Functional Study of Native and Synthetic Nuclei

描述（由申请人提供）：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、保护和调节的。每个人类细胞都包含一个基因组堡垒，即细胞核，细胞核上有复杂的入口，即核孔。在每一次细胞分裂中，在基因组复制之后，细胞必须拆除堡垒并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，将基因组包围在具有核孔的核膜中。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。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AIM 1中，基于我们广泛的专业知识和工具，我们解决了核孔组装的机制。因为孔是巨大的（120兆道尔顿），由30个核孔蛋白组成，并需要在长寿命的膜通道内独特地创建1200 A结构，所以我们的任务并不简单。它已经并将继续涉及设计创新的检测方法，新药和新的中间体。在AIM 1中，我们询问孔起始蛋白ELYS如何结合有丝分裂后染色质，这如何启动在内核膜上产生早期中间体，这然后如何起始外膜对应物，以及潜在的融合酶和/或介体，例如在人扭转蛋白肌张力障碍中突变的扭转蛋白A，用于产生膜通道。在AIM II和III中，我们转向核转运蛋白，已知的核输入和输出受体，并解决其在全球细胞调节中的扩展作用。现在从我们的工作和其他人的工作中可以清楚地看到，输入蛋白β和它的远亲核转运蛋白，转运蛋白，在有丝分裂中被细胞吸收，作为三个主要的有丝分裂组装事件的全局调节剂：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中不同点的染色质周围。在这种机制中，转运蛋白和输入蛋白β掩盖了在低RanGTP区域组装所需的因子，但在有丝分裂染色质附近释放这些因子以促进组装。空间"GPS"样方面的调节来自他们的"决斗"对应物，RanGTP，仅在染色质附近以活性形式产生。我们将探讨是否，正如我们所期望的那样，这种控制逻辑上延伸到其他17个不同的karyopherins，进口（AIM II）和出口（AIM III）的代表。如果是这样的话，正如我们完全预期的那样，潜在的靶点可能是如此之多，以至于大部分人类细胞在有丝分裂时都处于核转运蛋白的调控之下。这种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，为它们以前未被怀疑的控制增加空间方面。

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.