748. Requirements for Microtubule-DNA Interactions during Cytoplasmic Trafficking of Plasmids

Abstract

Relatively little is known about how naked DNA moves through the cytoplasm to the nucleus during transfections and other forms of gene delivery. It has been suggested that the dense latticework of the cytoskeleton impedes free diffusion of DNA, implying that plasmids would be unable to reach the nucleus. However, transfections do work, and thus there must be mechanisms by which DNA circumvents cytoplasmic obstacles. We have previously shown that in adherent cells after electroporation or microinjection, plasmid DNA requires intact microtubules to reach the nucleus. Further, we have demonstrated that inhibition of dynein impedes the ability of the plasmid to reach the nucleus. However, it remains unknown how plasmids interact with dynein and hence microtubules. In our past studies, all of the DNA used contained a promoter, a reporter gene and a downstream DNA Targeting Sequence (DTS). A DTS is a sequence in the DNA that is known to bind transcription factors. The transcription factors contain nuclear localization sequences (NLS) that facilitate nuclear import of the protein-DNA complex that is formed in the cytoplasm during transfection. One possible mechanism for cytoplasmic trafficking of transfected plasmids is that after microinjection or electoporation of adherent cells, the DTS on the plasmid is bound by transcription factors and it is via these transcription factors that the plasmid interacts with dynein and is transported to the nucleus via microtubules. Alternatively, a more general mechanism may occur, whereby factors that bind to any type of DNA (i.e. plasmids not containing a DTS) bind non-specifically to the plasmid, creating a different complex that mediates interaction between the plasmid and dynein. In order to test which of these possibilities is occurring, we used an in vitro microtubule spin-down assay. Plasmid, either with or without a DTS, was added to microtubules in the presence of cell extract and separated by centrifugation. Only plasmids that interact with microtubules should be found in the microtubule-containing pellet. As an alternative approach to the biochemical assays, fluorescently labeled PNA-DNA with and without a DTS was cytoplasmically microinjected and followed in real time to further determine if the DTS plays a role in trafficking to the nucleus. Our results suggest that the DTS facilitates an increased interaction between the microtubules and plasmid. These results are important in elucidating how naked DNA reaches the nucleus and an understanding of this process is imperative to improving the efficiency of gene transfer.