Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

• MeSH
• acetyltransferasy genetika   metabolismus   MeSH
• HeLa buňky MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• nádory genetika   MeSH
• nestabilita genomu MeSH
• poškození DNA MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• replikace DNA * MeSH
• transport RNA MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The nuclear pore complex (NPC) has emerged as a hub for the transcriptional regulation of a subset of genes, and this type of regulation plays an important role during differentiation. Nucleoporin TPR forms the nuclear basket of the NPC and is crucial for the enrichment of open chromatin around NPCs. TPR has been implicated in the regulation of transcription; however, the role of TPR in gene expression and cell differentiation has not been described. Here we show that depletion of TPR results in an aberrant morphology of murine proliferating C2C12 myoblasts (MBs) and differentiated C2C12 myotubes (MTs). The ChIP-Seq data revealed that TPR binds to genes linked to muscle formation and function, such as myosin heavy chain (Myh4), myocyte enhancer factor 2C (Mef2C) and a majority of olfactory receptor (Olfr) genes. We further show that TPR, possibly via lysine-specific demethylase 1 (LSD1), promotes the expression of Myh4 and Olfr376, but not Mef2C. This provides a novel insight into the mechanism of myogenesis; however, more evidence is needed to fully elucidate the mechanism by which TPR affects specific myogenic genes.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• exprese genu MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• kosterní svalová vlákna * cytologie   metabolismus   MeSH
• myoblasty kosterní * cytologie   metabolismus   MeSH
• myši MeSH
• protoonkogenní proteiny metabolismus   MeSH
• regulace genové exprese MeSH
• těžké řetězce myosinu metabolismus   MeSH
• vývoj svalů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-β interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-β pathway and implicate this mechanism in the maintenance of genome integrity.

• MeSH
• buněčné jádro metabolismus   MeSH
• genom lidský genetika   MeSH
• HeLa buňky MeSH
• imunoblotting MeSH
• imunoprecipitace MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• RNA interference fyziologie   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mutations in nucleoporin 93 (NUP93) gene have been shown recently to be one of the very rare causes of genetic steroid-resistant nephrotic syndrome (SRNS). Until now, none of the 7 published cases with NUP93-SRNS, experienced recurrence of nephrotic syndrome (NS) after transplantation. Here, we present the first case of recurrent NS in a patient with NUP93-SRNS ever reported. A 3-year-old boy with infantile SRNS was started on chronic peritoneal dialysis because of end-stage renal failure owing to biopsy-proven focal segmental glomerulosclerosis (FSGS). At the age of 6 years, the boy received a renal allograft. The posttransplant period was uncomplicated until 1.7 years after transplantation, when the patient developed nephrotic proteinuria during a respiratory tract infection. Renal graft biopsy showed subtotal fusion of podocytes, which was compatible with an early histopathologic sign of recurrence of FSGS. Immediate treatment with daily plasma exchange (PE) was started at the second day. The proteinuria disappeared completely after the second PE. However, it reappeared after stopping daily PE. It disappeared again after reintroduction of daily PE, therefore PE-dependent recurrent NS was diagnosed and treatment with rituximab was given. After the first dose, proteinuria never reappeared despite stopping PE therapy. Surprisingly, next-generation sequencing revealed compound heterozygous mutations in exons 16 and 18 of the NUP93 gene (c.1772G>T - European founder allele and 1916T>C) and his parents confirmed heterozygous asymptomatic carriers. This is the first case of recurrent NS in a patient with NUP93 gene mutations, suggesting a new pathomechanism possibly involving the nucleoporins.

• MeSH
• heterozygot MeSH
• imunologické faktory terapeutické užití   MeSH
• komplex proteinů jaderného póru genetika   MeSH
• lidé MeSH
• mutace MeSH
• nefrotický syndrom komplikace   genetika   chirurgie   MeSH
• plazmaferéza MeSH
• předškolní dítě MeSH
• recidiva MeSH
• rituximab terapeutické užití   MeSH
• transplantace ledvin * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

AIM: Heat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking. METHODS AND RESULTS: Hsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-β and Ran expression as well as cellular ATP levels compared to control. AdHsp60mito- treatment induced an up-regulation in importin-α, importin-β and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60. CONCLUSIONS: VSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• chaperon hsp60 antagonisté a inhibitory   genetika   metabolismus   MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• králíci MeSH
• kultivované buňky MeSH
• malá interferující RNA metabolismus   MeSH
• proliferace buněk * MeSH
• ran protein vázající GTP genetika   metabolismus   MeSH
• RNA interference MeSH
• svaly hladké cévní cytologie   metabolismus   MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The effects of acoustic waves on membrane structures, and any resulting consequences of this treatment on membrane subunit structures, remain poorly understood, as are the principals of related clinical effects. With a focus on morphological changes in the nuclear envelope, the current study presents detailed observations of membrane structures exposed to therapeutic ultrasound. Ultrasound treatment most commonly resulted in distinct changes in the distribution of nuclear pore complexes (NPCs) and mean NPC number per unit area after 30 min of repair, as well as alterations in NPC diameters on the protoplasmic face of fractured nuclear membranes after 10 min of repair. The greatest effects of ultrasound on nuclear envelope structure and NPCs were not to appear immediately, but became evident after repair processes were initiated. Results from the current study may contribute to the general view on the biophysical effects of therapeutic ultrasound on cell morphology and, particularly, the understanding of this effect in relation to the nuclear envelope.

• MeSH
• elektronová mikroskopie metody   využití   MeSH
• HL-60 buňky cytologie   ultrasonografie   MeSH
• intracelulární membrány ultrasonografie   MeSH
• jaderný obal * ultrasonografie   ultrastruktura   MeSH
• jaderný pór ultrastruktura   MeSH
• komplex proteinů jaderného póru * ultrastruktura   MeSH
• kultivační techniky metody   využití   MeSH
• leptání mrazem metody   využití   MeSH
• lidé MeSH
• mikrofotografie MeSH
• struktury buněčného jádra ultrasonografie   MeSH
• ultrasonografie metody   škodlivé účinky   využití   MeSH
• ultrazvuková terapie * metody   škodlivé účinky   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

This study was aimed at elucidating the fate of three important nuclear envelope components - lamins B and A/C and nucleoporin Nup160, during meiotic maturation of mouse oocytes. These proteins were localized by epifluorescence and confocal microscopy using specific antibodies in oocytes at different stages from prophase I (germinal vesicle) to metaphase II. In immature germinal vesicle oocytes, all three proteins were detected at the nuclear periphery. In metaphase I and metaphase II, lamin B co-localized with the meiotic spindle, lamin A/C was found in a diffuse halo surrounding the spindle and to a lesser degree throughout the cytoplasm, and Nup160 was concentrated to the spindle poles. To our knowledge, this is the first report on nucleoporin localization in mammalian oocytes and the first successful detection of lamins in mature oocytes. While the distribution patterns of both lamins closely paralleled the respective stages of mitosis, Nup160 localization in metaphase oocytes corresponded to that in mitotic prometaphase rather than metaphase. The peculiar distribution of this nucleoporin in oocytes may reflect its role in meiosis-specific mechanisms of spindle assembly and its regulation.

• MeSH
• buněčná diferenciace * MeSH
• jaderné proteiny metabolismus   MeSH
• konfokální mikroskopie MeSH
• laminy metabolismus   MeSH
• meióza * MeSH
• metafáze MeSH
• myši inbrední BALB C MeSH
• oocyty cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components, and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo, or real 3D at magnifications ranging from about 10× to 1,000,000×. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualized. In order to identify that protein at high resolution, or to see and quantify its distribution at lower magnifications, samples can be labeled with antibodies. Any surface that can be exposed can potentially be studied in this way. Presented here is a generic method for immunogold labeling for scanning electron microscopy, using two examples of specimens: isolated nuclear envelopes and the cytoskeleton of mammalian culture cells. Various parameters for sample preparation, fixation, immunogold labeling, drying, metal coating, and imaging are discussed so that the best immunogold scanning electron microscopy results can be obtained from different types of specimens.

• MeSH
• antigeny genetika   metabolismus   MeSH
• barvení a značení metody   MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• cytoskelet metabolismus   ultrastruktura   MeSH
• epoxidové pryskyřice chemie   MeSH
• exprese genu MeSH
• fixace tkání metody   MeSH
• fixativa chemie   MeSH
• formaldehyd chemie   MeSH
• imunohistochemie metody   MeSH
• jaderný obal metabolismus   ultrastruktura   MeSH
• koloidní zlato chemie   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• mikrotomie MeSH
• oocyty metabolismus   ultrastruktura   MeSH
• polymery chemie   MeSH
• protilátky chemie   MeSH
• Xenopus laevis MeSH
• zalévání tkání metody   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events.

• MeSH
• barvení a značení metody   MeSH
• epoxidové pryskyřice chemie   MeSH
• exprese genu MeSH
• fixace tkání metody   MeSH
• fixativa chemie   MeSH
• glutaraldehyd chemie   MeSH
• imunoelektronová mikroskopie metody   MeSH
• imunohistochemie metody   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• kryoprezervace metody   MeSH
• mikrotomie MeSH
• mrazová substituce metody   MeSH
• protilátky chemie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   ultrastruktura   MeSH
• zalévání tkání metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

• MeSH
• HeLa buňky MeSH
• jaderná lamina metabolismus   ultrastruktura   MeSH
• jaderný obal metabolismus   ultrastruktura   MeSH
• komplex proteinů jaderného póru antagonisté a inhibitory   genetika   metabolismus   MeSH
• lamin typ A antagonisté a inhibitory   genetika   metabolismus   MeSH
• lamin typ B genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• molekulární zobrazování MeSH
• protoonkogenní proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH