Functional Study of Native and Synthetic Nuclei

描述(申请人提供)：基因组的完整性是最重要的生物医学考虑因素。因此，必须确定和描述这种结构是如何形成、保护和监管的。每个人类细胞都包含一个基因组堡垒，即核，穿孔于复杂的进入入口，即核孔。在每一次细胞分裂中，基因组复制后，细胞必须拆除堡垒，组装一个大的有丝分裂纺锤体，以进行基因组分离。接下来，细胞必须再次构建细胞核，将基因组包裹在带有核孔的核膜中。因此，有丝分裂的特征是由三个巨大的结构组成：纺锤体、核膜和核孔。对任何步骤的干扰都可能是致命的。因此，了解这些机制是至关重要的。在《目标L》一书中，我们将以我们广泛的专业知识和工具为基础，探讨核孔的组装机制。因为这个孔很大(120兆吨)，由30个核孔素组成，并且需要在一个长寿命的膜通道内创造一个独特的1200A结构，所以我们的任务不是微不足道的。它已经并将继续包括设计创新的分析方法、新的药物和新的中间体。在L的研究中，我们探讨了毛孔起始蛋白Elys如何与有丝分裂后染色质结合，这如何启动内核膜上早期中间产物的产生，这如何启动外膜对应物，以及潜在的融合酶和/或介体，如Torsin A，在人Torsin Dystonia中突变，以创建膜通道。在目标11和11中，我们转向核粘附素，已知的核进出口受体，并讨论它们在全球细胞调控中的广泛作用。从我们和其他人的工作中可以清楚地看到，Importin Beta及其远亲Transportin已经被有丝分裂时的细胞所选择，作为三个主要的有丝分裂组装事件的全球调节因子：纺锤体组装、核膜组装和核孔组装，所有这些都发生在有丝分裂的不同点的染色质周围。在这一机制中，Transportin和Importinβ掩蔽因子需要在RanGTP较低的区域进行组装，但在有丝分裂染色质附近释放这些因子以促进组装。该调控的空间“GPS”方面来自于它们的“决斗”对手RanGTP，它只在染色质附近以活性形式产生。我们将探讨是否如我们预期的那样，这种控制在逻辑上扩展到其他17种不同的核粘附素的代表，包括重要蛋白(AIM 11)和出口蛋白(AIM III)。如果是这样的话，正如我们完全预期的那样，潜在的靶点可能如此之多，以至于人类细胞的大部分在有丝分裂时都受到核粘附素的调控。这一令人兴奋的调控模式的扩展将为许多众所周知的基因调控、癌症和信号蛋白开辟新的控制途径，为它们的控制增加一个以前未被怀疑的空间方面。

DESCRIPTION (provided by applicant): The integrity of the genome is the most important biomedical consideration. It is therefore essential to identify and to characterize how this structure is formed, protected, and regulated. Each human cell contains a genomic fortress, the nucleus, perforated with complex entry portals, the nuclear pores. At every cell division, after th genome is duplicated, the cell must take down the fortress and assemble a large mitotic spindle for genome separation. Following, the cell must once again build the nucleus, enclosing the genomes in nuclear membranes with nuclear pores. Mitosis is thus characterized by the construction of three massive structures: the spindle, the nuclear membranes, and the nuclear pores. Interference with any step can be lethal. Thus, understanding these mechanisms is essential. In AIM l, we address the mechanism of assembly of the nuclear pore, building on our extensive expertise and tools. Because the pore is massive (120 megadaltons), consists of 30 nucleoporins, and requires the unique creation of a 1200 A structure within a long-lived membrane channel, our task is not trivial. It has and continues to involve devising innovative assays, novel drugs, and new intermediates. In AIM l, we ask how the pore- initiating protein ELYS binds post-mitotic chromatin, how this sets in motion creation of an early intermediate on the inner nuclear membrane, how this then initiates an outer membrane counterpart, and potential fusases and/or mediators, such as Torsin A, mutated in human Torsin Dystonia, for creating the membrane channel. In AIMs ll and lll, we turn to karyopherins, the known nuclear import and export receptors, and address their expanded role in global cellular regulation. It is now clear from our work and others that Importin Beta and its distant karyopherin relative, Transportin, have been co-opted by the cell at mitosis to act as global regulators of the three major mitotic assembly events: spindle assembly, nuclear membrane assembly, and nuclear pore assembly, all of which occur around chromatin at different points in mitosis. In this mechanism, Transportin and Importin beta mask factors required for assembly in areas of low RanGTP, but free these factors near mitotic chromatin to promote assembly. The spatial "GPS"-like aspect of the regulation derives from their "dueling" counterpart, RanGTP, produced in active form only near chromatin. We will probe whether, as we expect, this control logically extends to representatives of the other 17 disparate karyopherins, both importins (AIM ll) and exportins (AIM III). If so, as we fully expect, the potential targets could be so numerous that much of the human cell would be under karyopherin regulation at mitosis. Expansion of this exciting regulatory paradigm would open new avenues of control for many well-known gene regulatory, cancer, and signaling proteins, adding a spatial aspect to their control previously unsuspected.