Nucleolar Separation from Chromosomes during Aspergillus nidulans Mitosis Can Occur Without Spindle Forces

How the nucleolus is segregated during mitosis is poorly understood and occurs by very different mechanisms during closed and open mitosis. Here we report a new mechanism of nucleolar segregation involving removal of the nucleolar-organizing regions (NORs) from nucleoli during Aspergillus nidulans mitosis. This involves a double nuclear envelope (NE) restriction which generates three NE-associated structures, two daughter nuclei (containing the NORs), and the nucleolus. Therefore, a remnant nucleolar structure can exist in the cytoplasm without NORs. In G1, this parental cytoplasmic nucleolus undergoes sequential disassembly releasing nucleolar proteins to the cytoplasm as nucleoli concomitantly reform in daughter nuclei. By depolymerizing microtubules and mutating spindle assembly checkpoint function, we demonstrate that a cycle of nucleolar “segregation” can occur without a spindle in a process termed spindle-independent mitosis (SIM). During SIM physical separation of the NOR from the nucleolus occurs, and NE modifications promote expulsion of the nucleolus to the cytoplasm. Subsequently, the cytoplasmic nucleolus is disassembled and rebuilt at a new site around the nuclear NOR. The data demonstrate the existence of a mitotic machinery for nucleolar segregation that is normally integrated with mitotic spindle formation but that can function without it.

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A Screen for Dynein Synthetic Lethals in Aspergillus nidulans Identifies Spindle Assembly Checkpoint Genes and Other Genes Involved in Mitosis

Genetics ◽

10.1093/genetics/149.1.101 ◽

1998 ◽

Vol 149 (1) ◽

pp. 101-116

Author(s):

Vladimir P Efimov ◽

N Ronald Morris

Keyword(s):

Aspergillus Nidulans ◽

Spindle Assembly Checkpoint ◽

Genetic Interaction ◽

Synthetic Lethality ◽

Spindle Assembly ◽

Nuclear Migration ◽

Cytoplasmic Dynein ◽

Vesicle Transport ◽

Synthetic Lethal ◽

Checkpoint Genes

Abstract Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0628 ◽

2009 ◽

Vol 20 (2) ◽

pp. 616-630 ◽

Cited By ~ 73

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e15-08-0577 ◽

2016 ◽

Vol 27 (11) ◽

pp. 1753-1763 ◽

Cited By ~ 19

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.

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Regulation of Mitotic Spindle Asymmetry by SUMO and the Spindle-Assembly Checkpoint in Yeast

Current Biology ◽

10.1016/j.cub.2008.07.091 ◽

2008 ◽

Vol 18 (16) ◽

pp. 1249-1255 ◽

Cited By ~ 34

Author(s):

Christian Leisner ◽

Daniel Kammerer ◽

Annina Denoth ◽

Mirjam Britschi ◽

Yves Barral ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly

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Interactions between N-Terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

10.1101/438903 ◽

2018 ◽

Author(s):

Spyridon T. Pachis ◽

Yoshitaka Hiruma ◽

Anastassis Perrakis ◽

Geert J.P.L. Kops

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Intramolecular Interactions ◽

Mitotic Progression ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Regulatory Input ◽

Tpr Domain

ABSTRACTFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

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EFFECTORS OF THE SPINDLE ASSEMBLY CHECKPOINT BUT NOT THE MITOTIC EXIT NETWORK ARE CONFINED WITHIN THE NUCLEUS OF SACCHAROMYCES CEREVISIAE

10.1101/340091 ◽

2018 ◽

Author(s):

Lydia R Heasley ◽

Jennifer G DeLuca ◽

Steven M Markus

Keyword(s):

Nuclear Envelope ◽

Mammalian Cells ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Mitotic Exit ◽

Cell Cycle Checkpoints ◽

Anaphase Promoting Complex ◽

Mitotic Exit Network ◽

And Function

The Spindle Assembly Checkpoint (SAC) prevents erroneous chromosome segregation by delaying mitotic progression when chromosomes are incorrectly attached to the mitotic spindle. This delay is mediated by Mitotic Checkpoint Complexes (MCCs), which assemble at unattached kinetochores and repress the activity of the Anaphase Promoting Complex/Cyclosome (APC/C). The cellular localizations of MCCs are likely critical for proper SAC function, yet remain poorly defined. We recently demonstrated that in mammalian cells, in which the nuclear envelope disassembles during mitosis, MCCs diffuse throughout the spindle region and cytoplasm. Here, we employed binucleate yeast zygotes to examine the localization dynamics of SAC effectors required for MCC assembly and function in budding yeast, in which the nuclear envelope remains intact throughout mitosis. Our findings indicate that in yeast MCCs are confined to the nuclear compartment and excluded from the cytoplasm during mitosis. In contrast, we find that effectors of the Mitotic Exit Network (MEN) - a pathway that initiates disassembly of the anaphase spindle only when it is properly oriented - are in fact freely exchanged between multiple nuclei within a shared cytoplasm. Our study provides insight into how cell cycle checkpoints have evolved to function in diverse cellular contexts.

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Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

The Journal of Cell Biology ◽

10.1083/jcb.200808130 ◽

2009 ◽

Vol 184 (3) ◽

pp. 373-381 ◽

Cited By ~ 181

Author(s):

Thomas J. Maresca ◽

Edward D. Salmon

Keyword(s):

Drosophila Melanogaster ◽

Green Fluorescent Protein ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Fluorescent Protein ◽

Spindle Assembly ◽

Centromeric Chromatin ◽

Cell Assays ◽

Green Fluorescent

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a “wait anaphase” signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.

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Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e11-11-0927 ◽

2012 ◽

Vol 23 (5) ◽

pp. 930-944 ◽

Cited By ~ 31

Author(s):

Eduardo Ródenas ◽

Cristina González-Aguilera ◽

Cristina Ayuso ◽

Peter Askjaer

Keyword(s):

Caenorhabditis Elegans ◽

Nuclear Envelope ◽

Molecular Mechanisms ◽

Spindle Assembly Checkpoint ◽

Protein Import ◽

Spindle Assembly ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Pore Complexes

Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly, and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in a metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5 independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation.

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