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Molecular Transport in Membranes
Published in Allen J. Bard, Michael V. Mirkin, Scanning Electrochemical Microscopy, 2022
SECM was used to investigate molecular transport through nuclear pore complexes (NPCs), which perforate the double-membrane nuclear envelope (NE) to solely transport both small molecules and macromolecules between the cytoplasm and nucleus of a eukaryotic cell. A greater mechanistic understanding of nucleocytoplasmic transport through the NPC is required urgently and broadly in various research fields. The NPC is crucial to gene expression regulation [49, 50] and gene delivery [51] and is linked to many human diseases and therapeutics of genetic disorders [52]. Moreover, the NPC is an attractive pathway for the efficient nuclear delivery of genetic macromolecules and nanomaterials, e.g., the large conjugates of nucleic acids with polymers [51] and nanoparticles [53] as vectors for gene therapy and nanomedicine. The NPC comprises multiple copies of the distinct 30 proteins called nucleoporins (nups) to represent one of the largest known protein complexes (in total ∼120 MDa). The transport barriers of the NPC prevent the passive transport of a macromolecule with a molecular weight of >40 kDa. Passively impermeable nuclear proteins are tagged with nuclear localization signal (NLS) peptides and chaperoned through the NPC by nuclear transport receptors, i.e., importins. Intriguingly, the even larger importin–protein complex permeates through the NPC because of the binding of importins to the repeats of phenylalanine and glycine (FG), which are abundant in transport barriers [54].
Reactive oxygen species and neuroepithelial interactions during wound healing
Published in David M. Gardiner, Regenerative Engineering and Developmental Biology, 2017
A hallmark of wound repair in vertebrates is the regeneration of cutaneous sensory axons into the wounded area. In rats, Dorsal root ganglion (DRG) axons were found to hyperinnervate the wound, and this hyperinnervation pattern persisted up to 12 weeks after injury (Reynolds and Fitzgerald 1995). Most of our current knowledge about the molecular mechanisms leading to somatosensory axon regeneration stems from rodent studies in which axotomies were performed. During an axotomy, either the brachial or sciatic nerve is exposed via an incision made into the limb, and axons are subsequently severed via nerve crush or transection. These injuries result in the disconnection of the distal axonal portion from the cell body, and the distal axon subsequently undergoes rapid degeneration. This process was originally identified by Augustus Volney Waller in 1850 and is defined by a stereotypic sequence of events leading to fragmentation, degradation, and clearance of axon debris. Nerve injury by axotomy triggers the release of two calcium waves at the injury site: a fast wave and a slow wave. These waves propagate along the proximal axon toward the soma. The fast wave leads to the release of internal calcium stores from the endoplasmic reticulum, which promotes nuclear export of histone deacetylase (HDAC5) in the soma (Cho et al. 2013). This primes the chromatin for transcription via acetylation (Ac) of histone 3 (H3). A second calcium wave stimulates local protein translation. One pathway involves importin-mediated retrograde transport of nuclear localization sequence (NLS)-containing transcription factors into the nucleus, which activate the expression of downstream genes. In addition, dual leucine zipper kinase (DLK-1) is locally translated, which leads to retrograde STAT3 transport into the soma. This process also involves Jun N-terminal kinase (JNK), leading to activation of C-JUN and activating transcription factor-3 (ATF3) (Rishal and Fainzilber 2014).
Nonviral Therapeutic Approaches for Modulation of Gene Expression: Nanotechnological Strategies to Overcome Biological Challenges
Published in Ana Rute Neves, Salette Reis, Nanoparticles in Life Sciences and Biomedicine, 2018
Ana M. Cardoso, Ana L. Cardoso, Maria C. Pedroso de Lima, Amalia S. Jurado
Contrary to siRNA or miRNA, pDNA needs to reach the nuclei of cells in order to be transcribed into the desired RNA molecule, either a protein-coding mRNA or a small RNA precursor. Thus, the nuclear membrane appears as an additional challenge to pDNA delivery. Like the cytoplasmic membrane, the nuclear membrane does not pose an insurmountable barrier, but, in fact, has nuclear pores that are responsible for the controlled trafficking of mRNA molecules from the nucleus to the cytosol for further processing and of nuclear proteins that are synthesized in the cytoplasm and are addressed to the nucleus. For fast dividing cells, it is usually assumed that nuclear membrane fission during the mitotic process facilitates the translocation of nanocarriers from the cytoplasm to the nucleus. Nonetheless, even in actively dividing cells, only 1%-10% of the pDNA copies internalized by the cells reach the nucleus (reviewed in “Progress and Prospects: Nuclear Import of Nonviral Vectors” [77]). However, the efficiency of this process can be enhanced using different strategies to modify gene delivery systems. In particular, advantage can be taken of cellular signaling sequences that target proteins at the nucleus. Peptide NLSs are used by cells to address proteins to the nuclear compartment, through binding to karyopherins (importins), which mediate their translocation across the nuclear pore complex (NPC) (reviewed in “Progress and Prospects: Nuclear Import of Nonviral Vectors” [77]). Typical examples of NLSs are the SV40 large T-antigen (PKKKRKV) and the 38-amino acid sequence M9 of the heterogeneous nuclear ribonucleoprotein-A1, a mRNA-binding protein that mediates nuclear uptake of otherwise cytoplasmic proteins [78]. This peptide, covalently linked to a scrambled version of the SV40 large T-antigen, has been preincubated with pDNA and subsequently with lipofectamine. Fluorescence microscopy images taken of murine 3T3 fibroblasts transfected with the resulting ternary complexes carrying a rhodamine-labeled pDNA showed colocalization of the pDNA with cell nuclei, whereas the control system lacking the peptide was found in the cytoplasm. The transfection efficiency of these systems correlated with their ability to mediate nuclear entry of the pDNA [79].
Nuclear targeting peptide-modified, DOX-loaded, PHBV nanoparticles enhance drug efficacy by targeting to Saos-2 cell nuclear membranes
Published in Journal of Biomaterials Science, Polymer Edition, 2018
Ayla Şahin, Gozde Eke, Arda Buyuksungur, Nesrin Hasirci, Vasif Hasirci
The nucleus is separated from the rest of cytoplasm by the double membrane structure of the nuclear envelope (NE) which constitutes a major barrier and the main rate limiting step in the transport of drugs into the nucleus [6,10]. The NE contains nuclear pore complexes (NPC) which are channels that allow the passive diffusion of small molecules (ca. 9 nm in diameter) or transport of larger molecules (ca. 39 nm in diameter) in an energy dependent manner [4]. The transfer of drugs through the energy dependent pathway into the nucleus is mediated by homologous proteins called importins. This transfer mechanism is divided into two steps. In the first step, importin α binds to Nuclear Localization Signal (NLS), which is a special oligopeptide, and importin β binds to the cytoplasmic filaments that bring the target molecule to the nuclear pore. In the second step, importin α is transported through the nuclear pore into the nucleus together with its cargo [11].