scholarly journals Nup98-Homeodomain Fusions Interact with Endogenous Nup98 during Interphase and Localize to Kinetochores and Chromosome Arms during Mitosis

2010 ◽  
Vol 21 (9) ◽  
pp. 1585-1596 ◽  
Author(s):  
Songli Xu ◽  
Maureen A. Powers
Keyword(s):  
Nuclear Pore Complex ◽  
Fusion Proteins ◽  
Cellular Transformation ◽  
Myelogenous Leukemia ◽  
Chromosomal Translocations ◽  
Nuclear Pore ◽  
Dynamic Interactions ◽  
C Terminus ◽  
Repeat Domain ◽  
Acute Myelogenous

Chromosomal translocations involving the Nup98 gene are implicated in leukemias, especially acute myelogenous leukemia. These translocations generate chimeric fusion proteins, all of which have in common the N-terminal half of Nup98, which contains the nucleoporin FG/GLFG repeat motifs. The homeodomain group of Nup98 fusion proteins retain the C-terminus of a homeodomain transcription factor, including the homeobox responsible for DNA binding. Current models for Nup98 leukemogenesis invoke aberrant transcription resulting from recruitment of coregulators by the Nup98 repeat domain. Here we have investigated the behavior of Nup98-homeodomain fusion proteins throughout the cell cycle. At all stages, the fusion proteins exhibit a novel localization distinct from the component proteins or fragments. During interphase, there are dynamic interactions between the Nup98 fusions and endogenous Nup98 that lead to mislocalization of the intranuclear fraction of Nup98, but do not alter the level of Nup98 at the nuclear pore complex. During mitosis, no interaction between the fusion proteins and endogenous Nup98 is observed. However, the fusions are entirely concentrated at kinetochores and on chromosome arms, sites where the APC/C, a target of Nup98 regulation, is also found. Our observations suggest new possibilities for misregulation by which Nup98 translocations may contribute to cellular transformation and leukemogenesis.

scholarly journals An RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans reveals the involvement of unexpected processes

2021 ◽  
Author(s):  
Richa Maheshwari ◽  
Mohammad M Rahman ◽  
Daphna Joseph-Strauss ◽  
Orna Cohen-Fix
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Pore Complex ◽  
Cellular Transformation ◽  
Rnai Screen ◽  
Nuclear Shape ◽  
Nuclear Pore ◽  
Nuclear Morphology ◽  
Anaphase Bridges ◽  
Early Embryos ◽  
Embryonic Viability

Abstract Aberration in nuclear morphology is one of the hallmarks of cellular transformation. However, the processes that, when mis-regulated, result aberrant nuclear morphology are poorly understood. In this study we carried out a systematic, high-throughput RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans embryos. The screen employed over 1700 RNAi constructs against genes required for embryonic viability. Nuclei of early embryos are typically spherical, and their NPCs are evenly distributed. The screen was performed on early embryos expressing a fluorescently tagged component of the nuclear pore complex (NPC), allowing visualization of nuclear shape as well as the distribution of NPCs around the nuclear envelope. Our screen uncovered 182 genes whose down-regulation resulted in one or more abnormal nuclear phenotypes, including multiple nuclei, micronuclei, abnormal nuclear shape, anaphase bridges and abnormal NPC distribution. Many of these genes fall into common functional groups, including some that were not previously known to affect nuclear morphology, such as genes involved in mitochondrial function, the vacuolar ATPase and the CCT chaperonin complex. The results of this screen add to our growing knowledge of processes that affect nuclear morphology and that may be altered in cancer cells that exhibit abnormal nuclear shape.

scholarly journals An RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans reveals the involvement of unexpected processes

2021 ◽  
Author(s):  
Richa Maheshwari ◽  
Mohammad M Rahman ◽  
Daphna Joseph-Strauss ◽  
Orna Cohen-Fix
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Pore Complex ◽  
Cellular Transformation ◽  
Rnai Screen ◽  
Nuclear Shape ◽  
Nuclear Pore ◽  
Nuclear Morphology ◽  
Anaphase Bridges ◽  
Early Embryos ◽  
Embryonic Viability

Aberration in nuclear morphology is one of the hallmarks of cellular transformation. However, the processes that, when mis-regulated, result aberrant nuclear morphology are poorly understood. In this study we carried out a systematic, high-throughput RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans embryos. The screen employed over 1700 RNAi constructs against genes required for embryonic viability. Nuclei of early embryos are typically spherical and their NPCs are evenly distributed. The screen was performed on early embryos expressing a fluorescently tagged component of the nuclear pore complex (NPC), allowing visualization of nuclear shape as well as the distribution of NPCs around the nuclear envelope. Our screen uncovered 182 genes whose down-regulation resulted in one or more abnormal nuclear phenotypes, including multiple nuclei, micronuclei, abnormal nuclear shape, anaphase bridges and abnormal NPC distribution. Many of these genes fall into common functional groups, including some that were not previously known to affect nuclear morphology, such as genes involved in mitochondrial function, the vacuolar ATPase and the CCT chaperonin complex. The results of this screen add to our growing knowledge of processes that affect nuclear morphology and that may be altered in cancer cells that exhibit abnormal nuclear shape.

FASN Is Critical for Maintenance of NUP98 Fusion-Induced Leukemias

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2377-2377
Author(s):  
Yutaka Shima ◽  
Issay Kitabayashi
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Fusion Proteins ◽  
Fatty Acid Synthase ◽  
Fusion Gene ◽  
Mass Spectrometry Analysis ◽  
Transformed Cells ◽  
Nuclear Pore ◽  
Repeat Domain

Abstract NUP98 is a component of the nuclear pore complex (NPC), which plays an important role in molecular traffic between the cytoplasm and the nucleus. The NUP98 gene is rearranged and fused to several partner genes, such as HOXA9 and DDX10, in acute myeloid leukemia and myelodysplastic syndromes and the leukemia with NUP98 rearrangement has a poor prognosis. The role of the NUP98 moiety of NUP98 fusion proteins is not well investigated. NUP98 has two Phe-Gly (FG) repeat domains, which are characteristic of the NPC proteins. To investigate the role of the NUP98 moiety of NUP98 fusion proteins, we purified the protein complexes formed with wild-type NUP98-HOXA9 and its mutants that lack the each FG repeat domain and looked for the proteins that interact with the NUP98 moiety. Mass spectrometry analysis identified that fatty acid synthase (FASN) interacted with the FG repeat domain of NUP98-HOXA9. These data suggest two possibilities. One is that NUP98-HOXA9 may affect the activity of FASN. The other is that FASN may affect activity of NUP98-HOXA9. To test the former possibility, we tested the effect of NUP98-HOXA9 on the activity of FASN in vitro, and we found that NUP98-HOXA9 reduced the FASN activity. FASN is known to be essential for cell growth. In fact, the colony formation of the NUP98-HOXA9-immortalized mouse myeloid stem/progenitor cells was inhibited by shRNA for FASN.　Because NUP98-HOXA9 reduced FASN activity, we hypothesized that FASN activity is low in NUP98 fusion gene-transformed cells and FASN inhibition selectively inhibits the colony formation of these cells. To test this hypothesis, we examined the effect of orlistat, which is the inhibitor of FASN on the colony formation. Orlistat strongly inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells, but it did not severely affect, the colony-forming activities of other fusion genes-transformed cells and normal progenitor cells. Other FASN inhibitors inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells. NUP98-HOXA9- and NUP98-DDX10 did not affect the expression level of FASN. The inhibition of FASN did not affect the expression of Hoxa genes and Meis1 gene, which are upregulated by NUP98 fusions. These results suggest that FASN activity is low but is essential for NUP98 fusion-mediated leukemia cells, and that FASN can be a therapeutic target for NUP98 fusion gene-mediated leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex

Cell ◽  
1995 ◽  
Vol 81 (2) ◽  
pp. 215-222 ◽  
Author(s):  
Aurelian Radu ◽  
Mary Shannon Moore ◽  
Günter Blobel
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Docking Site ◽  
Repeat Domain

scholarly journals The Nuclear Pore Complex and mRNA Export in Cancer

Cancers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 42
Author(s):  
Katherine L. B. Borden
Keyword(s):  
Nuclear Pore Complex ◽  
Molecular Basis ◽  
Human Cancer ◽  
Mrna Export ◽  
Therapeutic Strategies ◽  
Chromosomal Translocations ◽  
Nuclear Pore ◽  
Specific Effects ◽  
Rna Export ◽  
Aberrant Rna

Export of mRNAs from the nucleus to the cytoplasm is a key regulatory step in the expression of proteins. mRNAs are transported through the nuclear pore complex (NPC). Export of mRNAs responds to a variety of cellular stimuli and stresses. Revelations of the specific effects elicited by NPC components and associated co-factors provides a molecular basis for the export of selected RNAs, independent of bulk mRNA export. Aberrant RNA export has been observed in primary human cancer specimens. These cargo RNAs encode factors involved in nearly all facets of malignancy. Indeed, the NPC components involved in RNA export as well as the RNA export machinery can be found to be dysregulated, mutated, or impacted by chromosomal translocations in cancer. The basic mechanisms associated with RNA export with relation to export machinery and relevant NPC components are described. Therapeutic strategies targeting this machinery in clinical trials is also discussed. These findings firmly position RNA export as a targetable feature of cancer along with transcription and translation.

scholarly journals Destabilization of Ran C-terminus promotes GTP loading and occurs in multiple Ran cancer mutations

2018 ◽  
Author(s):  
Yuqing Zhang ◽  
Jinhan Zhou ◽  
Yuping Tan ◽  
Qiao Zhou ◽  
Aiping Tong ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Mitotic Spindle ◽  
Nuclear Export ◽  
Cellular Transformation ◽  
Nuclear Pore ◽  
Ras Proteins ◽  
Transformation Mechanism ◽  
C Terminus ◽  
Cytoplasmic Transport ◽  
Cancer Mutations

AbstractRan (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other diverse functions in the cytoplasm, as well as in cellular transformation when activated. Unlike other Ras superfamily proteins, Ran contains an auto-inhibitory C-terminal tail, which packs against its G domain and bias Ran towards binding GDP over GTP. The biological importance of this C-terminal tail is not well understood. By disrupting the interaction between the C-terminus and the G domain, we were able to generate Ran mutants that are innately active and potently bind to RanBP1 (Ran Binding Protein 1), nuclear export factor CRM1 and nuclear import factor KPNB1. In contrast to previously reported activated Ran mutants, the C-terminus destabilized mutants are hydrolysis competent in cells, support nuclear transport, and do not form nuclear rim staining. Crystal structures show that one of these C-terminal mutations slightly changes its mode of binding to RanBP1. Finally, a high percentage of Ran C-terminus mutations from cancer patients were found to be destabilizing and hyperactivating, suggesting that Ran C-destabilization might be an unprecedented cellular transformation mechanism in affected cancers. This study also highlights a new drug design strategy towards treating patients with hyperactivated Ras proteins including K-Ras.

scholarly journals Recruitment of Host Nuclear Pore Components to the Vicinity of Theileria Schizonts

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Sandra Huber ◽  
Anina Bär ◽  
Selina Epp ◽  
Jacqueline Schmuckli-Maurer ◽  
Naja Eberhard ◽  
...  
Keyword(s):  
Host Cell ◽  
Nuclear Pore Complex ◽  
Cell Biology ◽  
Metaphase Plate ◽  
Cellular Transformation ◽  
Small Gtpase ◽  
Nuclear Pore ◽  
Annulate Lamellae ◽  
Nuclear Trafficking ◽  
Host Parasite

ABSTRACT Parasitic protozoans of the genus Theileria are intracellular pathogens that induce the cellular transformation of leukocytes, causing uncontrolled proliferation of the infected host cell. The transforming stage of the parasite has a strictly intracellular lifestyle and ensures its distribution to both daughter cells during host cell cytokinesis by aligning itself across the metaphase plate and by binding tightly to central spindle and astral microtubules. Given the importance of the parasite surface in maintaining interactions with host microtubules, we analyzed the ultrastructure of the host-parasite interface using transmission electron microscopy combined with high-resolution fluorescence microscopy and live-cell imaging. We show that porous membranes, termed annulate lamellae (AL), closely associate with the Theileria surface in infected T cells, B cells, and macrophages and are not detectable in noninfected bovine cell lines such as BL20 or BoMACs. AL are membranous structures found in the cytoplasm of fast-proliferating cells such as cancer cells, oocytes, and embryonic cells. Although AL were first observed more than 60 years ago, the function of these organelles is still not known. Indirect immunofluorescence analysis with a pan-nuclear pore complex antibody, combined with overexpression of a panel of nuclear pore proteins, revealed that the parasite recruits nuclear pore complex components close to its surface. Importantly, we show that, in addition to structural components of the nuclear pore complex, nuclear trafficking machinery, including importin beta 1, RanGAP1, and the small GTPase Ran, also accumulated close to the parasite surface. IMPORTANCE Theileria schizonts are the only known eukaryotic organisms capable of transforming another eukaryotic cell; as such, probing of the interactions that occur at the host-parasite interface is likely to lead to novel insights into the cell biology underlying leukocyte proliferation and transformation. Little is known about how the parasite communicates with its host or by what route secreted parasite proteins are translocated into the host, and we propose that nuclear trafficking machinery at the parasite surface might play a role in this. The function of AL remains completely unknown, and our work provides a basis for further investigation into the contribution that these porous, cytomembranous structures might make to the survival of fast-growing transformed cells.

scholarly journals Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

2011 ◽  
Vol 22 (5) ◽  
pp. 661-672 ◽  
Author(s):  
Marie K. Cross ◽  
Maureen A. Powers
Keyword(s):  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Nuclear Pore ◽  
Bipolar Spindle ◽  
Terminal Domain ◽  
C Terminus ◽  
Mitotic Spindle Assembly

During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere–associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

scholarly journals GEF-independent Ran activation shifts a fraction of the protein to the cytoplasm and promotes cell proliferation

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Jinhan Zhou ◽  
Yuping Tan ◽  
Yuqing Zhang ◽  
Aiping Tong ◽  
Xiaofei Shen ◽  
...  
Keyword(s):  
Biological Significance ◽  
Cellular Transformation ◽  
Nuclear Pore ◽  
Missense Mutations ◽  
Nucleotide Exchange ◽  
C Terminus ◽  
Cytoplasmic Transport ◽  
Cancer Mutations ◽  
The Impact

AbstractRan (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other functions in the cytoplasm, as well as in cellular transformation when switched on. Unlike other members of the GTPase superfamily, Ran binds more tightly to GDP than to GTP due to the presence of an auto-inhibitory C-terminal tail. Multiple missense mutations in the C-terminus of Ran occur in cancers, but their biological significance remains unclear. Here, the quantitative GDP/GTP binding preference of four engineered mutations with unstable C-termini was analyzed using a devised mant-GDP dissociation assay. The results showed that the impact of different C-terminal mutations depends on multiple factors. Although these mutants were more GTP-loaded in human cells, they were shown to be more cytoplasmic, and to support nuclear transport with minimally or partially reduced efficiency. Further, several Ran cancer mutants were compromised in autoinhibition, slightly more GTP-bound, more cytoplasmic, and enhanced the proliferation of A549 and HeLa cells in vitro. Thus, our work reveals a new route of Ran activation independent of guanine nucleotide exchange factor (GEF), which may account for the hyper-proliferation induced by Ran cancer mutations.

scholarly journals 8 Å structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex solved by cryo-EM and AI

2021 ◽  
Author(s):  
Linhua Tai ◽  
Yun Zhu ◽  
He Ren ◽  
Xiaojun Huang ◽  
Chuanmao Zhang ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Pore Complex ◽  
Protein Complexes ◽  
Complex Structure ◽  
Nucleocytoplasmic Transport ◽  
Structural Features ◽  
Nuclear Pore ◽  
Electron Microscopic ◽  
C Terminus ◽  
Parallel Pattern

As one of the largest protein complexes in eukaryotes, the nuclear pore complex (NPC) forms a conduit regulating nucleocytoplasmic transport. Here, we determined 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the cytoplasmic ring (CR) from the Xenopus laevis NPC. With the aid of AlphaFold2, we managed to build a most comprehensive and accurate pseudoatomic model of the CR to date, including the Y complexes and flanking components of Nup358, Nup214 complexes, Nup205 and Nup93. Comparing with previously reported CR model, the Y complex structure in our model exhibits much tighter interactions in the hub region mediated by α-solenoid domain in Nup160 C-terminus. Five copies of Nup358 are identified in each CR subunit to provide rich interactions with other Nups in stem regions of Y complexes. Two copies of Nup214 complexes lay in a parallel pattern and attach to the short arm region of Y complexes towards the central channel of NPC. Besides, the structural details of two copies of Nup205 on the side of the short arm region and one copy of Nup93 on the stem region of Y complexes in each CR subunit are also revealed. These in-depth novel structural features represent a great advance in understanding the assembly of NPCs.

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