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

Abstract

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. Gene delivery by non-viral vectors is a complex and inefficient process that involves cellular internalization by endocytosis, escape from endosomes, DNA-polycation dissociation, and diffusion of non-complexed DNA in the cytoplasm to reach the nucleus (1-3). DNA diffusion in the cytoplasm and nuclear import are believed to be rate-limiting determinants of transgene delivery, extending the time that non-complexed DNA is exposed to cytosolic DNases. Remarkably, Ͻ0.1% of DNA microinjected into the cytosol is ultimately expressed (4, 5). We measured previously the mobility of differently sized DNAs in cytoplasm by photobleaching cells after microinjection with fluorescently labeled DNAs (6). The principal finding was that DNA diffusion in cytoplasm (D cyto ) was modestly slowed compared with that in saline (D o ) (D cyto /D o ϳ 0.2) for small DNA fragments of Ͻ250 bp but was greatly reduced (D cyto /D o Ͻ 0.05) for larger DNAs of Ͼ250 bp such as plasmid-sized DNAs. In contrast, the diffusion of dextrans and Ficolls, which are considered to be non-interacting macromolecules, was only mildly dependent on their size up to 500 -1000 kDa (7), which is equivalent in molecular mass to a 750 -1500-bp DNA fragment. Identification of the mechanism of reduced mobility of large DNAs in cytoplasm has important consequences regarding intrinsic limitations of non-viral gene delivery and in developing strategies to improve its efficacy.Several factors can in principle reduce the diffusion of a solute in cytoplasm versus saline, including fluid phase viscosity, binding, and crowding by mobile and immobile macromolecules (8). Systematic analysis of the diffusion of a small fluorescein-like molecule in cytoplasm indicated that reduced diffusion in cytoplasm (D cyto /D o ϳ 0.25) resulted mainly from macromolecular crowding by the relatively high concentration of proteins in cytoplasm of ϳ100 -150 mg/ml (reviewed in Refs. 9 and 10). Fluid phase viscosity, defined as the effective viscosity sensed by a small molecule that does not undergo binding or other macromolecular interactions, has little influence on cytoplasmic diffusion (11,12). Binding can greatly reduce apparent diffusion, as was found for some enzymes that assemble into macromolecular complexes (13). As a densely charged polyanion, DNA binding to cytoplasmic components could be an important factor in reducing its diffusion in cytoplasm as was found in the nucleus, where DNA is nearly immobile probably because of binding to positively charged histones (6,14). DNA diffusion in heterogeneous polydispers...

Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer

“…Verkman and co-workers recently reported that the actin cytoskeleton is the principle determinant for passive diffusion through the cytoplasm, and that disruption of the actin cytoskeleton through the addition of either cytochalasin D or latrunculin B was sufficient in vitro to dissipate the retarding sieving effect seen in the diffusion of large macromolecules or linear DNA fragments. 32 This result echoes the earlier findings of Popov and Poo, 33 suggesting that disruption or dissipation of the actin cytoskeleton could improve cytoplasmic trafficking of macromolecules. Viruses such as SV40 take a similar approach in disrupting the actin cytoskeleton through the activation of tyrosine kinases to aid in their movement through the cytoplasm.…”

110 Years of the Meyer–Overton Rule: Predicting Membrane Permeability of Gases and Other Small Compounds

“…The difference between P UL,1 and P UL,2 results from a fourfold reduction of the solute diffusion coefficient in cytoplasm versus saline. [56,57] Detection of an aquaporin-mediated increase in P M would require that P M of the lipid matrix is ≤10 −4 cm s −1 , that is, several orders of magnitude below the value measured with a tracer technique for planar lipid membranes. [58] In the absence of any experimental proof for this very unlikely anticipation, Xenopus oocytes may not be used for P M measurements of CO 2 .…”

Monitoring intracellular degradation of exogenous DNA using diffusion properties

“…On the other hand, physical approaches such as microinjection [7,8] and electroporation [9] are performed to incorporate naked DNAs directly into cytoplasm across the plasma membrane. Some naked DNAs diffuse in the cytoplasm to the nucleus [10]. The diffusion of exogenous DNAs in cytoplasm is relatively slow compared with that in solution [10,11], and it is hard for large DNA to cross the nuclear membrane [11].…”

“…The diffusion of exogenous DNAs in cytoplasm is relatively slow compared with that in solution [10,11], and it is hard for large DNA to cross the nuclear membrane [11]. Under such conditions in cytoplasm, the existence of nuclease degradation in cytoplasm is suggested [10][11][12][13][14][15]. It is presumed that the translocation and nuclear uptake of transfected DNA compete with degradation by cytoplasmic nucleases [11].…”

“…The purpose of this study was to investigate the mechanism of exogenous DNA degradation in situ, in cytoplasm, by analyses of diffusion properties of DNAs and to determine the effects of cytoplasmic degradation on the gene expression rate. Thus we employed FCS [10,[16][17][18] to measure the diffusion properties of DNAs and FCCS [18][19][20][21][22] to monitor the degradation of DNAs by cytoplasmic nucleases at the single molecule level. Furthermore, we predicted that exonucleases would work as the main barrier in cytoplasm, so a capped structure was attached to the transfected DNA to enhance the expression of the protein EGFP.…”