scholarly journals Importin-β negatively regulates multiple aspects of mitosis including RANGAP1 recruitment to kinetochores

2012 ◽  
Vol 196 (4) ◽  
pp. 435-450 ◽  
Author(s):  
Emanuele Roscioli ◽  
Laura Di Francesco ◽  
Alessio Bolognesi ◽  
Maria Giubettini ◽  
Serena Orlando ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Binding Sites ◽  
Protein Import ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Microtubule Dynamic ◽  
Nuclear Envelope Breakdown ◽  
Microtubule Attachment ◽  
Importin Α ◽  
Mitotic Spindle Pole

Importin-β is the main vector for interphase nuclear protein import and plays roles after nuclear envelope breakdown. Here we show that importin-β regulates multiple aspects of mitosis via distinct domains that interact with different classes of proteins in human cells. The C-terminal region (which binds importin-α) inhibits mitotic spindle pole formation. The central region (harboring nucleoporin-binding sites) regulates microtubule dynamic functions and interaction with kinetochores. Importin-β interacts through this region with NUP358/RANBP2, which in turn binds SUMO-conjugated RANGAP1 in nuclear pores. We show that this interaction continues after nuclear pore disassembly. Overexpression of importin-β, or of the nucleoporin-binding region, inhibited RANGAP1 recruitment to mitotic kinetochores, an event that is known to require microtubule attachment and the exportin CRM1. Co-expressing either importin-β–interacting RANBP2 fragments, or CRM1, restored RANGAP1 to kinetochores and rescued importin-β–dependent mitotic dynamic defects. These results reveal previously unrecognized importin-β functions at kinetochores exerted via RANBP2 and opposed by CRM1.

scholarly journals The Three Fungal Transmembrane Nuclear Pore Complex Proteins of Aspergillus nidulans Are Dispensable in the Presence of an Intact An-Nup84-120 Complex

2009 ◽  
Vol 20 (2) ◽  
pp. 616-630 ◽  
Author(s):  
Hui-Lin Liu ◽  
Colin P.C. De Souza ◽  
Aysha H. Osmani ◽  
Stephen A. Osmani
Keyword(s):  
High Temperature ◽  
Nuclear Envelope ◽  
Aspergillus Nidulans ◽  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Spindle Pole Bodies ◽  
Pore Complexes

In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup.

scholarly journals Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly

2016 ◽  
Vol 27 (11) ◽  
pp. 1753-1763 ◽  
Author(s):  
Hirohisa Masuda ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Synthetic Lethality ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Spindle Microtubule ◽  
Spindle Pole Bodies ◽  
Mitotic Spindle Pole

In fission yeast, γ-tubulin ring complex (γTuRC)–specific components Gfh1GCP4, Mod21GCP5, and Alp16GCP6 are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1MOZART1, a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16GCP6 promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1GCP4 and Mod21GCP5 are not required for Alp16GCP6-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16GCP6 and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation.

scholarly journals The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

2002 ◽  
Vol 158 (1) ◽  
pp. 63-77 ◽  
Author(s):  
Tobias C. Walther ◽  
Helen S. Pickersgill ◽  
Volker C. Cordes ◽  
Martin W. Goldberg ◽  
Terry D. Allen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Slight Reduction ◽  
Cytoplasmic Filaments ◽  
Localization Sequence ◽  
Importin Α

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin α/β– or transportin-dependent import.

Probing The Mechanisms Underlying Kinetochore Behavior In Vertebate Cells Using Combinations of Advanced Light and 3-D Electron Microscopy

1997 ◽  
Vol 3 (S2) ◽  
pp. 217-218
Author(s):  
B. F. McEwen ◽  
A.B. Heagle ◽  
C.L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Mitotic Spindle ◽  
Attachment Site ◽  
Spindle Pole ◽  
Nuclear Envelope Breakdown ◽  
Daughter Cells ◽  
Primary Constriction ◽  
Sister Kinetochore ◽  
To Receive ◽  
Chromosome Motion

For daughter cells to receive equal copies of the genome during mitosis, the replicated chromosomes must attach to and move bi-directionally on the mitotic spindle. A chromosome becomes attached to the spindle via a pair specialized structures, known as kinetochores, that are positioned on opposite sides of its primary constriction (one on each of the two chromatids). In addition to being the spindle attachment site, kinetochores are also involved in producing and/or transmitting the forces for chromosome motion. In vertebrates the kinetochore closest to a spindle pole at the time of nuclear envelope breakdown usually is the first to attach to the spindle. As a result of this attachment the now “monooriented” chromosome moves toward the closest pole where its only attached kinetochore initiates oscillatory motions toward and away from that pole until the unattached sister kinetochore acquires microtubules (Mts) from the opposite spindle pole.

scholarly journals Influence of cargo size on Ran and energy requirements for nuclear protein import

2002 ◽  
Vol 159 (1) ◽  
pp. 55-67 ◽  
Author(s):  
Susan K. Lyman ◽  
Tinglu Guan ◽  
Janna Bednenko ◽  
Harald Wodrich ◽  
Larry Gerace
Keyword(s):  
Biochemical Analysis ◽  
Protein Import ◽  
Small Gtpase ◽  
Nucleoside Triphosphate ◽  
Nuclear Pore ◽  
Central Regions ◽  
Importin Α ◽  
Direct Binding ◽  
Gtpase Ran

Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin α/β and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin β and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.

scholarly journals Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance

Cell Cycle ◽  
2013 ◽  
Vol 12 (16) ◽  
pp. 2643-2655 ◽  
Author(s):  
Adam M Corrigan ◽  
Roshan L Shrestha ◽  
Ihsan Zulkipli ◽  
Noriko Hiroi ◽  
Yingjun Liu ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Automated Tracking ◽  
Spindle Rotation ◽  
Mitotic Spindle Pole

scholarly journals Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces

2011 ◽  
Vol 10 (1) ◽  
pp. 131 ◽  
Author(s):  
Italia A Asteriti ◽  
Maria Giubettini ◽  
Patrizia Lavia ◽  
Giulia Guarguaglini
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Aurora A ◽  
Mitotic Spindle Pole

scholarly journals Importin β contains a COOH-terminal nucleoporin binding region important for nuclear transport

2003 ◽  
Vol 162 (3) ◽  
pp. 391-401 ◽  
Author(s):  
Janna Bednenko ◽  
Gino Cingolani ◽  
Larry Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Binding Sites ◽  
Nuclear Transport ◽  
Terminal Region ◽  
Nuclear Pore ◽  
Binding Region ◽  
Similar Extent ◽  
Importin Α ◽  
Import Receptor

Proteins containing a classical NLS are transported into the nucleus by the import receptor importin β, which binds to cargoes via the adaptor importin α. The import complex is translocated through the nuclear pore complex by interactions of importin β with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin β. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin β to a similar extent (∼50%). An importin β mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin β possesses two nucleoporin binding sites, both of which are important for its nuclear import function.

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