Actin Cytoskeleton as the Principal Determinant of Size-dependent DNA Mobility in Cytoplasm
Open accessAbstract
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/D(o)) was approximately independent of DNA size (20-4500 bp), quite different from the strong reduction in D/D(o) in the cytoplasm of living cells. However, the reduced D/D(o) 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/D(o). In intact cells microinjected with fluorescent DNAs and studied by fluorescence correlation spectroscopy or photobleaching methods, D/D(o) was reduced by 5-150-fold (20-6000 bp); however, the size-dependent reduction in D/D(o) 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.
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Disruption or reorganization of actin cytoskeleton, or depletion of keratin and vimentin, significantly increases diffusion of gene payloads.
“Disruption or reorganization of the actin cytoskeleton, or depletion of keratin and vimentin in cells, significantly increases the diffusion of gene payloads [190–192].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [191]
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- Authors
- Dauty, E., & Verkman, A. S.
- Affiliations
- University of California, San Francisco (US)
- Corresponding author
- Verkman, A.S.
- ORCID
- Verkman, A.S.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2005-03
- Volume
- 280
- Issue
- 9
- Pages
- 7823-7828
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2078392705
- Cited by
- 215
- References
- 41
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 6 · 2023: 5 · 2022: 4 · 2021: 4 · 2020: 7 · 2019: 9 · 2018: 9 · 2017: 8 · 2016: 10 · 2015: 13 · 2014: 11 · 2013: 19 · 2012: 9
- FWCI
- 7.05
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Cystic Fibrosis Foundation (EY13574)
- National Institutes of Health (DK35124, EB00415)
- National Heart, Lung, and Blood Institute (HL75856, R01HL059198, HL59198)
- National Eye Institute (R01EY013574, EY 13574)
- National Institute of Diabetes and Digestive and Kidney Diseases (DK 35124)
- National Institute of Biomedical Imaging and Bioengineering (EB 00415)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
- Physical Sciences › Chemistry › Spectroscopy › Diffusion Coefficients in Liquids
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- actin cytoskeleton, DNA diffusion, cytoplasmic transport, size-dependent diffusion, non-viral gene delivery, gelsolin, actin filament cross-linking, DNA microinjection, endosomal escape
- MeSH terms
- Actins, Cations, Cell Nucleus, Cytoplasm, Cytoskeleton, Cytosol, Diffusion, DNA, Dose-Response Relationship, Drug, Genetic Vectors, HeLa Cells, Humans, Light, Endosomes, Spectrometry, Fluorescence, Time Factors, Protein Biosynthesis, Gene Transfer Techniques
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