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，有必要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，细胞核孔是由复杂的入口孔穿孔而成。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，将基因组包裹在有核孔的核膜中。因此，有丝分裂的特点是由三个巨大的结构构成：纺锤体、核膜和核孔。干扰任何一步都可能是致命的。因此，理解这些机制是必要的。在AIM 1，我们解决核孔的组装机制，建立在我们广泛的专业知识和工具。由于这个孔是巨大的（120兆道尔顿），由30个核孔蛋白组成，并且需要在一个长寿命的膜通道内独特地创造一个1200 a的结构，我们的任务不是微不足道的。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，我们研究了孔隙启动蛋白ELYS如何结合有丝分裂后的染色质，它如何启动核膜上早期中间体的产生，然后如何启动外膜对应物，以及潜在的fusase和/或介质，如在人类Torsin肌张力障碍中突变的Torsin A，以创建膜通道。在AIMs ii和ii中，我们转向核细胞蛋白，已知的核输入和输出受体，并讨论它们在全球细胞调节中的扩大作用。从我们和其他人的工作中可以清楚地看出，在有丝分裂时，细胞选择了输入蛋白β和它的远缘核丝蛋白亲戚转运蛋白，作为三个主要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的不同时间点染色质周围。在该机制中，转运蛋白和进口蛋白β屏蔽因子需要在低RanGTP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。这种空间“GPS”式的调控来自于它们的“决斗”对应物RanGTP，后者仅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，这种控制在逻辑上延伸到其他17种不同的核细胞蛋白的代表，包括进口蛋白（AIM ii）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，潜在的目标可能是如此之多，以至于人类细胞的大部分在有丝分裂时都受到核丝蛋白的调节。这一令人兴奋的调控范式的扩展将为许多众所周知的基因调控、癌症和信号蛋白的控制开辟新的途径，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.