Intracellular trafficking of plasmids during transfection is mediated by microtubules

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.

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

(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.

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

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.).

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

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.

Figure 4. Quantitative analysis of DNA association with microtubules

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.