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

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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.