scholarly journals The fission yeast nucleoporin Alm1 is required for proteasomal degradation of kinetochore components

2017 ◽  
Vol 216 (11) ◽  
pp. 3591-3608 ◽  
Author(s):  
Silvia Salas-Pino ◽  
Paola Gallardo ◽  
Ramón R. Barrales ◽  
Sigurd Braun ◽  
Rafael R. Daga
Keyword(s):  
Nuclear Envelope ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Essential Role ◽  
Spindle Assembly Checkpoint ◽  
Proteasomal Degradation ◽  
Nuclear Pore ◽  
Checkpoint Activation ◽  
Centromeric Chromatin

Kinetochores (KTs) are large multiprotein complexes that constitute the interface between centromeric chromatin and the mitotic spindle during chromosome segregation. In spite of their essential role, little is known about how centromeres and KTs are assembled and how their precise stoichiometry is regulated. In this study, we show that the nuclear pore basket component Alm1 is required to maintain both the proteasome and its anchor, Cut8, at the nuclear envelope, which in turn regulates proteostasis of certain inner KT components. Consistently, alm1-deleted cells show increased levels of KT proteins, including CENP-CCnp3, spindle assembly checkpoint activation, and chromosome segregation defects. Our data demonstrate a novel function of the nucleoporin Alm1 in proteasome localization required for KT homeostasis.

scholarly journals Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

2008 ◽  
Vol 182 (2) ◽  
pp. 277-288 ◽  
Author(s):  
Ayumu Yamamoto ◽  
Kenji Kitamura ◽  
Daisuke Hihara ◽  
Yukinobu Hirose ◽  
Satoshi Katsuyama ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Related Factor ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Anaphase Onset ◽  
Yeast Meiosis ◽  
Meiosis I

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/CCdc20), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.

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

2009 ◽  
Vol 184 (3) ◽  
pp. 373-381 ◽  
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.

scholarly journals Dual Regulation of Mad2 Localization on Kinetochores by Bub1 and Dam1/DASH that Ensure Proper Spindle Interaction

2008 ◽  
Vol 19 (9) ◽  
pp. 3885-3897 ◽  
Author(s):  
Shigeaki Saitoh ◽  
Yasuyo Kobayashi ◽  
Yuki Ogiyama ◽  
Kohta Takahashi
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
The State ◽  
Abnormal Chromosome ◽  
Mitotic Progression ◽  
Checkpoint Activation ◽  
Molecular Sensors ◽  
Checkpoint Proteins ◽  
Spindle Attachment

The spindle assembly checkpoint monitors the state of spindle–kinetochore interaction to prevent premature onset of anaphase. Although checkpoint proteins, such as Mad2, are localized on kinetochores that do not interact properly with the spindle, it remains unknown how the checkpoint proteins recognize abnormalities in spindle–kinetochore interaction. Here, we report that Mad2 localization on kinetochores in fission yeast is regulated by two partially overlapping but distinct pathways: the Dam1/DASH and the Bub1 pathways. We show that Mad2 is localized on “unattached” as well as “tensionless” kinetochores. Our observations suggest that Bub1 is required for Mad2 to detect tensionless kinetochores, whereas Dam1/DASH is crucial for Mad2 to detect unattached kinetochores. In cells lacking both Bub1 and Dam1/DASH, Mad2 localization on kinetochores is diminished, and mitotic progression appears to be accelerated despite the frequent occurrence of abnormal chromosome segregation. Furthermore, we found that Dam1/DASH is required for promotion of spindle association with unattached kinetochores. In contrast, there is accumulating evidence that Bub1 is involved in resolution of erroneous spindle attachment on tensionless kinetochores. These pathways may act as molecular sensors determining the state of spindle association on each kinetochore, enabling proper regulation of the checkpoint activation as well as promotion/resolution of spindle attachment.

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 Centromere-localized Aurora B kinase is required for the fidelity of chromosome segregation

2019 ◽  
Vol 219 (2) ◽  
Author(s):  
Cai Liang ◽  
Zhenlei Zhang ◽  
Qinfu Chen ◽  
Haiyan Yan ◽  
Miao Zhang ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Essential Role ◽  
Spindle Assembly Checkpoint ◽  
Histone H3 ◽  
Spindle Assembly ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Chromosome Missegregation ◽  
Proper Timing ◽  
Segregation Of Chromosomes

Aurora B kinase plays an essential role in chromosome bi-orientation, which is a prerequisite for equal segregation of chromosomes during mitosis. However, it remains largely unclear whether centromere-localized Aurora B is required for faithful chromosome segregation. Here we show that histone H3 Thr-3 phosphorylation (H3pT3) and H2A Thr-120 phosphorylation (H2ApT120) can independently recruit Aurora B. Disrupting H3pT3-mediated localization of Aurora B at the inner centromere impedes the decline in H2ApT120 during metaphase and causes H2ApT120-dependent accumulation of Aurora B at the kinetochore-proximal centromere. Consequently, silencing of the spindle assembly checkpoint (SAC) is delayed, whereas the fidelity of chromosome segregation is negligibly affected. Further eliminating an H2ApT120-dependent pool of Aurora B restores proper timing for SAC silencing but increases chromosome missegregation. Our data indicate that H2ApT120-mediated localization of Aurora B compensates for the loss of an H3pT3-dependent pool of Aurora B to correct improper kinetochore–microtubule attachments. This study provides important insights into how centromeric Aurora B regulates SAC and kinetochore attachment to microtubules to ensure error-free chromosome segregation.

scholarly journals csi2p modulates microtubule dynamics and organizes the bipolar spindle for chromosome segregation

2014 ◽  
Vol 25 (24) ◽  
pp. 3900-3908 ◽  
Author(s):  
Judite Costa ◽  
Chuanhai Fu ◽  
V. Mohini Khare ◽  
Phong T. Tran
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Microtubule Dynamics ◽  
High Rate ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Eukaryotic Chromosome ◽  
Relative Contribution ◽  
Multiple Phenotypes

Proper chromosome segregation is of paramount importance for proper genetic inheritance. Defects in chromosome segregation can lead to aneuploidy, which is a hallmark of cancer cells. Eukaryotic chromosome segregation is accomplished by the bipolar spindle. Additional mechanisms, such as the spindle assembly checkpoint and centromere positioning, further help to ensure complete segregation fidelity. Here we present the fission yeast csi2+. csi2p localizes to the spindle poles, where it regulates mitotic microtubule dynamics, bipolar spindle formation, and subsequent chromosome segregation. csi2 deletion (csi2Δ) results in abnormally long mitotic microtubules, high rate of transient monopolar spindles, and subsequent high rate of chromosome segregation defects. Because csi2Δ has multiple phenotypes, it enables estimates of the relative contribution of the different mechanisms to the overall chromosome segregation process. Centromere positioning, microtubule dynamics, and bipolar spindle formation can all contribute to chromosome segregation. However, the major determinant of chromosome segregation defects in fission yeast may be microtubule dynamic defects.

scholarly journals A quantitative analysis of cohesin decay in mitotic fidelity

2018 ◽  
Vol 217 (10) ◽  
pp. 3343-3353 ◽  
Author(s):  
Sara Carvalhal ◽  
Alexandra Tavares ◽  
Mariana B. Santos ◽  
Mihailo Mirkovic ◽  
Raquel A. Oliveira
Keyword(s):  
Quantitative Analysis ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Mitotic Spindle ◽  
Sister Chromatid Cohesion ◽  
Force Balance ◽  
Chromosome Organization ◽  
Centromeric Chromatin ◽  
Chromatid Cohesion ◽  
Chromosome Alignment

Sister chromatid cohesion mediated by cohesin is essential for mitotic fidelity. It counteracts spindle forces to prevent premature chromatid individualization and random genome segregation. However, it is unclear what effects a partial decline of cohesin may have on chromosome organization. In this study, we provide a quantitative analysis of cohesin decay by inducing acute removal of defined amounts of cohesin from metaphase-arrested chromosomes. We demonstrate that sister chromatid cohesion is very resistant to cohesin loss as chromatid disjunction is only observed when chromosomes lose >80% of bound cohesin. Removal close to this threshold leads to chromosomes that are still cohered but display compromised chromosome alignment and unstable spindle attachments. Partial cohesin decay leads to increased duration of mitosis and susceptibility to errors in chromosome segregation. We propose that high cohesin density ensures centromeric chromatin rigidity necessary to maintain a force balance with the mitotic spindle. Partial cohesin loss may lead to chromosome segregation errors even when sister chromatid cohesion is fulfilled.

scholarly journals Selective Nuclear Pore Complex Removal Drives Nuclear Envelope Division in Fission Yeast

2020 ◽  
Vol 30 (16) ◽  
pp. 3212-3222.e2 ◽  
Author(s):  
María Expósito-Serrano ◽  
Ana Sánchez-Molina ◽  
Paola Gallardo ◽  
Silvia Salas-Pino ◽  
Rafael R. Daga
Keyword(s):  
Nuclear Envelope ◽  
Fission Yeast ◽  
Nuclear Pore Complex ◽  
Nuclear Pore

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 LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

2017 ◽  
Vol 114 (11) ◽  
pp. E2166-E2175 ◽  
Author(s):  
Mingyu Gu ◽  
Dollie LaJoie ◽  
Opal S. Chen ◽  
Alexander von Appen ◽  
Mark S. Ladinsky ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Fission Yeast ◽  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Human Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Loss Of Function

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. InSchizosaccharomyces pombe, deletion of the ATPasevps4leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously inlem2orcmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.

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