Nuclear envelope assembly and disassembly are fundamental events in higher eukaryotes whose mechanisms are not understood. Extracts of Xenopus eggs provide a versatile system in which the interphase assembly and mitotic disassembly of the nucleus can be examined in vitro. We have used Xenopus extracts to study the mechanism by which nuclear vesicles fuse. In testing for a possible role of Ca2+, we made a seminal discovery: nuclear vesicle fusion involves Ca2+ mobilization. Our preliminary results suggest that Ca2+ is mobilized from within nuclear vesicles by IP3 receptors, which are ligand-gated calcium channels that respond to the second messenger, inositol, 1,4,5-triphosphate (IP3). IP3 receptors classically release Ca2+ in response to signalling events at the plasma membrane. Our findings point to an unexpected role for IP3 receptors in vesicle fusion and as potential components of the fusion complex, and further suggest that phosphoinositide signalling may regulate nuclear vesicle fusion. In Projects One and Two we will test the proposed roles of IP3 receptors and phosphoinositide signalling using antisense ablation and inhibition studies. We already have an antibody of the IP3 receptor (directed against a region critical for Ca2+ flux) that inhibits nuclear vesicle fusion in vitro, suggesting that our systematic antibody inhibition approach will be successful. The next part of the proposal is focussed on ARF, a member of the Ras superfamily of GTP-binding proteins. We discovered that ARF binds nuclear vesicles and inhibits their fusion in a GTPgammaS-dependent manner. In the secretory pathway, ARF regulates the assembly of coat complexes onto transport vesicles. We will use antisense ablation and immunodepletion methods in Project Three to determine if ARF promotes coating of nuclear vesicles, identify these putative coasts, and test the mechanism of nuclear disassembly by asking if ARF is required for vesicle budding from the nuclear envelop at mitosis. We will study the regulation of ARF binding to nuclear vesicles, and determine if the guanine nucleotide exchange factor (GNEF) for ARF is negatively regulated (inactive) on nuclear vesicles arrested at the Ca2+-mobilization (BAPTA-arrested) stage, to which ARF does not bind. These studies will lead to a fundamental understanding of how cells regulate nuclear envelope structure, and may also be relevant to the regulation of fusion during membrane trafficking.
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