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. Author manuscript; available in PMC: 2014 Sep 2.
Published in final edited form as: Mol Ther. 2005 Nov 21;13(2):422–428. doi: 10.1016/j.ymthe.2005.10.004

Intracellular trafficking of plasmids during transfection is mediated by microtubules

Erin E Vaughan 1, David A Dean 1,*
PMCID: PMC4150869  NIHMSID: NIHMS48139  PMID: 16301002

Abstract

Little is known about how plasmids move through the cytoplasm to the nucleus. It has been suggested that the dense latticework of the cytoskeleton impedes free diffusion of large macromolecules, including DNA. However, since transfections do work, there must be mechanisms by which DNA circumvents cytoplasmic obstacles. One possibility is that plasmids become cargo on cytoskeletal motors, much like viruses do, and move to the nucleus in a directed fashion. Using microinjection and electroporation approaches in the presence of drugs that alter the dynamics and organization of the cytoskeleton, we show that microtubules are involved in plasmid trafficking to the nucleus. Further, by co-injecting inhibitory antibodies, we find that dynein likely facilitates this movement. These results were confirmed using an in vitro spin-down assay that demonstrated that plasmids bind to microtubules through adaptor proteins provided by cytoplasmic extracts. Taken together, these results suggest that plasmids, like most viruses, utilize the microtubule network and its associated motor proteins to traffic through the cytoplasm to the nucleus.

Keywords: electroporation, gene therapy, transfection, lipoplex, dynein, cytoskeleton

INTRODUCTION

Gene therapy, specifically non-viral gene therapy, has been greatly limited by the relative inability to achieve high levels of expression. To date, most attempts to improve non-viral gene therapy have focused on overcoming the barriers presented by the cell membrane and nuclear envelope or increasing the ability of the plasmids to escape the endosome in lipid mediated transfections [1]. However, it has been shown that in liposome transfected cells, after endosomal escape, a significant amount of plasmid remains free in the cytoplasm and does not reach the nucleus, suggesting that it may be a contributing factor to the observed low expression levels [2, 3]. Further, when DNA is delivered to the cytoplasm directly by techniques such as electroporation or microinjection, it must navigate through the cytoplasm to reach the nucleus. It has been shown previously that molecules of DNA larger than 2000 base pairs are unable to diffuse freely in the cytoplasm and that microinjected plasmids do not diffuse far from the site of injection [4, 5]. However, it is known that despite this apparent inability of plasmids to diffuse throughout the cytoplasm, transfections do result in expression. Hence, plasmids must be able to traverse the cytoplasm via a means other than diffusion. Understanding how plasmids move through the cytoplasm is imperative to better understand and enhance transfections, as well as the field of gene therapy as a whole.

One possibility is that plasmids utilize the cell’s own machinery for transporting molecules through the cytoplasm: the cytoskeletal network. For instance, when cellular DNA in the nucleus is damaged, p53, translocates to the nucleus via dynein, a retrograde molecular motor that travels on microtubules [6–10]. However, it is not only endogenous proteins that use the cytoskeleton to reach the nucleus. Many viruses also utilize microtubules to reach the nucleus including cytomegalious virus, human immunodeficiency virus, herpes simplex virus, adenovirus, and parvovirus [11–17]. Alternatively, other viruses and certain bacterial pathogens use actin for intracellular movement [18, 19].

Despite the realization that DNA movement to the nucleus is possibly one of the most important barriers to transfection, little work has been done to characterize the mechanisms by which plasmids traverse the dense cytoplasm to reach the nucleus [3]. In the present study, we use electroporation and microinjection to show that plasmids move along the microtubule network and likely use dynein as the molecular motor that facilitates movement toward the nucleus. Biochemical experiments confirm the interaction between plasmids and microtubules in the presence of cell extract, suggesting the need for adaptor proteins.

MATERIALS AND METHODS

Cell Culture and Electroporation

TC7 cells, a sub line of African Green Monkey kidney epithelium, and human adenocarcinoma A549 cells (ATCC, #CCL-185, Rockville, MD) were grown in DMEM supplemented with 10% fetal bovine serum (FBS). For electroporations, cells were grown to confluencey in 6-well dishes, and rinsed twice in serum- and antibiotic-free media. Ten micrograms of plasmids in one ml of serum- and antibiotic-free DMEM were added to each well and one 165 mV square wave electric pulse was applied using a PetriPulser electrode (BTX, San Diego, CA). Immediately following electroporation (within 15 seconds), the indicated drug was added to the cells in 1 ml DMEM with 10% fetal bovine serum and antibiotic. Drugs were used at the following final concentrations: nocodazole 20 µM, taxol 10 µM, latrunculin B 2.5 µM, and jasplankinolide 125 nM. Two hours after electroporation, cells were lysed in Promega lysis buffer and luciferase activity was measured using the Promega Luciferase Assay System, as previously described [20]. All luciferase measurements were normalized to total cell protein and reported as fold-increases in expression over DMSO (vehicle-treated). All experiments were performed in triplicate wells and the experiments were repeated at least three times. For statistical analyses, student’s t-tests were used.

Plasmids

Plasmids pCMV-lux-DTS and pCMV-GFP-DTS express either luciferase or green fluorescent protein (GFP) from the CMV immediate early promoter and contain the SV40 DNA nuclear targeting sequence, downstream of the reporter gene [21]. The NFkB-reponsive luciferase plasmid, pNFkB-Luc, was from Clontech (BD biosciences, Mountain View, CA). A derivative of pCMV-GFP-DTS that contains tandem binding sites for the GeneGrip Cy3-labeled peptide nucleic acid (PNA; Gene Therapy Systems, San Digeo, CA) was used for live cell imaging and was labeled with Cy3-PNA as described [22]. All plasmids were purified from E. coli using Qiagen Gigaprep kits as described by the manufacturer (Qiagen, Chatsworth, CA).

Real-time quantitative PCR

Quantitative, real-time PCR was performed in a 20 µl reaction volume, using the DyNAmoTM SYBRR Green qPCR Kit as described by the manufacturer (Finnzymes, Espoo, Finland). Reactions were carried out and quantified with the MJ Research Opticon 2. The supernatants and pellets from the microtubule and actin spin-down assays were diluted 1:1 in water and 4 µl were used for the reactions. The primers amplified a 116 base-pair region of the beta-lactamase gene present in the plasmids. Standard curves were generated using seven 10-fold dilutions of pCMV-Lux-DTS. The threshold was set manually by determining the best fit line for the quantitation standards, All samples were run in duplicate and amounts were determined based on the standard curve. A melting curve analysis was preformed to ensure reaction specificity. Data were normalized by using the percent DNA found in the pellet versus percent DNA in the supernatant. All experiments were preformed at least three times.

Microinjection

TC7 cells were grown on cover slips and microinjected with pCMV-GFP-DTS (0.5mg/ml in phosphate buffered saline) using an Eppendorf Femtojet system as previously described [22]. Drugs were added immediately following microinjection (20 µM nocodazole or 10 µM taxol) and maintained in the medium throughout the course of the experiment. GFP-expressing cells were counted 5 hours after microinjection.

Microtubule Spin-down assay

Tubulin protein, 5 mg/ml (Cytoskeleton Inc., Denver, CO) was placed on ice and 2.5 µl of PEM buffer (80 mM PIPES pH 7.0, 1 mM MgCl2, 1 mM EGTA, and 50% glycerol) was added. The tubulin was allowed to polymerize for 20 min at 35°C and then stabilized by the addition of 20 µM taxol (Cytoskeleton, Inc.). Tubulin (10 µg) was incubated with DNA (10 ng) and/or cell extract (12 – 24 µg) in PEM buffer containing 20 µM taxol for 30 minutes, placed over a cushion of PEM with 50% (vol/vol) glycerol with added and taxol (20 µM) and centrifuged at 100,000 × g for 40 min in an Airfuge (Beckman Instruments Inc., Palo Alto, CA). The pellet was resuspended in cushion buffer for PCR. Cell extract was prepared as described [23].

Actin Spin down

Non-muscle actin (250 µg; Cytoskeleton, Inc., CO) was resuspended to a final concentration of 1 mg/ml in General Actin Buffer (Cytoskeleton, Inc. CO) and incubated on ice for thirty minutes. After incubation, 25 µl of Actin Polymerization Buffer (Cytoskeleton, Inc. CO) was added and the actin was incubated for one hour at room temperature. Reactions containing actin, plasmid DNA (10 ng), and/or and cell extract were centrifuged for 1.5 hours at 150,000 × g, the supernatant was removed, the pellet then resuspended in an equal volume of buffer, and quantitative PCR was performed.

RESULTS

Taxol Stabilization of Microtubules Enhances Expression of Electroporated Plasmids

To determine whether the cytoskeleton plays a role in the intracellular trafficking of plasmids during transfections, adherent A549 cells were electroporated with luciferase expressing plasmids in the presence or absence of various cytoskeletal-altering drugs. Since these drugs are known to affect endocytosis, if lipoplex reagents were used for transfections in these studies it would be impossible to discern the drugs’ effects on endocytosis from those on cytoplasmic trafficking. By contrast, electroporation induces small, transient pores in the cell membrane thereby allowing entry of DNA into the cytoplasm independent of endocytosis [24], ensuring that the drug effects are due to events occurring in the cytoplasm and not the ability of the plasmids to enter the cytoplasm. Two hours after electroporation of the plasmids, the cells were harvested and luciferase expression was determined. The 2 hour time point was chosen to minimize any additional changes that could occur with prolonged drug treatment of the cells. However, the same results were noted following 24 hour incubation in the presence of drugs (data not shown). When jasplankinolide, which stabilizes the actin cytoskeleton, or latrunculin B, which disrupts the actin cytoskeleton, were used there was no change in luciferase expression over vehicle treated cells, suggesting that the actin cytoskeleton is not involved in transport of the plasmids to the nucleus. Disruption of the microtubule network with nocodazole also did not significantly alter luciferase expression levels. By contrast, stabilization of the microtubule network by taxol increased luciferase expression approximately a 4.5-fold over baseline (Figure 1).

Figure 1. Stabilization of the microtubule network increases intracellular trafficking and gene expression of plasmids during transfection.

Figure 1

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(A) A549 cells were electroporated with pCMV-Lux-DTS and immediately treated with vehicle alone (DMSO), jasplankinolide (125 nM), latrunculin B (2.5 µM), nocodazole (20 µM), or taxol (10 µM) for two hours, after which cells were harvested and luciferase activity was measured. Mean luciferase activities ± st. dev. (RLU/mg cell protein) were normalized to control transfected cells (no drug or vehicle treatment) and experiments were performed in triplicate and repeated three times. * p < 0.001 by paired student’s t-test. (B) Plasmid transcription is not greatly altered by drug treatment. A549 cells were electroporated with either pCMV-Lux-DTS or pNFkB-Lux and 24 hours later, the same drugs were added to the cells. Two hours after the addition of drugs, luciferase activities were measured and normalized to control cells as in A.

To ensure that the observed changes in luciferase expression seen upon incubation of cells with taxol were a result of cytoplasmic plasmid trafficking and not altered transcriptional activity, we electroporated cells with the same luciferase-expressing plasmids and waited 24 hours to ensure that the plasmids had reached the nucleus before drug treatment. The cells were then treated with the various drugs for 2 hours and luciferase expression was measured. Using this approach, any changes in luciferase expression would be due to drug effects on gene expression, independent of cytoplasmic trafficking. Neither disruption nor stabilization of the actin cytoskeleton affected the ability of the plasmids to express once in the nucleus (Figure 1b). It has been reported that NFκB can be activated by translocating to the nucleus in some cell lines when cytoskeletal altering compounds are used [25, 26]. Since there is an NFκB binding site in the CMV promoter in pCMV-lux-DTS, to confirm that the drug effects were not at the transcriptional level, an NFκB responsive, luciferase-expressing plasmid was used in a set of transfections. Using a similar strategy to that just described, pNFκB-luc was transfected into cells and 24 hours later the cells were treated with jasplankinolide, latrunculin B, nocodazole, or taxol for 2 or 24 hours. Again, no changes in luciferase expression were noted (Figure 1b). Similar results were obtained in both A549 and TC7 cells (not shown). Taken together, these results suggest that stabilization of the microtubule network increases intracellular trafficking of plasmids and subsequent gene expression.

Disruption of Microtubules Decreases Expression of Microinjected Plasmids

To confirm that microtubules are involved in the trafficking of plasmids to the nucleus, we microinjected green fluorescent protein (GFP) expressing plasmids into the cytoplasm or nucleus of TC7 cells and treated the cells with either taxol or nocodazole. When the plasmids were injected directly into the nuclei of cells, neither nocodazole nor taxol altered the number of cells expressing GFP or the amount of GFP expressed per cell, confirming that these drugs are not greatly affecting transcription in these cells (Figure 2a). By contrast, when plasmids were injected cytoplasmically and GFP expression was examined 5 hours post-injection, nocodazole treatment was found to significantly decrease the ability of the plasmids to express (Figure 2b). Similar results were seen in A549 cells (data not shown). These results again suggest that the microtubule network plays a role in DNA trafficking.

Figure 2. Disruption of the microtubule network or inhibition of dynein results in decreased DNA trafficking and gene expression following cytoplasmic microinjection of plasmids.

Figure 2

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TC7 cells were microinjected with pCMV-GFP-DTS (0.5 mg/ml) into the nucleus (A) or cytoplasm (B) and five hours later, the percentage of GFP-expressing cells was determined. Cells were incubated with vehicle (DMSO) or nocodazole (20 µM) immediately following microinjection of plasmid or co-injected with a control IgM (IgM) or the anti-dynein 70.1 antibody (anti-dynein) along with pCMV-GFP-DTS. At least 100 cells were injected for each condition and the experiment was repeated three times (mean % expressing cells ± st. dev.).

Dynein Inhibition Decreases Expression from Microinjected Plasmids

Most trafficking of cargo along microtubules toward the nucleus uses the major minus end directed motor dynein. As such, we sought to determine if inhibition of dynein would also inhibit intracellular plasmid movement. To address this, we co-injected the GFP-expressing plasmids with either anti-dynein or control antibodies and measured GFP expression 5 hours later (Figure 2). When the plasmids and antibodies were co-injected into the nucleus, no change in GFP expression was noted (Figure 2A). However, when the plasmids were cytoplasmically injected, co-injection with anti-dynein but not the control antibody, significantly decreased GFP expression levels, suggesting that dynein plays a role in DNA movement (Figure 2B).

An Intact Microtubule Network is Required for Efficient Nuclear Localization of Plasmids

The previous data suggest that plasmids utilize the microtubule network and the retrograde motor dynein for intracellular trafficking to the nucleus. However, this data is based on the downstream readout of gene expression. To visualize plasmid trafficking directly, plasmids were labeled with fluorescent PNAs and cytoplasmically microinjected into TC7 cells. Two hours prior to injection, cells were treated with either nocodazole to disrupt the microtubule network or vehicle alone (DMSO), and the drugs were maintained in the culture medium following microinjection. Immediately following microinjection, the labeled DNA was evenly distributed either throughout the cytoplasm at or near the site of injection (not shown). At 5 hours post-injection, the majority of the plasmids in cells treated with vehicle alone were found localized either within the nucleus or directly around it (Figure 3A). In fact, strong nuclear localization was observed in most vehicle treated cells by 3 hours. By contrast, in cells treated with nocodazole, plasmids were located diffusely throughout the cell and did not demonstrate any specific pattern of localization at 5 hours post-injection (Figure 3B). These results suggest that the effects of microtubule organization altering drugs directly affect plasmid trafficking through the cytoplasm.

Figure 3. Disruption of the microtubule network inhibits trafficking of plasmids to the nucleus.

Figure 3

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A derivative of pCMV-GFP-DTS was fluorescently labeled with Cy3-PNA and the labeled plasmid (0.5 mg/ml) was microinjected into the cytoplasm of TC7 cells that had been pre-treated for one hour with DMSO (vehicle)(A) or nocodazole (20 µM)(B). Five hours after injection and continued drug treatment, plasmids were visualized in the living cells. Images are representative of over 200 injected cells in three separate experiments.

Plasmids Interact with Microtubules in the Presence of Cellular Proteins

Taken together, these findings suggest that plasmids interact with microtubules via dynein and other proteins. Consequently, we sought to confirm in vitro that plasmids could, in fact, interact with microtubules. A microtubule spin-down assay was used, in which plasmid DNA, cell extracts, and stabilized microtubules were allowed to interact and then centrifuged to separate polymerized microtubules and any proteins or DNA interacting with the microtubules from the reaction. After separating the pellet (containing polymerized microtubules) and the supernatant, quantitative PCR was performed to determine the location of the DNA (Figure 4). When the assays were performed in the absence of microtubules or in the presence of unstabilized tubulin (in the absence of taxol), plasmid was found only in the supernatant, confirming that it does not pellet on its own. DNA also failed to pellet when incubated with either stabilized microtubules or cytoplasmic extracts alone. However, when cell extract was added to stabilized microtubules, the plasmid was found in the pellet with the microtubules. Further, increasing amounts of DNA pelleted with the microtubules as the amounts of cell extract were increased. When a similar spin-down assay was used to determine whether plasmids could also interact with actin filaments, no interaction under any condition, with or without cell extracts, was detected (not shown). Taken together, these results demonstrate that plasmid can interact, most likely through adapter proteins, with microtubules for movement through the cytoplasm.

Figure 4. Quantitative analysis of DNA association with microtubules.

Figure 4

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Non-polymerized tubulin or taxol-stabilized microtubules were incubated with pCMV-Lux-DTS in the presence or absence of cell extract (10 mg/ml) and subsequently separated over a glycerol cushion by centrifugation. The plasmid content of the pellets (containing polymerized microtubules and associated proteins/DNA) and supernatants was determined by real time quantitative PCR to determine where the plasmid localized. Mean DNA concentration from three independent experiments, performed in duplicate are shown ± st. dev.

DISCUSSION

When plasmids enter the cytoplasm by means such as electroporation or microinjection, they need to travel at distances ranging from 1 µm in small cells to hundreds of micrometers in larger cells. Yet, since diffusion through the dense meshwork of the cytoplasm is not likely, there must be other mechanisms by which plasmids navigate through the cytoplasm[4]. Understanding this intracellular trafficking is imperative to improve the ability of plasmids to reach the nucleus following not only electroporation or direct injection, but for all methods of transfection. For instance, it has been shown that lipid-DNA complexes can accumulate in the perinuclear region after endocytosis, but following endosomal escape, the DNA must still travel some distance to enter the nucleus [3, 27]. Therefore, regardless of the method by which DNA is introduced into a cell, at some point it must traverse the cytoplasm to reach the nucleus.

In this study, we found that reorganization of the microtubule network greatly affected plasmid trafficking and expression. Stabilization of the microtubule network lead to enhanced plasmid expression in electroporated cells, while disassembly of the microtubules decreased expression and trafficking in microinjected cells. Further, inhibition of dynein, the microtubule-associated retrograde motor, decreased DNA trafficking and subsequent gene expression in microinjected cells. The ability of plasmids to interact with microtubules was confirmed using in vitro binding assays. These results demonstrate that plasmids, like various cellular and exogenous proteins and viruses, use the microtubule network for retrograde transport to the nucleus.

It was rather surprising that the effects of drug treatment were not identical in both gene transfer systems. Whereas microtubule disassembly inhibited gene expression following microinjection, it had no effect on expression in electroporated cells, and similarly, while microtubule stabilization increased gene expression in electroporated cells, it did not in injected cells. One possibility for these seemingly disparate results could be due to the initial intracellular distribution of plasmids following the two different delivery methods. When cells are electroporated, DNA enters the cell across the entire plasma membrane and is distributed throughout the cytoplasm, relatively uniformly [28, 29]. Consequently, some of the plasmids enter the cell in close proximity to the nucleus, if one assumes that epithelial cells in culture resemble fried eggs. The expression measured in nocodazole treated cells may be simply a reflection of plasmid that enters the cytoplasm in the immediate vicinity of the nucleus, whereas plasmid on the inner face of the plasma membrane at locations far from the nucleus may indeed fail to traffic in the absence of microtubules in these cells. Microinjection, on the other hand, deposits plasmids at one point far from the nucleus (based on our method of microinjection) and as such would be more affected by the loss of the microtubule network. Similar logic could account for why stabilization of the microtubule network did not increase expression in microinjected cells: with the majority of plasmids concentrated at one place which are largely unable to diffuse, access to microtubules is limited. Thus, even with an increased number of stabilized microtubules, the plasmids may be unable to reach and utilize them for increased trafficking.

Verkman and colleagues have shown that microinjected DNA fragments greater than 2000 base pairs in length show no translational diffusion through the cytoplasm [4]. More recent work from this group has shown that the actin cytoskeleton is the principle structure that limits this passive diffusion through the cytoplasm, and that disruption of the actin cytoskeleton by drugs results in an increase in the diffusion of large macromolecules or linear DNA fragments [30]. Similar earlier findings from Popov and Poo support these studies [31]. Although we see no effect on intracellular plasmid trafficking upon stabilization or disassembly of the actin cytoskeleton, this may not be counter to these reports. Indeed, our study has focused not on measuring rates of diffusion through the cytoplasm on small time scales (seconds to minutes), but rather the directed movement of DNA on longer time scales (hours) as measured by gene expression and subcellular localization. Thus, disruption of the actin network may play a significant role in local movement over very short times, but this role may be lessened over time, as microtubules play the predominant role in DNA movement.

Our results strongly suggest that plasmid DNA uses microtubules and dynein to reach the nucleus, but the mechanisms of DNA-dynein complex formation is as yet unknown. Recent work by Mesika et. al. has shown that the addition of protein nuclear localization signals (NLSs) to plasmids facilitates their transport in the cytoplasm, based on analysis of fluorescently labeled plasmids following microinjection [32]. When plasmids containing binding sites for the p50 subunit of NF-kB were complexed with the protein and microinjected into cells, the plasmids localized to the area around the nuclear envelope and even in the nucleus. Further, this movement appeared to be dependent on dynein, since its inhibition decreased the nuclear localization of the complexes. By contrast, when uncomplexed plasmids were injected into the cells, very little movement was detected. In other studies, Hanz et. al. demonstrated that importinα was constitutively bound to dynein in neurons and could form a complex with importinβ and NLS containing proteins to transport the complex to the nucleus along microtubules [33]. More recent work from this group has reported that the protein vimentin can bind to importinβ through an NLS-independent mechanism, and that this complex can interact with dynein and microtubules, through importinα, for transport through the axon to the cell body, demonstrating that multiprotein complexes can be formed for transport along the microtubule network [34].

We and others have demonstrated that nuclear import of plasmids is sequence-dependent, and we have identified several DNA sequences that mediate DNA nuclear import [35–39]. All of these DNA nuclear targeting sequences (DTSs), including the SV40 DTS, bind to multiple transcription factors, and since transcription factors function in the nucleus and thus, contain NLSs, the DTS becomes coated with NLSs provided by these proteins. Our current model is that these NLSs on the DNA-bound transcription factors can then facilitate entry of the DNA-protein complex into the nucleus in an importinα/β- and RAN-dependent process [37, 40–42]. Based on this model, it is also possible that these same transcription factors could serve as adapters between the plasmid, importinα/β, and dynein, to promote cytoplasmic trafficking of the DNA complexes toward the nuclear envelope. However, all of the plasmids used in this study contain not only the SV40 DTS, but also other eukaryotic control elements such as promoters, introns, and polyadenylation sites, suggesting that perhaps these sequences may act as scaffolds for the adapter proteins. Thus, whether microtubule trafficking of plasmids requires specific sequences or not, and what these sequences may be, remains to be seen.

ACKNOWLEDGEMENTS

We would like to thank R. Chris Geiger and Josh Gasiorowski for insightful discussions and technical advice. This work was supported in part by grants HL59956 and HL71863 and by a predoctoral fellowship from the American Heart Association, Midwest Affiliate (EEV).

REFERENCES

  • 1.Elouahabi A, Ruysschaert JM. Formation and intracellular trafficking of lipoplexes and polyplexes. Mol Ther. 2005;11:336–347. doi: 10.1016/j.ymthe.2004.12.006. [DOI] [PubMed] [Google Scholar]
  • 2.Gao X, Huang L. Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes. Nucleic Acids Res. 1993;21:2867–2872. doi: 10.1093/nar/21.12.2867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem. 1995;270:18997–19007. doi: 10.1074/jbc.270.32.18997. [DOI] [PubMed] [Google Scholar]
  • 4.Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS. Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem. 2000;275:1625–1629. doi: 10.1074/jbc.275.3.1625. [DOI] [PubMed] [Google Scholar]
  • 5.Dowty ME, Williams P, Zhang G, Hagstrom JE, Wolff JA. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes. Proc Natl Acad Sci U S A. 1995;92:4572–4576. doi: 10.1073/pnas.92.10.4572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell. 1998;95:5–8. doi: 10.1016/s0092-8674(00)81774-2. [DOI] [PubMed] [Google Scholar]
  • 7.Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991;51:6304–6311. [PubMed] [Google Scholar]
  • 8.Lane DP. Cancer. p53, guardian of the genome. Nature. 1992;358:15–16. doi: 10.1038/358015a0. [DOI] [PubMed] [Google Scholar]
  • 9.Giannakakou P, Sackett DL, Ward Y, Webster KR, Blagosklonny MV, Fojo T. p53 is associated with cellular microtubules and is transported to the nucleus by dynein. Nat Cell Biol. 2000;2:709–717. doi: 10.1038/35036335. [DOI] [PubMed] [Google Scholar]
  • 10.Giannakakou P, Nakano M, Nicolaou KC, O'Brate A, Yu J, Blagosklonny MV, Greber UF, Fojo T. Enhanced microtubule-dependent trafficking and p53 nuclear accumulation by suppression of microtubule dynamics. Proc Natl Acad Sci U S A. 2002;99:10855–10860. doi: 10.1073/pnas.132275599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Vihinen-Ranta M, Yuan W, Parrish CR. Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport. J Virol. 2000;74:4853–4859. doi: 10.1128/jvi.74.10.4853-4859.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ogawa-Goto K, Tanaka K, Gibson W, Moriishi E, Miura Y, Kurata T, Irie S, Sata T. Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid. J Virol. 2003;77:8541–8547. doi: 10.1128/JVI.77.15.8541-8547.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Bukrinskaya A, Brichacek B, Mann A, Stevenson M. Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton. J Exp Med. 1998;188:2113–2125. doi: 10.1084/jem.188.11.2113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lycke E, Kristensson K, Svennerholm B, Vahlne A, Ziegler R. Uptake and transport of herpes simplex virus in neurites of rat dorsal root ganglia cells in culture. J Gen Virol. 1984;65(Pt 1):55–64. doi: 10.1099/0022-1317-65-1-55. [DOI] [PubMed] [Google Scholar]
  • 15.Lycke E, Hamark B, Johansson M, Krotochwil A, Lycke J, Svennerholm B. Herpes simplex virus infection of the human sensory neuron. An electron microscopy study. Arch Virol. 1988;101:87–104. doi: 10.1007/BF01314654. [DOI] [PubMed] [Google Scholar]
  • 16.Penfold ME, Armati P, Cunningham AL. Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly. Proc Natl Acad Sci U S A. 1994;91:6529–6533. doi: 10.1073/pnas.91.14.6529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Suomalainen M, Nakano MY, Keller S, Boucke K, Stidwill RP, Greber UF. Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. J Cell Biol. 1999;144:657–672. doi: 10.1083/jcb.144.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Forest T, Barnard S, Baines JD. Active intranuclear movement of herpesvirus capsids. Nat Cell Biol. 2005;7:429–431. doi: 10.1038/ncb1243. [DOI] [PubMed] [Google Scholar]
  • 19.Gouin E, Welch MD, Cossart P. Actin-based motility of intracellular pathogens. Curr Opin Microbiol. 2005;8:35–45. doi: 10.1016/j.mib.2004.12.013. [DOI] [PubMed] [Google Scholar]
  • 20.Vacik J, Dean BS, Zimmer WE, Dean DA. Cell-specific nuclear import of plasmid DNA. Gene Ther. 1999;6:1006–1014. doi: 10.1038/sj.gt.3300924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Dean DA, Dean BS, Muller S, Smith LC. Sequence requirements for plasmid nuclear entry. Exp. Cell Res. 1999;253:713–722. doi: 10.1006/excr.1999.4716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Gasiorowski JZ, Dean DA. Postmitotic Nuclear Retention of Episomal Plasmids Is Altered by DNA Labeling and Detection Methods. Mol Ther. 2005;12:460–467. doi: 10.1016/j.ymthe.2005.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology. New York: 1994. [Google Scholar]
  • 24.Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, Malone RW. Theory and in vivo application of electroporative gene delivery. Mol Ther. 2000;2:178–187. doi: 10.1006/mthe.2000.0124. [DOI] [PubMed] [Google Scholar]
  • 25.Rosette C, Karin M. Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kappa B. J Cell Biol. 1995;128:1111–1119. doi: 10.1083/jcb.128.6.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Wang L, MacDonald RC. Effects of microtubule-depolymerizing agents on the transfection of cultured vascular smooth muscle cells: enhanced expression with free drug and especially with drug-gene lipoplexes. Mol Ther. 2004;9:729–7237. doi: 10.1016/j.ymthe.2004.02.009. [DOI] [PubMed] [Google Scholar]
  • 27.Coonrod A, Li FQ, Horwitz M. On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Ther. 1997;4:1313–1321. doi: 10.1038/sj.gt.3300536. [DOI] [PubMed] [Google Scholar]
  • 28.Golzio M, Teissie J, Rols MP. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci U S A. 2002;99:1292–1297. doi: 10.1073/pnas.022646499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Golzio M, Rols MP, Teissie J. In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression. Methods. 2004;33:126–135. doi: 10.1016/j.ymeth.2003.11.003. [DOI] [PubMed] [Google Scholar]
  • 30.Dauty E, Verkman AS. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery. J Biol Chem. 2005;280:7823–7828. doi: 10.1074/jbc.M412374200. [DOI] [PubMed] [Google Scholar]
  • 31.Popov S, Poo MM. Diffusional transport of macromolecules in developing nerve processes. J Neurosci. 1992;12:77–85. doi: 10.1523/JNEUROSCI.12-01-00077.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Mesika A, Kiss V, Brumfeld V, Ghosh G, Reich Z. Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein. Hum Gene Ther. 2005;16:200–208. doi: 10.1089/hum.2005.16.200. [DOI] [PubMed] [Google Scholar]
  • 33.Hanz S, Perlson E, Willis D, Zheng JQ, Massarwa R, Huerta JJ, Koltzenburg M, Kohler M, van-Minnen J, Twiss JL, Fainzilber M. Axoplasmic importins enable retrograde injury signaling in lesioned nerve. Neuron. 2003;40:1095–1104. doi: 10.1016/s0896-6273(03)00770-0. [DOI] [PubMed] [Google Scholar]
  • 34.Perlson E, Hanz S, Ben-Yaakov K, Segal-Ruder Y, Seger R, Fainzilber M. Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve. Neuron. 2005;45:715–726. doi: 10.1016/j.neuron.2005.01.023. [DOI] [PubMed] [Google Scholar]
  • 35.Dean DA. Import of plasmid DNA into the nucleus is sequence specific. Exp. Cell Res. 1997;230:293–302. doi: 10.1006/excr.1996.3427. [DOI] [PubMed] [Google Scholar]
  • 36.Langle-Rouault F, Patzel V, Benavente A, Taillez M, Silvestre N, Bompard A, Sczakiel G, Jacobs E, Rittner K. Up to 100-fold increase of apparent gene expression in the presence of Epstein-Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids. J Virol. 1998;72:6181–6185. doi: 10.1128/jvi.72.7.6181-6185.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Mesika A, Grigoreva I, Zohar M, Reich Z. A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei. Mol Ther. 2001;3:653–657. doi: 10.1006/mthe.2001.0312. [DOI] [PubMed] [Google Scholar]
  • 38.Vacik J, Dean BS, Zimmer WE, Dean DA. Cell-specific nuclear import of plasmid DNA. Gene Therapy. 1999;6:1006–1014. doi: 10.1038/sj.gt.3300924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Dean DA. Nucleocytoplasmic trafficking. In: Mahato RI, editor. Pharmaceutical perspectives of nucleic acid-based therapeutics. London: Harwood Academic Publishers; 2002. pp. 229–260. [Google Scholar]
  • 40.Wilson GL, Dean BS, Wang G, Dean DA. Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J. Biol. Chem. 1999;274:22025–22032. doi: 10.1074/jbc.274.31.22025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Dean DA, Strong DD, Zimmer WE. Nuclear entry of nonviral vectors. Gene Ther. 2005;12:881–890. doi: 10.1038/sj.gt.3302534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Vaysse L, Harbottle R, Bigger B, Bergau A, Tolmachov O, Coutelle C. Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein. J Biol Chem. 2004;279:5555–5564. doi: 10.1074/jbc.M311894200. [DOI] [PubMed] [Google Scholar]

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