Functional Study of Native and Synthetic Nuclei

描述（由申请人提供）：信息不断地从细胞外世界传递到细胞内部。这些信息中的许多必须传递到细胞核才能影响细胞的变化。一个必要的步骤是通过精心设计的，高度调节的核通道，核孔复合体。当这种交通受到干扰时，癌症，发育缺陷或异常的细胞功能可能随之而来。脊椎动物核孔的大小为1.2亿道尔顿，具有复杂的结构体系。构成这个通道的亚基中的关键是Nup 107 -160复合物，即所谓的核孔关键。对这个关键亚基的结合伴侣的分析向我们揭示了蛋白质ELYS，一种特异性靶向染色质表面的孔组装的蛋白质。在没有ELYS的情况下，孔不能在细胞核处形成，而是在细胞质膜堆叠中形成。 显然，ELYS对我们作为真核生物的存在至关重要。没有它，我们的基因组将无法与细胞的外部世界进行交流。目前的资助有四个主要目标：（1）第一是确定ELYS的分子相互作用：其染色质结合靶点，其与Nup 107 -160复合物的接合规则，以及其包含关键完整膜孔蛋白的囊泡的募集。这些知识是理解这些关键网关如何形成的关键。(2)下一个目标是确定核孔组装的物理步骤，具体分析核内外膜之间发生的融合事件，以启动孔的形成。为此，我们将使用我们最近开发的新型核中间体和检测方法。(3)我们的第三个目标将是探索核膜形成和核孔组装的负调节剂importin的作用机制。我们将具体询问上面确定的核孔组装步骤中的哪些步骤是其调节的目标。(4)最后，我们将攻击一个新的挑战，我们最近发现与脊椎动物核孔相关的蛋白质中心蛋白的作用。我们发现中心蛋白突变体阻断mRNA和蛋白质从细胞核输出。我们将询问哪些输出因素或途径显示与 centrin，以确定其在核孔中的作用。这些目标的成功完成将为深入了解核孔的组装和功能提供必要的信息，核孔是通往细胞核的重要门户。公共卫生相关性：我们每个人的基因组都包含在一个堡垒中，即细胞核，只能通过称为核孔的错综复杂的通道进入。没有信号可以到达基因组，也没有信息可以从它发出，除非通过这些孔。了解这些通道是如何构建的以及它们是如何发挥作用的，不仅对了解基因组是如何被获取的至关重要，而且对了解基因组是如何被保护的也至关重要。

DESCRIPTION (provided by applicant): Messages pass constantly from the extracellular world into the interior of the cell. Many of these messages must travel to the nucleus to affect cellular change. A necessary step is passage through elaborate, highly regulated nuclear gateways, the nuclear pore complexes. When this traffic is perturbed, cancer, developmental defects, or aberrant cellular function can ensue. The vertebrate nuclear pore is 120 million daltons in size with a complex structural architecture. Key among the subunits that make up this gateway is the Nup107-160 complex, the so-called linchpin of the nuclear pore. Analysis of the binding partners of this key subunit revealed to us the protein ELYS, a protein that specifically targets pore assembly to the surface of the chromatin. Without ELYS pores fail to form at the nucleus and instead form in cytoplasmic membrane stacks. Clearly, ELYS is of primary importance to our existence as eukaryotes. Without it, our genomes would have no communication with the outer world of the cell. The present grant has four major goals: (1) The first is to determine the molecular interactions of ELYS: its chromatin binding targets, its rules of engagement with the Nup107-160 complex, and its recruitment of vesicles containing key integral membrane pore proteins. This knowledge is key to understanding how these key gateways are formed. (2) A next goal is to determine the physical steps of nuclear pore assembly, with specific analysis of the fusion event that occurs between the inner and outer membrane of the nucleus to initiate pore formation. For this, we will use novel nuclear intermediates and assays that we have recently developed. (3) Our third goal will be to probe the mechanism of action of a negative regulator of both nuclear membrane formation and nuclear pore assembly, importin ¿. We will specifically ask which of the steps of nuclear pore assembly identified above are targeted by its regulation. (4) Lastly, we will attack a new challenge, the role of the protein centrin which we have recently discovered to be associated with vertebrate nuclear pores. We find centrin mutants to block both mRNA and protein export from the nucleus. We will ask which of the export factors or pathways show interaction with centrin in order to define its role at the nuclear pore. Successful completion of these goals will provide essential insight into the assembly and function of the nuclear pore, vital gateway to the nucleus. PUBLIC HEALTH RELEVANCE: The genome of each of us is contained within a fortress, the nucleus, and can be accessed only through intricate gateways called nuclear pores. No signals can reach the genome, nor messages be sent from it except through these pores. Understanding how the gateways are built and how they function is essential not only to understanding how the genome can be reached, but also how it is protected.