Role of the microtubule network in nuclear transport: potential use in gene delivery

调控生长和其他过程的关键蛋白质进出细胞核，这是基因转录的位置，对真核细胞的功能至关重要。然而，对这一过程的了解很大程度上是基于使用半完整细胞系统的研究，这意味着细胞骨架元素在核运输中的作用在很大程度上被忽视了。有趣的是，体内研究，包括我们自己与癌症调控中重要的信号分子有关的研究，清楚地表明细胞微管(MT)网络在核输入中发挥着不可或缺的作用。我们建议对核进口依赖于由3个调节癌症的分子组成的MT网络的机制基础进行详细的研究。我们将比较这些分子与其他核定位分子的性质，并检查从病毒和其他蛋白质中介导类似MT相关序列的核输入的能力。这一结果将首次确立核蛋白进口依赖于细胞骨架元素的普遍性。由于大DNA分子通过简单扩散移动是非病毒基因传递方法中的一个限制因素，因此也将首次评估应用这一知识促进编码治疗性基因产品的DNA运输到靶细胞核的可能性。我们将使用与MT网络相互作用的模块序列，通过将它们包括在我们考虑到这一点的模块结构中来帮助基因转移。因此，我们将能够第一次直接测试MT相互作用是否可以增强DNA到细胞核的输送和报告基因的表达，这在基因治疗方法中有明显的应用。

Transport of key proteins that regulate growth and other processes, into and out of the nucleus, the site of gene transcription, is central to the function of eukaryotic cells. Knowledge of the process is largely based on studies using semi-intact cell systems, however, meaning that the role of cytoskeletal elements in nuclear transport has been largely overlooked. Intriguingly, in vivo studies, including our own relating to signalling molecules important in the regulation of cancer, clearly implicate the cellular microtubule (MT) network as playing an integral role in nuclear import. We propose to carry out a detailed examination of the mechanistic basis of the dependence of nuclear import on the MT network of 3 molecules regulating cancer. We will compare the properties of these molecules to those of other nuclear localizing molecules, as well as examine the ability to mediate nuclear import of similar MT-associating sequences from viral and other proteins. The results will establish for the first time, the generality of the dependence of nuclear protein import on cytoskeletal elements. Since the movement of large DNA molecules by simple diffusion is a limiting factor in non-viral gene delivery approaches, the possibility of applying this knowledge to facilitate the transport of DNA encoding therapeutic gene products to the nucleus of target cells, will also be assessed for the first time. We will use the modular sequences that confer interaction with the MT network to assist gene transfer by including them in modular constructs we have designed with this in mind. We will thus be able to test directly for the first time whether MT-interaction can enhance the delivery of DNA to the nucleus and reporter gene expression, with obvious application in gene therapy approaches.