Cytoplasmic transport and nuclear import of plasmid DNA

Abstract

Productive transfection and gene transfer require not simply the entry of DNA into cells and subsequent transcription from an appropriate promoter, but also a number of intracellular events that allow the DNA to move from the extracellular surface of the cell into and through the cytoplasm, and ultimately across the nuclear envelope and into the nucleus before any transcription can initiate. Immediately upon entry into the cytoplasm, naked DNA, either delivered through physical techniques or after disassembly of DNA-carrier complexes, associates with a large number of cellular proteins that mediate subsequent interactions with the microtubule network for movement toward the microtubule organizing center and the nuclear envelope. Plasmids then enter the nucleus either upon the mitotic disassembly of the nuclear envelope or through nuclear pore complexes in the absence of cell division, using a different set of proteins. This review will discuss our current understanding of these pathways used by naked DNA during the transfection process. While much has been elucidated on these processes, much remains to be discerned, but with the development of a number of model systems and approaches, great progress is being made.

Keywords: cell nucleus; cytoskeleton; gene therapy; intracellular transport; microtubule; nuclear protein transport.

Figure 1. Intracellular trafficking of plasmids

Most plasmids enter the cell by either endocytosis and/or direct entry into the cytosol at which point they must traverse the cortical actin layer, perhaps using actin-based movement [25,119]. Once free in the cytoplasm, plasmids are rapidly complexed by a number of DNA-binding proteins in the cytoplasm which in turn bind to other proteins to form large protein–DNA complexes [2]. Transcription factors bound to the DNA interact with importin β and other proteins that link the complex to dynein and kinesin for movement along microtubules toward the nucleus [19]. During this process, the DNA–protein complexes appear to be dynamic, with various proteins coming on and off the DNA at different times, perhaps to mediate different processes. Nuclear entry is then mediated by importin β in a sequence- and importin-dependent manner through the nuclear pore complex (NPC) in non-dividing cells or independent of importins and any DNA sequence requirement during mitosis and the associated dissolution of the nuclear envelope.

Figure 2. Strategies to increase nuclear targetting of plasmids

A number of different approaches have been used to improve the nuclear import of plasmids, all of which center around attaching NLS peptides, or NLS proteins, or other importin interacting peptides to the DNA. This can be also done by increasing the functional diameter of the NPC itself by enhancing non-selective gating of the pore with the drug TCHD [120,121].

Figure 3. Model for general and cell-specific DNA nuclear import in non-dividing cells

If plasmids containing sequences that act as scaffolds for transcription factors and other DNA-binding proteins (termed DTS or DNA nuclear targetting sequences) are deposited into the cytoplasm during transfection, they can form complexes with these proteins, thereby attaching NLSs to the DNA. Some, but not all, of these NLSs may be in a conformation able to interact with importins for transport of the DNA–protein complex into the nucleus through nuclear pores. In the case of general DTSs, the transcription factors that interact with the DTS are ubiquitously expressed and thus allow DNA nuclear import in all the cell types. By contrast, cell-specific DTSs bind to a group of cell-specific transcription factors as well as some general transcription factors to form import competent complexes in corresponding cell types (the cell type X DTS binds cell type X-specific transcription factors in cell type X). In other cell types, the specific transcription factors are absent and consequently, plasmids fail to be imported into the nuclei for expression.