678. Cytoskeleton as the Principal Determinant of the Size-Dependent DNA Mobility in Cytoplasm: A New Barrier for Non-Viral Gene Transfer

Abstract

Gene delivery by synthetic vectors is a multi-step process that relies on the endocytosis of the DNA particles, escape from endosomes, and diffusion of the DNA through the cytosol before the import in the nucleus. The cytoplasm of mammalian cells is a complex environment containing a fluid phase, a high concentration of soluble proteins, and a network of cytoskeletal filaments. We found previously that the translational diffusion of small DNAs ( 250 bp) were greatly slowed (Dcyto/Dw/Dw< 0.05) (J. Biol. Chem. 275:1625–1629, 2000). To investigate the mechanisms responsible for size-dependent DNA diffusion, we measured by fluorescence correlation spectroscopy the diffusion of fluorescent linear, double-stranded DNAs in saline and artificial crowded solutions, cytosol extracts, actin network and cell cytoplasm. DNA fragments containing a single rhodamine green molecule were generated by PCR. In saline solution, DNA diffusion coefficients decreased with DNA size according to: Dw = 8.1 × 10−6cm2/s × (base pair) −0.71. In solutions containing the inert crowding agent Ficoll-70, DNA mobility was reduced exponentially with the Ficoll-70 concentration (5-fold at 18 wt% Ficoll-70); however, neither Ficoll-70 nor concentrated cytosolic soluble fractions reproduced size-dependent reduction in Dcyto/Dw as found in intact cells. In contrast, reduced Dcyto/Dw with increasing DNA size was reproduced in saline solutions crowded with actin filaments, the principal component of the cytoskeleton. Compact globular macromolecules of same size were not slowed, implicating the extended conformation of the DNA in its impaired diffusion. In intact cells microinjected with the fluorescent DNAs, Dcyto/Dw was greatly reduced as found in prior photobleaching measurements; however, the size-dependent reduction in Dcyto/Dw was eliminated with cytoskeletal disrupters (cytochalasin D, latrunculin B) and was dependent on cell volume. Our results implicate actin cytoskeleton as the principal determinant of the restricted DNA mobility in cytoplasm, thus identifying a new barrier in restricting DNA movement to the nucleus.