Actin Cytoskeleton as the Principal Determinant of Size-dependent DNA Mobility in Cytoplasm

The cytosol of mammalian cells is a crowded environment containing soluble proteins and a network of cytoskeletal filaments. Gene delivery by synthetic vectors involves the endocytosis of DNA-polycation complexes, escape from endosomes, and diffusion of non-complexed DNA through the cytosol to reach the nucleus. We found previously that the translational diffusion of large DNAs (>250 bp) in cytoplasm was greatly slowed compared with that of smaller DNAs (Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. (2000) J. Biol. Chem. 275, 1625–1629). To determine the mechanisms responsible for size-dependent DNA diffusion, we used fluorescence correlation spectroscopy to measure the diffusion of single fluorophore-labeled DNAs in crowded solutions, cytosol extracts, actin network, and living cells. DNA diffusion (D) in solutions made crowded with Ficoll-70 (up to 40 weight percentage) or soluble cytosol extracts (up to 100 mg/ml) relative to diffusion of the same sized DNAs in saline (D/Do) was approximately independent of DNA size (20–4500 bp), quite different from the strong reduction in D/Do in the cytoplasm of living cells. However, the reduced D/Do with increasing DNA size was closely reproduced in solutions containing cross-linked actin filaments assembled with gelsolin, whereas soluble macromolecules of the same size and concentration did not reduce D/Do. In intact cells microinjected with fluorescent DNAs and studied by fluorescence correlation spectroscopy or photobleaching methods, D/Do was reduced by 5–150-fold (20–6000 bp); however, the size-dependent reduction in D/Do was abolished after actin cytoskeleton disruption. Our results identify the actin cytoskeleton as a major barrier restricting cytoplasmic transport of non-complexed DNA in non-viral gene transfer.