REGULATION OF NUCLEAR ENVELOPE ASSEMBLY AND DISASSEMBLY

核包膜的组装和拆解是核科学中的基本事件 高等真核生物，其机制尚不清楚。植物精华 非洲爪蛙卵提供了一种多功能系统，在该系统中，相间组装 并且核的有丝分裂解体可以在体外进行检测。我们有 用非洲爪哇提取物研究核泡的作用机制 保险丝。在测试钙离子可能的作用时，我们做了一个开创性的 发现：核泡融合涉及钙动员。我们的 初步结果表明，钙离子是从细胞核内动员起来的。 通过IP3受体，这是配体门控的钙通道， 对第二信使肌醇，1，4，5-三磷酸(IP3)作出反应。IP3 受体经典地释放Ca~(2+)以响应信号传递事件 质膜。我们的发现指出了IP3出乎意料的作用 囊泡融合中的受体及其作为融合的潜在成分 复杂，并进一步表明，肌醇磷脂信号可能 调节核泡融合。在项目一和项目二中，我们将测试 IP3受体和肌醇磷脂信号转导机制的研究进展 反义消融和抑制研究。我们已经有一种抗体 IP3受体(针对钙离子流动的关键区域) 在体外抑制核泡融合，这表明我们的 系统的抗体抑制方法将会成功。下一个 该提案的一部分集中在RAS超级家族的成员ARF身上 GTP结合蛋白。我们发现ARF结合核泡 并以GTP-GammaS依赖的方式抑制它们的融合。在 分泌途径，ARF调节衣物复合体在 运输囊泡。我们将使用反义消融和免疫耗竭 方案三中确定ARF是否促进核包膜的方法 小泡，确定这些假定的海岸，并测试 通过询问囊泡萌发是否需要ARF来进行核拆解 有丝分裂时的核膜。我们将研究ARF的监管 与核泡结合，并确定鸟嘌呤核苷酸是否 ARF的交换因子(GNEF)被负调节(无效) 钙动员时核泡受阻(BAPTA受阻) 阶段，ARF不绑定到该阶段。这些研究将导致 对细胞如何调节核膜的基本认识 结构，也可能与融合的调节有关 膜贩子。

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.