The potential dangers of using viruses to deliver and integrate DNA into host cells in gene therapy have been poignantly highlighted in recent clinical trials. Safer, non-viral gene delivery approaches have been largely ignored in the past because of their inefficient delivery and the resulting transient transgene expression. However, recent advances indicate that efficient, long-term gene expression can be achieved by non-viral means. In particular, integration of DNA can be targeted to specific genomic sites without deleterious consequences and it is possible to maintain transgenes as small episomal plasmids or artificial chromosomes. The application of these approaches to human gene therapy is gradually becoming a reality.

Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormonerelated protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110 Rb tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.Key words: actin filaments, FRAP, importins, microtubules, nuclear import, retinoblastoma protein Transport into the nucleus through the nuclear envelopelocalized nuclear pore complexes (NPCs) is dependent on modular nuclear localization sequences (NLSs) (1-3) within cargoes that are typically recognized by members of the importin (IMP) superfamily. Most commonly, the IMPa/b1 heterodimer or IMPb1 alone (4-6) act to effect NLSdependent translocation through the NPC, and release within the nucleus. Understanding of the detailed mechanisms of nuclear protein import, however, is largely based on the study of semi-intact cell systems (7-10), where cytoskeletal elements are either lacking or severely damaged, meaning that the role of the cytoskeleton has been largely overlooked. Many viruses are known to exploit the microtubule (MT) network for efficient nuclear targeting (11), while there is evidence for negative regulation of the nuclear import of several viruses through association with the actin cytoskeleton (12). Intriguingly, IMPa has been demonstrated to associate with MTs in mammalian and plant cells (13,14), while NLS-comprising peptides have been reported to invoke active transport along MTs (15). Significantly, a role for the MT network in facilitating nuclear import (16) is strongly implicated in the case of the tumor suppressor protein p53 and the cancer-related signaling molecule parathyroid hormone-related protein (PTHrP) (17,18). Treatment with the MT-depolymerizing agent nocodazole (NCZ) reduces both p53 and PTHrP nuclear accumulation in vivo (17,18), with application of the flu...

Gene delivery into the nucleus of eukaryotic cells is inefficient, largely because of the significant barriers within the target cell of the plasma membrane and nuclear envelope. Recently, a group of basic proteins, including the HIV-1 Tat protein and the four core histones, have been shown to enter cells through a novel energy- and receptor-independent manner. Here, we show that engineered histone H2B proteins are able to mediate the efficient delivery of either green fluorescent protein or DNA into HeLa cells through the process of "Histone-Mediated Transduction" (HMT), with further enhancement achieved by utilizing a dimer of histones H2B and H2A. Subsequent nuclear delivery was accelerated approximately two-fold by the addition of an optimized nuclear localization signal to histone H2B, thereby increasing the affinity of interaction with components of the cellular nuclear import machinery, resulting in increased expression of a reporter gene. Further, we demonstrate that the domains responsible for this histone transduction are located in the N-terminal tail and globular regions of histone H2B. HMT represents a new, efficient, and technically non-demanding means to deliver DNA to the nucleus of intact cells, including embryonic stem cells, which has important applications in gene therapy and cancer therapeutics.

Background The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression.

The fabrication of nanoscale devices requires architectural templates on which to position functional molecules in complex arrangements. Protein scaffolds are particularly promising templates for nanomaterials due to inherent molecular recognition and self-assembly capabilities combined with genetically encoded functionalities. However, difficulties in engineering protein quaternary structure into stable and well-ordered shapes have hampered progress. Here we report the development of an ultrastable biomolecular construction kit for the assembly of filamentous proteins into geometrically defined templates of controllable size and symmetry. The strategy combines redesign of protein–protein interaction specificity with the creation of tunable connector proteins that govern the assembly and projection angles of the filaments. The functionality of these nanoarchitectures is illustrated by incorporation of nanoparticles at specific locations and orientations to create hybrid materials such as conductive nanowires. These new structural components facilitate the manufacturing of nanomaterials with diverse shapes and functional properties over a wide range of processing conditions.