This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The main aim of the project will be to develop our understanding of the role of the microtubule and actin networks in pDNA transport during liposomal transfection. To elucidate the role of the cytoskeleton networks in the trafficking of plasmids to the cell nucleus, inhibitors of polymerization of actin or microtubule networks will be employed. We will investigate whether disruption of either of these networks lead to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and/or accumulation of plasmid DNA in large aggregates at the cell periphery. Cells will be stained with fluorescent dyes that will bind to the cytoskeleton networks. When in the cytosol unforced diffusion of pDNA-loaded lipid nanoparticles (as detected by co-localization of fluorescence signals arising from fluorescent lipids and labeled DNA) throughout the cell cytoplasm and their spontaneous binding to cytoskeletal networks will be imaged in real time. + Actin filaments. Inhibition of actin polymerization will be achieved by treating cell cultures with latrunculin B or cytochalasin D. + Microtubule network. Nocodazole will disrupt the microtubule network. Experimental strategy. The nanoparticle trajectory (MSD) will be calculated by confocal images as reported previously (Ondrej V. et al., Acta Biochim. Pol. 2007, 54, 657-663). Such strategy will also enable us to track pDNA to assign dynamic parameters to it. The precise role of cytoskeleton networks will therefore be investigated by comparing results (lipid nanoparticle trajectory in the cytoplasm, diffusion parameters of both the pDNA and the MENS nanoparticle) obtained with and without inhibitors of polymerization of actin or microtubule networks. Cytoplasmic transport of plasmid DNA after network disruption using different drug treatments can be also followed. Changes in positions of the fluorescence signals (particle tracking) will be determined using dedicated softwares (developed by Prof. Enrico Gratton), which will allow us to assign 3D coordinates to the fluorescence signals. The diffusion coefficient (D) and the average velocity (v) of the nanoparticles bound to both the networks can be calculated. To quantitatively analyze the motion of the particles, it is helpful to classify the trajectories in terms of simple models. Analytical forms of the equations for various types of motion have been derived (Saxton and Jacobson, Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373). These models will be fitted to the experimental data, and the best fitting model provides the parameters D, v, and/or, for the particle motion.
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