Trafficking Of DNA Research Articles

Evaluation of cationic liposomes composed of an amino acid–based lipid for neuronal transfection

We investigated the ability of cationic liposomes composed of 1,5-dihexadecyl N-arginyl-L-glutamate (Arg-Glu2C 16) to carry nucleic acids into neuronal cells. Such liposomes have been shown to have a remarkable capacity for transfecting immortalized cell lines. Lipoplexes between the Arg-Glu2C 16 liposomes and plasmid DNA encoding green fluorescent protein (GFP) were analyzed in terms of lipoplex formation, intracellular DNA trafficking, transfection efficiency, and cytotoxicity in neuronal SH-SY5Y cells. A maximum number of cells expressing GFP was obtained with lipoplexes at a lipid-to-DNA ratio of 15. With these lipoplexes, 16% of the cells were GFP-positive, which was approximately fourfold higher than the level obtained with a commercially available transfection reagent, Lipofectamine 2000. Furthermore, as a result of the low cytotoxicity of the Arg-Glu2C 16 lipoplexes, the proportion of GFP-positive cells could be increased to 25% by increasing the concentration of lipoplexes that was applied to the cells. We have demonstrated that Arg-Glu2C 16, as a model cationic amino acid–based lipid, has a high capability as a gene carrier, even for neuronal transfection. From the Clinical Editor In this study, specific cationic liposomes were characterized as nucleic acid transfection agents for neuronal cells. A fourfold higher transfection rate with low cytotoxicity was reported compared to Lipofectamine 2000, a commercial reagent. The authors conclude that the studied cationic liposomes have a high capability as a gene carrier for neuronal transfection. This may become clinically significant in future gene therapy efforts of neuronal diseases.

HIV-1 exploits importin 7 to maximize nuclear import of its DNA genome.

BackgroundNuclear import of the HIV-1 reverse transcription complex (RTC) is critical for infection of non dividing cells, and importin 7 (imp7) has been implicated in this process. To further characterize the function of imp7 in HIV-1 replication we generated cell lines stably depleted for imp7 and used them in conjunction with infection, cellular fractionation and pull-down assays.ResultsImp7 depletion impaired HIV-1 infection but did not significantly affect HIV-2, simian immunodeficiency virus (SIVmac), or equine infectious anemia virus (EIAV). The lentiviral dependence on imp7 closely correlated with binding of the respective integrase proteins to imp7. HIV-1 RTC associated with nuclei of infected cells with remarkable speed and knock down of imp7 reduced HIV-1 DNA nuclear accumulation, delaying infection. Using an HIV-1 mutant deficient for reverse transcription, we found that viral RNA accumulated within nuclei of infected cells, indicating that reverse transcription is not absolutely required for nuclear import. Depletion of imp7 impacted on HIV-1 DNA but not RNA nuclear import and also inhibited DNA transfection efficiency.ConclusionAlthough imp7 may not be essential for HIV-1 infection, our results suggest that imp7 facilitates nuclear trafficking of DNA and that HIV-1 exploits imp7 to maximize nuclear import of its DNA genome. Lentiviruses other than HIV-1 may have evolved to use alternative nuclear import receptors to the same end.

Microtubule Acetylation Through HDAC6 Inhibition Results in Increased Transfection Efficiency

The success of viral and nonviral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm and reach the nucleus. We, as well as other researchers, have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules with nocodazole was shown to greatly inhibit cytoplasmic plasmid trafficking, it did not abolish it. It has been demonstrated that a pool of stabilized post-translationally acetylated microtubules exists in cells, and that this acetylation may play a role in protein trafficking. In order to determine whether this modification could account for the residual DNA trafficking in nocodazole-treated cells, we inhibited or knocked down the levels of the tubulin deacetylase, histone deacetylase 6 (HDAC6), thereby generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not because of increased transcriptional activity, but rather, because of increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild-type cells. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.

NSP-Interacting GTPase

Despite the significant progress in the identification of essential components of the nuclear transport machinery, some events of this process are still unclear. Particularly, functional information about the release of nuclear-exported macromolecules at the cytoplasmic side of the nuclear pore complex and their subsequent trans-cytoplasmic movement is lacking. Recently, we identified a cytoplasmic GTPase, designated NIG (NSP-interacting GTPase), which may play a relevant role in these processes. NIG interacts in vivo with the geminivirus NSP and promotes the translocation of the viral protein from the nucleus to the cytoplasm where it is redirected to the cell surface to interact with the viral movement protein, MP. Here we position the NIG function into the mechanistic model for the intracellular trafficking of viral DNA and discuss the putative role of NIG in general cellular nucleocytoplasmic transport of nucleic acid-protein complexes.Addendum to: Carvalho CM, Fontenelle MR, Florentino LH, Santos AA, Zerbini FM, Fontes EPB. A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein. Plant J 2008; DOI: 10.1111/j.1365-313X.2008.03556.x.

Fluorescence in situ hybridization to monitor the intracellular location and accessibility of plasmid DNA delivered by cationic polymer-based gene carriers

Information about the intracellular trafficking of exogenous DNA delivered by nonviral gene delivery systems is of major importance for optimization of such gene carriers. We used fluorescence in situ hybridization (FISH) as a tool to visualize polyplex-delivered pDNA inside cells. This avoids the need to directly label DNA inside the polyplexes, which may influence their cellular behavior and fate. Using FISH the introduced plasmid DNA could be detected in the cytosol and nucleus of different cell lines. The FISH probe itself did not interact with cells nor different polymers used for condensing the DNA. We further demonstrate differences in accessibility of polyplex-delivered DNA when different polymers were used for DNA complexation. Therefore, FISH is a valuable tool to detect location and accessibility of exogenous plasmid DNA delivered in the cell by cationic polymers.

Intranuclear trafficking of plasmid DNA is mediated by nuclear polymeric proteins lamins and actin.

Functions of nuclear polymeric proteins such as lamin A/C and actin in transport of plasmid DNA were studied. The results show that the lamina plays an important role in plasmid DNA's entry into the cell nucleus from the cytoplasm. Selective disruption of lamin A/C led to a halt in plasmid DNA transport through the nuclear envelope. Inside the nucleus, plasmid DNA was frequently localized at sites with impaired genome integrity, such as DNA double-strand breaks (DSBs), occurring spontaneously or induced by ionizing radiation. Polymeric actin obviously participates in nuclear transport of plasmid DNA, since inhibition of actin polymerization by latrunculin B disturbed plasmid transport inside the cell nucleus. In addition, precluding of actin polymerization inhibited plasmid co-localization with newly induced DSBs. These findings indicate the crucial role of polymeric actin in intranuclear plasmid transport.

Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed From Quantum Dot-FRET

A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.

Endocytosis of DNA-Hsp65 Alters the pH of the Late Endosome/Lysosome and Interferes with Antigen Presentation

BackgroundExperimental models using DNA vaccine has shown that this vaccine is efficient in generating humoral and cellular immune responses to a wide variety of DNA-derived antigens. Despite the progress in DNA vaccine development, the intracellular transport and fate of naked plasmid DNA in eukaryotic cells is poorly understood, and need to be clarified in order to facilitate the development of novel vectors and vaccine strategies.Methodology and Principal FindingsUsing confocal microscopy, we have demonstrated for the first time that after plasmid DNA uptake an inhibition of the acidification of the lysosomal compartment occurs. This lack of acidification impaired antigen presentation to CD4 T cells, but did not alter the recruitment of MyD88. The recruitment of Rab 5 and Lamp I were also altered since we were not able to co-localize plasmid DNA with Rab 5 and Lamp I in early endosomes and late endosomes/lysosomes, respectively. Furthermore, we observed that the DNA capture process in macrophages was by clathrin-mediated endocytosis. In addition, we observed that plasmid DNA remains in vesicles until it is in a juxtanuclear location, suggesting that the plasmid does not escape into the cytoplasmic compartment.Conclusions and SignificanceTaken together our data suggests a novel mechanism involved in the intracellular trafficking of plasmid DNA, and opens new possibilities for the use of lower doses of plasmid DNA to regulate the immune response.

Bacterial Cytoskeleton: Not Your Run-of-the-Mill Tubulin

Large plasmids of some Bacillus species encode a distinct tubulin homolog, TubZ, implicated in maintenance of the host plasmid. A recent study has shown that TubZ polymers exhibit treadmilling behavior in vivo, suggesting that they are involved in mitotic activity.

Prenatal screening and diagnosis—An introduction

Prenatal screening and diagnosis—An introduction

Modeling DNA and Virus Trafficking in the Cell Cytoplasm

Cytosol trafficking is a limiting step of viral infection or DNA delivery. Starting from the cell surface, most viruses have to travel through a crowded and risky environment in order to reach a small nuclear pore. This work is dedicated to estimating the probability p N of a viral arrival success and, in that case, the mean time τ N it takes. Viral movement is described by a stochastic equation, containing both a drift and a Brownian component. The drift part represents the movement along microtubules, while the Brownian component corresponds to the free diffusion. The success of a viral infection is limited by a killing activity occurring inside the cytoplasm. We model the killing activity by a steady state killing rate k. Because nuclear pores occupy a small fraction of the nuclear area, we use this property to obtain asymptotic estimates of p N and τ N as a function of the diffusion constant D, the amplitude of the drift B and the killing rate k.

Uptake, Biodistribution, and Time Course of Naked Plasmid DNA Trafficking After Intratumoral In Vivo Jet Injection

Uptake, Biodistribution, and Time Course of Naked Plasmid DNA Trafficking After Intratumoral In Vivo Jet Injection

The bacterial segrosome: a dynamic nucleoprotein machine for DNA trafficking and segregation

The genomes of unicellular and multicellular organisms must be partitioned equitably in coordination with cytokinesis to ensure faithful transmission of duplicated genetic material to daughter cells. Bacteria use sophisticated molecular mechanisms to guarantee accurate segregation of both plasmids and chromosomes at cell division. Plasmid segregation is most commonly mediated by a Walker-type ATPase and one of many DNA-binding proteins that assemble on a cis-acting centromere to form a nucleoprotein complex (the segrosome) that mediates intracellular plasmid transport. Bacterial chromosome segregation involves a multipartite strategy in which several discrete protein complexes potentially participate. Shedding light on the basis of genome segregation in bacteria could indicate new strategies aimed at combating pathogenic and antibiotic-resistant bacteria.

688. The Effects of Cyclic Stretch on Microtubule Stability and Exogenous Gene Expression in the Alveolar Epithelium

Top of pageAbstract Cells in the lung undergo cyclic deformation under both normal breathing processes as well as mechanical ventilation. While large deformations due to disease or mechanical ventilation can cause cellular damage such as that seen in ventilator induced lung injury, our lab has found that smaller, physiologically relevant deformations have a positive effect on gene transfer in an in vitro pulmonary epithelium model, increasing reporter gene products up to 12-fold in A549 cells, a lung adenocarcinoma cell line frequently used in cellular models of the pulmonary epithelium. This result is not that surprising considering exogenous mechanical forces have been shown by a number of groups to alter the same barriers efficient gene transfer faces as a vector moves from the outside of the cell to the nucleus; namely alterations in the cytoskeleton and extracellular matrix, activation of cell signaling pathways and changes in transcription factor activation. We have previously shown that this increase in exogenous gene expression is due to increased trafficking of the plasmid DNA through the cytosol toward the nucleus, and that cytoskeletal rearrangement, including depolymerization of the microtubule network, is required for stretch-enhanced gene transfer and expression to occur. These results are somewhat puzzling as our lab and others have also shown that the cytoskeleton, specifically the microtubule network, is required for cytoplasmic trafficking of plasmid DNA. One type of post-polymerization modification that microtubules undergo is acetylation, an activity that is not well understood, but has been shown through the use of a variety of drugs to potentially increase microtubule stability against depolymerization from cold or drug-treatment. We wanted to see if this stabilization also applied to exogenous mechanical forces, and if this stabilization allowed for stretch-enhanced gene transfer and expression to occur. To do this, A549 cells were exposed to equibiaxial stretch (10% change in membrane surface area, 15 cycles per minute). Significant increases in gene expression were seen within 15 minutes of the applied stretch. Overexpression of acetylated microtubules through the addition of either tricostatin A, a drug known to inhibit all histone deacetylases (HDACs), or tubacin, a small-molecule inhibitor of only HDAC6, the specific deacetylase for microtubules, caused a significant increase in gene expression over untreated controls. Changes in acetylated microtubule levels were examined via western blot analysis and immunofluoresent microscopy in both cells treated with drugs that affected HDAC activity as well as cells that were stretched. These data demonstrated that a majority of the microtubules in cells that were stretched were also acetylated. Taken together, these results suggest that acetylated microtubules may be resistant to depolymerization due to mechanical forces, a result not previously demonstrated in the literature, and that acetylation may also enhance gene expression through enhanced trafficking of the plasmid DNA through the cytosol.

518. Mechanism of Difference in the Transcription Activity of Nucleus-Delivered Transgene Introduced by Adenovirus and Lipoplex

To develop a highly potent artificial vector, which represents a comparable transfection activity to the viral vectors, we must comprehend why and to what extent artificial vectors are inferior to the viral ones. Recently, we demonstrated a systematic and quantitative comparison of intracellular trafficking of exogenous DNA transfected by viral and non-viral, using a combination of TaqMan PCR and a recently developed confocal image-assisted 3-dimensionally integrated quantification (CIDIQ) method. As a result, we found that LipofectAMINE PLUS (LFN) requires three orders of magnitude more intra-nuclear gene copies to exhibit a trans-gene expression comparable to the adenovirus (Ad), suggesting that difference of transfection efficiency principally arises from differences in nuclear transcription (Hama et al. Mol Ther, in press). Here, we address the mechanism for the prominent differences of nuclear transcription.

748. Quantitative Comparison of the Intracellular Trafficking and Expression Pattern of the Exogenous Genes between the Adenovirus and Artificial Vector

Top of pageAbstract Purpose: Despite great advantages such as low immunogenicity and cytotoxicity, in general, the utility of an artificial vector is hampered because trans-gene expression of artificial vectors is less efficient than that of viral one. To improve the artificial vectors to the comparable level of the viral ones in terms of the trans-gene expression, we must comprehend why and to what extent artificial vectors are inferior to the viral ones. Here, we quantitatively compared the cellular uptake and following intracellular trafficking of exogenous DNA transfected by adenovirus (Ad) and LipofectAMINE PLUS (LFN), highly potent and widely used virus and artificial vectors, respectively. Methods: Trans-gene expression by both vectors was quantified by luciferase assay. The intracellular distribution of the exogenous genes (i.e. endosome/lysosome, cytosol and nucleus) was evaluated in terms of the number of the luciferase gene copies by combination of real time PCR and recently developed technique of confocal image-assisted 3-dimensionally integrated quantification (CIDIQ). In brief, total cellular uptake of exogenous DNA transfected by Ad and LFN is quantified by real time PCR. In LFN, Fraction of DNA in cytosol, endosomes/ lysosomes (end/lys) or nucleus is estimated by CIDIQ. In Ad, nuclear DNA was first assessed by nuclear isolation followed by real time PCR and the distribution of Ad in cytosol or end/lys is estimated by CIDIQ. Results: At 1hr post-transfection, both Ad and LFN were found at nucleus. Futhermore, Consistent with the rapid nuclear delivery of the DNA, trans-gene expression was observed within 3hr and the time profiles of the trans-gene expression were quite comparable thereafter. However, LFN needs approximately three orders of magnitude more gene copies in the culture medium than that of Ad to achieve the comparable level trans-gene expression. The efficiency of cellular uptake in LFN was significantly higher than in Ad. However, once taken up into cell, nuclear fraction of Ad was significantly higher than that of pDNA transfected by LFN, considering that the efficiency of endosomal escape was about comparable in both vectors, the potential of Ad to deliver the its DNA from cytosol to the nucleus is higher than that of LFN. Moreover, LFN needs three orders of magnitude more intra-nuclear gene copies to exhibit the comparable trans-gene expression to the Ad. In other words, pDNA transfected by LFN need much larger amount of gene copies than Ad to represent a comparable level of trans-gene expression. Conclusion: The differences of transfection efficiency between Ad and LFN were derived from the differences of the nuclear translocation and especially transcriptional processes. Improving these process would lead to the development of high efficient gene vector.

Potocytosis and cellular exit of complexes as cellular pathways for gene delivery by polycations

Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified. The trafficking of plasmid DNA complexed to glycosylated polylysines or polyethylenimine (PEI) derivatives was studied by electron microscopy of human airway epithelial cells. The cellular processing of complexes varied with their size and the polycation derivative used: large complexes (> 200 nm) made with all polycationic vectors studied were internalized by macropinocytosis. In contrast, intermediate (100-200 nm) ligand-coupled polylysine and PEI complexes primarily entered through clathrin-coated pits. Complexes were then found in endosomal vesicles, accumulated in lysosomes or vesicles near the nucleus and their nuclear entry was limited. For the population of small complexes (< or = 100 nm) obtained with PEI derivatives, they were internalized through caveolae and pursued a traffic pattern of potocytosis to the endoplasmic reticulum where their fate remains unclear. Finally, some complexes exited the cells either by regurgitation when PEI derivatives were used or through an exosome-like pathway for glycosylated-polylysine complexes. The different pathways of complex trafficking observed in relation with complex size imply the development and study of vectors forming complexes with definite size. Moreover, the complex exit we describe may contribute to the well-established short-term efficiency of gene transfer based on synthetic vectors. It favors the engineering of vectors allowing repeated treatment.

Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus

Human herpesvirus 8 (HHV-8; also called Kaposi's sarcoma-associated herpesvirus), which is implicated in the pathogenesis of Kaposi's sarcoma (KS) and lymphoproliferative disorders, infects a variety of target cells both in vivo and in vitro. HHV-8 binds to several in vitro target cells via cell surface heparan sulfate and utilizes the alpha3beta1 integrin as one of its entry receptors. Interactions with cell surface molecules induce the activation of host cell signaling cascades and cytoskeletal changes (P. P. Naranatt, S. M. Akula, C. A. Zien, H. H. Krishnan, and B. Chandran, J. Virol. 77:1524-1539, 2003). However, the mechanism by which the HHV-8-induced signaling pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA is as yet undefined. Here we examined the role of HHV-8-induced cytoskeletal dynamics in the infectious process and their interlinkage with signaling pathways. The depolymerization of microtubules did not affect HHV-8 binding and internalization, but it inhibited the nuclear delivery of viral DNA and infection. In contrast, the depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, or infection. Early during infection, HHV-8 induced the acetylation of microtubules and the activation of the RhoA and Rac1 GTPases. The inactivation of Rho GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus. In contrast, the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor significantly augmented the nuclear delivery of viral DNA. Among the Rho GTPase-induced downstream effector molecules known to stabilize the microtubules, the activation of RhoA-GTP-dependent diaphanous 2 was observed, with no significant activation in the Rac- and Cdc42-dependent PAK1/2 and stathmin molecules. The nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA. HHV-8 capsids colocalized with the microtubules, as observed by confocal microscopic examination, and the colocalization was abolished by the destabilization of microtubules with nocodazole and by the phosphatidylinositol 3-kinase inhibitor affecting the Rho GTPases. These results suggest that HHV-8 induces Rho GTPases, and in doing so, modulates microtubules and promotes the trafficking of viral capsids and the establishment of infection. This is the first demonstration of virus-induced host cell signaling pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus. These results further support our hypothesis that HHV-8 manipulates the host cell signaling pathway to create an appropriate intracellular environment that is conducive to the establishment of a successful infection.

New technologies for vaccine development

AbstractVaccines offer protection against infectious diseases, but the successes from the years of development have been mixed. Some diseases have been effectively controlled or eradicated through vaccination, whereas treatments for a number of diseases, especially the reemerging diseases, currently do not exist. Improved understanding of pathogen variability as well as of the diversity of the human immune system, plus technological advances in genetic and molecular biology provide better insights in the host–pathogen interaction and new avenues for vaccine development. In this article we discuss the latest trends in vaccine research with focus on epitope‐based DNA vaccines and trafficking components for efficient vaccine delivery. These approaches are promising in dealing with host and pathogen diversity as well as with diversity within human populations. Drug Dev. Res. 62:383–392, 2004. © 2004 Wiley‐Liss, Inc.

Identification and characterization of traE genes of Spiroplasma kunkelii

Four traE homologs, designated traE1, traE2, traE3 and traE4, were identified and amplified from the genome of the leafhopper-transmitted corn stunt pathogen Spiroplasma kunkelii and were predicted to encode membrane-bound adenine tri-phosphatases (ATPases). Deduced proteins of all traE genes have 62.3% to 89.9% similarity to the conserved VirB4 domain that is frequently a component of type IV secretory pathways involved in intracellular trafficking and secretion of DNA and proteins. In phylogenetic analysis, TraE homologs of S. kunkelii, Mycoplasma pulmonis and Mycoplasma fermentans cluster together and are more similar to TraE proteins of Gram-positive bacteria than to those of Gram-negative bacteria, thereby resembling the 16S rRNA phylogeny. Gene traE2 was most conserved whereas the presence of the three other traE genes varied among S. kunkelii strains, M2, CS-2B, FL-80 and PU8-17. Further, traE1 and traE2 appeared to be located on the chromosome, and traE3 and traE4 genes on plasmids of S. kunkelii strain M2. Transcripts of the spiralin gene and traE2 genes were detected on Northern blots containing total ribonucleic acids (RNA) of S. kunkelii cultures and S. kunkelii-infected plants and insects, in which traE2 appeared to be of a larger transcription unit. Full-length expression products of the other traE genes were not detected. S. kunkelii traE genes could be involved in S. kunkelii cell morphogenesis, adhesion and DNA recombination.