Accurate chromosome segregation during cell division is essential to maintain genome integrity in all eukaryotic cells, and chromosome missegregation leads to aneuploidy and therefore represents a ...hallmark of many cancers. Accurate segregation requires sister kinetochores to attach to microtubules emanating from opposite spindle poles, known as bipolar attachment or biorientation. Recent studies have uncovered several mechanisms critical to chromosome bipolar attachment. First, a mechanism exists to ensure that the conformation of sister centromeres is biased toward bipolar attachment. Second, the phosphorylation of some kinetochore proteins destabilizes kinetochore attachment to facilitate error correction, but a protein phosphatase reverses this phosphorylation. Moreover, the activity of the spindle assembly checkpoint is regulated by kinases and phosphatases at the kinetochore, and this checkpoint prevents anaphase entry in response to faulty kinetochore attachment. The fine-tuned kinase/phosphatase balance at kinetochores is crucial for faithful chromosome segregation during both mitosis and meiosis. Here, we discuss the function and regulation of protein phosphatases in the establishment of chromosome bipolar attachment with a focus on the model organism budding yeast.
The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated ...Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Accurate chromosome segregation depends on bipolar chromosome–microtubule attachment and tension generation on chromosomes. Incorrect chromosome attachment results in chromosome missegregation, which ...contributes to genome instability. The kinetochore is a protein complex that localizes at the centromere region of a chromosome and mediates chromosome–microtubule interaction. Incorrect chromosome attachment leads to checkpoint activation to prevent anaphase onset. Kinetochore detachment activates the spindle assembly checkpoint (SAC), while tensionless kinetochore attachment relies on both the SAC and tension checkpoint. In budding yeast Saccharomyces cerevisiae, kinesin-5 motor proteins Cin8 and Kip1 are needed to separate spindle pole bodies for spindle assembly, and deletion of CIN8 causes lethality in the absence of SAC. To study the function of Cin8 and Kip1 in chromosome segregation, we constructed an auxin-inducible degron (AID) mutant, cin8-AID. With this conditional mutant, we first confirmed that cin8-AID kip1∆ double mutants were lethal when Cin8 is depleted in the presence of auxin. These cells arrested in metaphase with unseparated spindle pole bodies and kinetochores. We further showed that the absence of either the SAC or tension checkpoint was sufficient to abolish the cell-cycle delay in cin8-AID mutants, causing chromosome missegregation and viability loss. The tension checkpoint-dependent phenotype in cells with depleted Cin8 suggests the presence of tensionless chromosome attachment. We speculate that the failed spindle pole body separation in cin8 mutants could increase the chance of tensionless syntelic chromosome attachments, which depends on functional tension checkpoint for survival.
The accumulation of misfolded proteins is associated with multiple neurodegenerative disorders, but it remains poorly defined how this accumulation causes cytotoxicity. Here, we demonstrate that the ...Cdc48/p97 segregase machinery drives the clearance of ubiquitinated model misfolded protein Huntingtin (Htt103QP) and limits its aggregation. Nuclear ubiquitin ligase San1 acts upstream of Cdc48 to ubiquitinate Htt103QP. Unexpectedly, deletion of SAN1 and/or its cytosolic counterpart UBR1 rescues the toxicity associated with Cdc48 deficiency, suggesting that ubiquitin depletion, rather than compromised proteolysis of misfolded proteins, causes the growth defect in cells with Cdc48 deficiency. Indeed, Cdc48 deficiency leads to elevated protein ubiquitination levels and decreased free ubiquitin, which depends on San1/Ubr1. Furthermore, enhancing free ubiquitin levels rescues the toxicity in various Cdc48 pathway mutants and restores normal turnover of a known Cdc48-independent substrate. Our work highlights a previously unappreciated function for Cdc48 in ensuring the regeneration of monoubiquitin that is critical for normal cellular function.
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•Cdc48 segregase is required for the degradation of misfolded proteins in yeast•Cdc48 deficiency leads to a decreased pool of free ubiquitin that compromises the UPS•San1 and Ubr1 ubiquitinate misfolded proteins, reducing the free ubiquitin pool•Restoring free ubiquitin suppresses the toxicity associated with Cdc48 deficiency
Misfolded protein accumulation causes cytotoxicity, but the mechanism remains poorly understood. Using budding yeast as a model organism, Higgins et al. show that ubiquitination of misfolded proteins depletes free ubiquitin, which compromises ubiquitin-dependent cellular functions and causes cytotoxicity. The Cdc48/p97 segregase antagonizes this cytotoxicity by promoting ubiquitin recycling from misfolded proteins.
The conserved chromosomal passenger complex (CPC) consists of Ipl1
, Sli15
, Bir1
, and Nbl1
, and localizes at the kinetochore/centromere to correct kinetochore attachment errors and to prevent ...checkpoint silencing. After anaphase entry, the CPC moves from the kinetochore/centromere to the spindle. In budding yeast, CPC subunit Sli15 is phosphorylated by both cyclin-dependent kinase (CDK) and Ipl1 kinase. Following anaphase onset, activated Cdc14 phosphatase reverses Sli15 phosphorylation imposed by CDK to promote CPC translocation. Although abolished Sli15 phosphorylation imposed by Ipl1 also causes CPC translocation, the regulation of Ipl1-imposed Sli15 phosphorylation remains unclear. In addition to Sli15, Cdc14 also dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. Here, we present evidence supporting the notion that kinetochore-localized Fin1-PP1 likely reverses Ipl1-imposed Sli15 phosphorylation to promote CPC translocation from the kinetochore/centromere to the spindle. Importantly, premature Fin1 kinetochore localization or phospho-deficient
mutation causes checkpoint defects in response to tensionless attachments, resulting in chromosome missegregation. In addition, our data indicate that reversion of CDK- and Ipl1-imposed Sli15 phosphorylation shows an additive effect on CPC translocation. Together, these results reveal a previously unidentified pathway to regulate CPC translocation, which is important for accurate chromosome segregation.
Accurate chromosome segregation is vital to maintain genomic integrity. Chromosome missegregation, or aneuploidy, contributes to cancer development and conditions like down syndrome (Trisomy 21). ...Chromosomes are attached to microtubules during mitosis through centromere-localized kinetochores, and accurate chromosome segregation requires establishment of chromosome bipolar attachment. As a result of bipolar attachment, tension is generated across sister kinetochores. Failure in this process activates the spindle assembly checkpoint (SAC) to prevent anaphase entry and allow for correction of attachments errors. In contrast, tension generation is believed to silence the SAC for anaphase onset.One key regulator of the SAC is the conserved chromosomal passenger complex (CPC), composed of Ipl1 kinase, Sli15, Bir1, and Nbl1 in budding yeast. The CPC localizes to the kinetochore/centromere before anaphase entry. Kinetochore protein Ndc80 is one Ipl1 substrate, and its phosphorylation destabilizes kinetochore-microtubule interaction to promote error correction. After anaphase onset, the CPC is removed from the kinetochore/centromere and relocates to the spindle, where it assists in spindle stability. Meanwhile, Ipl1-dependent Ndc80 phosphorylation is reversed, but the phosphatase responsible for this reversal is unknown. In budding yeast, the phosphatase Cdc14 becomes active after anaphase onset and dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1). Fin1 dephosphorylation allows kinetochore recruitment of Fin1-PP1. In this work, we show that kinetochore recruitment of Fin1-PP1 triggers the removal of the SAC proteins Bub1/Bub3. Furthermore, Fin1-PP1 partially dephosphorylates Ndc80, which promotes kinetochore dissociation of Bub1/Bub3. In addition, phospho-deficient ndc80 mutants causes viability loss in response to syntelic chromosome attachment, wherein sister kinetochores are attached by microtubules from the same spindle pole, indicating the importance of timely Fin1-PP1 kinetochore localization in checkpoint regulation.Multiple mechanisms regulate CPC kinetochore/centromere localization. A main driver for localization is Sli15 phosphorylation by both cyclin-dependent kinase (CDK) and Ipl1 kinases. After anaphase onset, Sli15 is dephosphorylated, which triggers CPC translocation to anaphase spindle. Active Cdc14 reverses CDK-imposed Sli15 phosphorylation, but it is currently unclear which phosphatase reverses Ipl1-imposed Sli15 phosphorylation. Here we show that Fin1-PP1 partially reverses Sli15 phosphorylation. Interestingly, premature Fin1 kinetochore localization is sufficient to promote both CPC dissociation from the kinetochore/centromere in metaphase and CPC spindle association in anaphase. In addition, premature Fin1-kinetochore localization or phospho-deficient sli15 mutants result in SAC defects in response to tensionless attachments. These results indicate that Fin1-PP1 reverses Sli15 phosphorylation imposed by Ipl1 to promote CPC translocation in anaphase.The tension checkpoint is a subset of the SAC that prevents anaphase onset in response to tensionless attachments. In response to tension defects, Ipl1 kinase phosphorylates the kinetochore protein Dam1 to prevent anaphae onset. Budding yeast Cin8 is a kinesin-5 motor protein, and its synthetic lethality with SAC mutants indicates the role of Cin8 in accurate chromosome attachment. To study the function of Cin8 in chromosome attachment, we constructed an auxin-inducible degron (AID) mutant, cin8-AID. With this conditional mutant, we show that cells lacking Cin8 arrest in metaphase due to unseparated spindle pole bodies. This metaphase arrest depends on the SAC as well as Dam1 phosphorylation by Ipl1 kinase. In cells expressing phospho-deficient dam1-3A, the metaphase arrest induced by Cin8 depletion is abolished, causing chromosome missegregation. These results support the conclusion that the cell cycle delay in cin8 mutants is a result of tensionless chromsome attachments.Because of the critical role of CPC in chromosome bipolar attachment, its kinetochore/centromere localization must be tightly regulated during cell cycle. SUMOylation is a post-translational modification, wherein SUMO (Small Ubiquitin-like Modifier) proteins are covalently attached to other proteins. SUMOylation has been shown to promote protein subcellular localization through the interacteion between SUMO and SUMO-interacting motifs (SIMs). This interaction facilitates formation of molecular condensates through liquid-liquid phase separation (LLPS). On the other hand, formation of SUMO chains (polySUMOylation) can induce condensate disassembly by triggering a series downstream events, including ubiquitination by SUMO-dependent Ubiquitin ligases (STUbLs) and extraction by Cdc48 segregase. In mammalian cells, the CPC was shown to be enriched at kinetochores through LLPS. A recent work indicates that CPC components Bir1 is SUMOylated. One attractive idea is that CPC SUMOylation promotes it kinetochores localization, while polySUMOylation triggers CPC kinetochore delocalization for its translocation. In this work, we show that Bir1 phosphorylation is cell cycle regulated, and the absence of STUbL delays Bir1 dephosphorylation. Moreover, depletion of SUMO protease, Ulp2, increases Bir1 SUMOylation Interestingly, inducing polySUMOylation prevents proper CPC kinetochore localization in metaphase. These preliminary results support the idea that Bir1 SUMOylation may regulate Bir1 phosphorylation and CPC localization.
BIG 3-07/TROG 07.01 is an international, multicentre, randomised, controlled, phase 3 trial evaluating tumour bed boost and hypofractionation in patients with non-low-risk ductal carcinoma in situ ...following breast-conserving surgery and whole breast radiotherapy. Here, we report the effects of diagnosis and treatment on health-related quality of life (HRQOL) at 2 years.
The BIG 3-07/TROG 07.01 trial is ongoing at 118 hospitals in 11 countries. Women aged 18 years or older with completely excised non-low-risk ductal carcinoma in situ were randomly assigned, by use of a minimisation algorithm, to tumour bed boost or no tumour bed boost, following conventional whole breast radiotherapy or hypofractionated whole breast radiotherapy using one of three randomisation categories. Category A was a 4-arm randomisation of tumour bed boost versus no boost following conventional whole breast radiotherapy (50 Gy in 25 fractions over 5 weeks) versus hypofractionated whole breast radiotherapy (42·5 Gy in 16 fractions over 3·5 weeks). Category B was a 2-arm randomisation between tumour bed boost versus no boost following conventional whole breast radiotherapy, and category C was a 2-arm randomisation between tumour bed boost versus no boost following hypofractionated whole breast radiotherapy. Stratification factors were age at diagnosis, planned endocrine therapy, and treating centre. The primary endpoint, time to local recurrence, will be reported when participants have completed 5 years of follow-up. The HRQOL statistical analysis plan prespecified eight aspects of HRQOL, assessed by four questionnaires at baseline, end of treatment, and at 6, 12, and 24 months after radiotherapy: fatigue and physical functioning (EORTC QLQ-C30); cosmetic status, breast-specific symptoms, arm and shoulder functional status (Breast Cancer Treatment Outcome Scale); body image and sexuality (Body Image Scale); and perceived risk of invasive breast cancer (Cancer Worry Scale and a study-specific question). For each of these measures, tumour bed boost was compared with no boost, and conventional whole breast radiotherapy compared with hypofractionated whole breast radiotherapy, by use of generalised estimating equation models. Analyses were by intention to treat, with Hochberg adjustment for multiple testing. This trial is registered with ClinicalTrials.gov, NCT00470236.
Between June 1, 2007, and Aug 14, 2013, 1208 women were enrolled and randomly assigned to receive no tumour bed boost (n=605) or tumour bed boost (n=603). 396 of 1208 women were assigned to category A: conventional whole breast radiotherapy with tumour bed boost (n=100) or no boost (n=98), or to hypofractionated whole breast radiotherapy with tumour bed boost (n=98) or no boost (n=100). 447 were assigned to category B: conventional whole breast radiotherapy with tumour bed boost (n=223) or no boost (n=224). 365 were assigned to category C: hypofractionated whole breast radiotherapy with tumour bed boost (n=182) or no boost (n=183). All patients were followed up at 2 years for the HRQOL analysis. 1098 (91%) of 1208 patients received their allocated treatment, and most completed their scheduled HRQOL assessments (1147 95% of 1208 at baseline; 988 87% of 1141 at 2 years). Cosmetic status was worse with tumour bed boost than with no boost across all timepoints (difference 0·10 95% CI 0·05–0·15, global p=0·00014, Hochberg-adjusted p=0·0016); at the end of treatment, the estimated difference between tumour bed boost and no boost was 0·13 (95% CI 0·06–0·20; p=0·00021), persisting at 24 months (0·13 0·06–0·20; p=0·00021). Arm and shoulder function was also adversely affected by tumour bed boost across all timepoints (0·08 95% CI 0·03–0·13, global p=0·0033, Hochberg adjusted p=0·045); the difference between tumour bed boost and no boost at the end of treatment was 0·08 (0·01 to 0·15, p=0·021), and did not persist at 24 months (0·04 –0·03 to 0·11, p=0·29). None of the other six prespecified aspects of HRQOL differed significantly after adjustment for multiple testing. Conventional whole breast radiotherapy was associated with worse body image than hypofractionated whole breast radiotherapy at the end of treatment (difference –1·10 95% CI –1·79 to –0·42, p=0·0016). No significant differences were reported in the other PROs between conventional whole breast radiotherapy compared with hypofractionated whole breast radiotherapy.
Tumour bed boost was associated with persistent adverse effects on cosmetic status and arm and shoulder functional status, which might inform shared decision making while local recurrence analysis is pending.
National Health and Medical Research Council, Susan G Komen for the Cure, Breast Cancer Now, OncoSuisse, Dutch Cancer Society.
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