phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), diacylglycerols (DAG), and lipid-modifying enzymes in NE assembly.

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    • "Lytechinus pictus were purchased from Marinus Scientific (Long Beach, CA). Gamete shedding and collection took place as described [31], [32]. Fertilised egg cytoplasmic (S10) extracts and demembranated sperm nuclei (0.1% Triton X-100 extracted) were prepared as previously described [31]. "
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    ABSTRACT: Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P 3]. PLCε is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCε variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation. KeywordsPhospholipase C activity–reconstitution in vitro –phospholipase Cε–Ras–Rho–phospholipid vesicles
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