Intracellular tracking of single-plasmid DNA particles after delivery by electroporation.
Electroporation is a physical method of transferring molecules into cells and tissues. It takes advantage of the transient permeabilization of the cell membrane induced by electric field pulses, which gives hydrophilic molecules access to the cytoplasm. This method offers high transfer efficiency for small molecules that freely diffuse through electrically permeabilized membranes. Larger molecules, such as plasmid DNA, face several barriers (plasma membrane, cytoplasmic crowding, and nuclear envelope), which reduce transfection efficiency and engender a complex mechanism of transfer. Our work provides insight into the way electrotransferred DNA crosses the cytoplasm to reach the nucleus. For this purpose, single-particle tracking experiments of fluorescently labeled DNA were performed. Investigations were focused on the involvement of the cytoskeleton using drugs disrupting or stabilizing actin and tubulin filaments as the two relevant cellular networks for particle transport. The analysis of 315 movies (~4,000 trajectories) reveals that DNA is actively transported through the cytoskeleton. The large number of events allows a statistical quantification of the DNA motion kinetics inside the cell. Disruption of both filament types reduces occurrence and velocities of active transport and displacements of DNA particles. Interestingly, stabilization of both networks does not enhance DNA transport.
1] Department of Chemistry, University of Konstanz, Konstanz, Germany [2] CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France [3] University of Toulouse III Paul Sabatier, Toulouse, France.
Christelle
C
Rosazza
Annette
A
Buntz
Thorsten
T
Rieß
Dominik
D
Wöll
Andreas
A
Zumbusch
Marie-Pierre
MP
Rols
D000199:Actins / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D016466:CHO Cells; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D058948:Cell Tracking; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism*; D003599:Cytoskeleton; D047630:Depsipeptides / Q000494:pharmacology; D018274:Electroporation; D006801:Humans; D008856:Microscopy, Fluorescence; D017239:Paclitaxel / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D053778:Thiazolidines / Q000494:pharmacology; D014162:Transfection; D014404:Tubulin / Q000378:metabolism*
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D053778:Thiazolidines; D014404:Tubulin; C057531:jasplakinolide; C037068:latrunculin B; D017239:Paclitaxel