title: Highly acetylated tubulin permits enhanced interactions with and trafficking of plasmids along microtubules.
abstract: Microtubule-based transport is required for plasmid translocation to the nucleus during transfections, and having stable structures could enhance this movement. In previous studies, in which the cytoskeleton was disrupted, we found that populations of microtubules remain that are stable and highly acetylated. By increasing the levels of acetylated tubulin through inhibition of the tubulin deacetylase HDAC6, we observe more rapid plasmid nuclear localization of transfected plasmids and greater levels of gene transfer. In this study, we sought to understand plasmid movement in cells with enhanced tubulin acetylation. Using variations of a microtubule spin-down assay, we found that plasmids bound to hyper-acetylated microtubules to a greater degree than they did to unmodified microtubules. To determine whether microtubule acetylation also affects cytoplasmic trafficking, plasmid movement was evaluated in real time by particle tracking in cells with varying levels of acetylated microtubules. We found that plasmids display greater net rates of movement, spend more time in productive motion and display longer runs of continuous motion in cells with highly acetylated microtubules compared with those with fewer modifications. These results all suggest that plasmid movement is enhanced along highly acetylated microtubules, reducing the time spent in the cytoplasm before nuclear import. Taken together, these findings provide a foundation for determining how modulation of microtubule acetylation can be used as a means to increase intracellular trafficking of plasmids and enhance gene therapy.
authors.affiliation: Department of Pediatrics, Division of Neonatology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Badding
authors.forename: D A
authors.initials: DA
authors.lastname: Dean
mesh_terms: D000107:Acetylation; D021581:Active Transport, Cell Nucleus / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism
chemical_list: D014404:Tubulin; C433811:HDAC6 protein, human; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases