Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells

Daigle, Nathalie; Beaudouin, Joël; Hartnell, Lisa M.; Imreh, Gabriela; Hallberg, Einar; Lippincott‐Schwartz, Jennifer; Ellenberg, Jan

doi:10.1083/jcb.200101089

Cited by 363 publications

(366 citation statements)

References 60 publications

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“…The SalI/NotI insert was subcloned in a pGEX-4T2 vector (Amersham Biosciences, Piscataway, NJ) to produce the glutathione S-transferase (GST)-Nup160⌬C 92 plasmid, and into a pEGFP3-C1 vector that contains a triple GFP concatamer constructed in pEGFP-C1 to produce the pEGFP 3 -Nup160⌬C 92 plasmid (Daigle et al, 2001). Plasmids pGEX-4T2-Nup160-N (encoding aa 1-598 of Nup160) and pGEX-4T2-Nup160-C (encoding aa 598-1294 of Nup160) were generated using an internal BspE1 restriction site.…”

Section: Plasmidsmentioning

confidence: 99%

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

Loïodice

Alves

Rabut

et al. 2004

MBoC

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In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of ϳ30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

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Section: Plasmidsmentioning

confidence: 99%

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

Loïodice

Alves

Rabut

et al. 2004

MBoC

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254

240

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“…There are several fates for the disassembled NE components. The disassembled lamins and Nups either become dispersed in the cytoplasm or, as is the case for integral membrane Nups, are redistributed within the mitotic ER network (Yang et al, 1997;Daigle et al, 2001). In addition, Nups belonging to the Nup107-160 complex have been shown to interact with kinetochores during M phase of the cell cycle (Belgareh et al, 2001;Loiodice et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

Hawryluk-Gara

Platani

Santarella

et al. 2008

MBoC

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Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly. INTRODUCTIONThe nuclear envelope (NE) provides a physical barrier between the nucleus and cytoplasm and their unique metabolic tasks. The NE is defined by three morphologically distinct regions. The outer nuclear membrane (ONM) is continuous with the rough endoplasmic reticulum (ER) and contains a similar set of proteins (reviewed in Mattaj, 2004). The inner nuclear membrane (INM) lies adjacent to the nucleoplasm and contains a unique repertoire of proteins that, in part, mediate its interactions with the nuclear lamina and chromatin. Finally, at numerous locations along the NE, the ONM and INM are interrupted by pores where the INM and the ONM are bridged by a connecting membrane termed the pore membrane (POM) domain. Within these pores reside the nuclear pore complexes (NPCs), aqueous channels that provide portals for both passive and active transport of macromolecules.An NPC contains ϳ30 nucleoporins (Nups), many of which are evolutionarily conserved in structure and function (Tran and Wente, 2006). In many cases these Nups are organized into subcomplexes that are present in multiple copies, and they are distributed around the central axis of the NPC, contributing to its characteristic eightfold symmetry. Different groups of Nups and their respective subcomplexes contribute to distinct structural components of the NPC. For example, Nup214/Nup84 are components of the cytoplasmic fibrils (Kraemer et al., 1994;Bastos et al., 1997), the Nup53/Nup155 complex is part of the central core (Marelli et al., 1998), and Nup153 and Tpr contribute to the nucleoplasmic ring and fibrils (Krull et al., 2004).When metazoan cells enter mitosis, their NE is disassembled allowing for the mitotic spindle to access the condensed chromatin. Several models based on previous findings have been proposed to explain the mechanism by which this occurs (reviewed in Hetzer et al., 2005). NE disassembly is accompanied by the phosphorylation of multiple NE proteins including the lamins and several Nups (Burke and Ellenberg, 2002, and refer...

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“…13 NUP153 also facilitates nuclear export of mRNA and ribonucleoprotein particles 14,15 and may have additional functions within the nucleus. [16][17][18][19] Here, we performed an siRNA screen for novel factors involved in ionizing radiation (IR)-induced DDR foci formation, DNA repair; siRNA screen; 53BP1 nuclear import; nucleoporin NUP153; cell survival; ionizing radiation Abbreviations: 53BP1, p53-Binding Protein 1; ANAPC10, anaphase-promoting complex subunit 10; ANAPC4, anaphase-promoting complex subunit 4; APC/C, anaphase-promoting complex/cyclosome; APH, aphidicolin; ASB18, ankyrin repeat and SOCS box protein 18; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; ATXN7, ataxin-7; BRCA1, breast cancer type 1 susceptibility protein; DDR, DNA damage response; DNA-PK, DNA-dependent protein kinase; DSB, Double strand breaks ; ECS, elongin, Cullin, SOCS-box containing complex; FANCD2, Fanconi anemia group D2 protein; FG repeat, phenylalanine-glycine repeat; GFP, green fluorescent protein; gH2AX, phosphorylated histone H2AX at serine 139; HeLa, human adenocarcinoma cell line; HERC2, HECT domain and RCC1-like domain-containing protein 2; IR, ionizing radiation; MAP3K3, mitogen-activated protein kinase kinase kinase 3; MDC1, mediator of DNA damage checkpoint protein 1; Mec1, Mitosis entry checkpoint protein 1; MMSET, multiple myeloma SET domain-containing protein; NBS1, Nijmegen breakage syndrome protein 1; NPC, nuclear pore complex; NUP, nucleoporin; NUP153, nucleoporin 153kDa protein; NUP84, nucleoporin 84kDa protein; NUP93, nucleoporin 93kDa protein; PARP, poly [ADP-ribose] polymerase; PIAS4, protein inhibitor of activated STAT protein 4; Rad51, DNA repair protein RAD51; Rad53, Serine/threonineprotein kinase RAD53; RNF8, RING finger protein 8; RNF168, RING finger protein 168; SAGA, Spt-Ada-Gcn5 acetyltransferase; SCF, Skp, Cullin, F-box containing complex; SEC13L1, SEC13-like protein 1; SET8, SET domain-containing protein 8; Slx5, synthetic lethal of unknown function protein 5; Slx8, synthetic lethal of unknown function protein 8; TopBP1, DNA topoisomerase 2-binding protein 1; U2OS, human osteosarcoma cell line; UBA1, ubiquitin-activating enzyme E1; UBC9, ubiquitinconjugating enzyme 9 …”

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confidence: 99%

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1

et al. 2011

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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-b interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-b pathway and implicate this mechanism in the maintenance of genome integrity. Cell Death and Differentiation (2012) 19, 798-807; doi:10.1038/cdd.2011; published online 11 November 2011The cellular DNA damage response (DDR) machinery serves as a biological barrier against accumulation of genetic changes associated with aging, and guards against neurodegenerative and immunodeficiency disorders and cancer. 1 The proximal DDR kinases ATM, ATR and DNA-PK have a central role in response to various DNA lesions including DNA double strand breaks (DSBs), in part by phosphorylating histone H2AX (gH2AX) and its sensor MDC1, which in turn coordinates recruitment of signaling and repair factors to DNA damage foci. 1-4 These foci include 53BP1 5,6 whose retention at the DSB-flanking chromatin requires histone ubiquitylation by E3-ubiquitin ligases RNF8, RNF168 and HERC2, SUMOylation by the PIAS4/UBC9 complex, and histone methylation by MMSET and SET8 methyltransferases. [1][2][3]7 Apart from the role of 53BP1 in telomere maintenance, cell cycle checkpoints and DNA repair by non-homologous end joining of DSBs, 2,3,5,6 53BP1 is also involved in response to viruses, 8 and contributes to replication stress responses by protecting under-replicated genomic loci in G1 phase of the cell cycle. 9,10 Trafficking across the nuclear membrane occurs through nuclear pore complexes (NPCs), large channels consisting of over 30 nucleoporins (NUPs). The central channel of NPC is filled with hydrophobic phenylalanine-glycine (FG) repeats contained in many NUPs, which mediate mostly low-affinity interactions with nuclear transport factors. NUP153 is also an FG-repeat containing NUP, but contains a high-affinity site for importin-b. 11,12 NUP15...

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Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells

Cited by 363 publications

References 60 publications

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1

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