scholarly journals Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

2011 ◽  
Vol 22 (19) ◽  
pp. 3584-3594 ◽  
Author(s):  
Randy Wei ◽  
Bryan Ngo ◽  
Guikai Wu ◽  
Wen-Hwa Lee
Keyword(s):  
Cell Death ◽  
Rna Interference ◽  
Molecular Mechanism ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Distribution Characteristic ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Ndc80 Complex ◽  
Complex Protein

The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.

scholarly journals Molecular mechanism underlying the non-essentiality of Bub1 for the fidelity of chromosome segregation in human cells

2021 ◽  
Author(s):  
Qinfu Chen ◽  
Miao Zhang ◽  
Xuan Pan ◽  
Linli Zhou ◽  
Haiyan Yan ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Chromosome ◽  
Threshold Level ◽  
Underlying Mechanism ◽  
Human Cells ◽  
Strong Reduction ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Redundant Role

SUMMARYThe multi-task protein kinase Bub1 has long been considered important for chromosome alignment and spindle assembly checkpoint signaling during mitosis. However, recent studies provide surprising evidence that Bub1 may not be essential in human cells, with the underlying mechanism unknown. Here we show that Bub1 plays a redundant role with the non-essential CENP-U complex in recruiting Polo-like kinase 1 (Plk1) to the kinetochore. While disrupting either pathway of Plk1 recruitment does not affect the accuracy of whole chromosome segregation, loss of both pathways leads to a strong reduction in the kinetochore accumulation of Plk1 under a threshold level required for proper chromosome alignment and segregation. Thus, parallel recruitment of Plk1 to kinetochores by Bub1 and the CENP-U complex ensures high fidelity of mitotic chromosome segregation. This study may have implications for targeted treatment of cancer cells harboring mutations in either Bub1 or the CENP-U complex.

scholarly journals Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

2011 ◽  
Vol 22 (9) ◽  
pp. 1473-1485 ◽  
Author(s):  
Zuzana Storchová ◽  
Justin S. Becker ◽  
Nicolas Talarek ◽  
Sandra Kögelsberger ◽  
David Pellman
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Budding Yeast ◽  
Spindle Pole ◽  
Growth Defect ◽  
Aurora B ◽  
Mitotic Kinase ◽  
Sister Chromatids ◽  
Chromosome Alignment ◽  
Chromosomal Passenger

The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B–independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B–mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.

scholarly journals Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore–microtubule attachments

2012 ◽  
Vol 199 (2) ◽  
pp. 285-301 ◽  
Author(s):  
Ana R.R. Maia ◽  
Zaira Garcia ◽  
Lilian Kabeche ◽  
Marin Barisic ◽  
Stefano Maffini ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Regulatory Mechanism ◽  
Stable Condition ◽  
Spindle Assembly ◽  
Chromosome Alignment ◽  
Chromosome Movements

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)–microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT–MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT–MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 “phospho-switch” that temporally regulates KT–MT attachment stability.

scholarly journals Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore

2015 ◽  
Vol 211 (6) ◽  
pp. 1141-1156 ◽  
Author(s):  
Itaru Samejima ◽  
Christos Spanos ◽  
Flavia de Lima Alves ◽  
Tetsuya Hori ◽  
Marinela Perpelescu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Protein Complexes ◽  
Interacting Proteins ◽  
Quantitative Mass Spectrometry ◽  
Mitotic Chromosomes ◽  
Microtubule Binding ◽  
Checkpoint Signaling ◽  
Ndc80 Complex ◽  
Stable Association

Kinetochores orchestrate mitotic chromosome segregation. Here, we use quantitative mass spectrometry of mitotic chromosomes isolated from a comprehensive set of chicken DT40 mutants to examine the dependencies of 93 confirmed and putative kinetochore proteins for stable association with chromosomes. Clustering and network analysis reveal both known and unexpected aspects of coordinated behavior for members of kinetochore protein complexes. Surprisingly, CENP-T depends on CENP-N for chromosome localization. The Ndc80 complex exhibits robust correlations with all other complexes in a “core” kinetochore network. Ndc80 associated with CENP-T interacts with a cohort of Rod, zw10, and zwilch (RZZ)–interacting proteins that includes Spindly, Mad1, and CENP-E. This complex may coordinate microtubule binding with checkpoint signaling. Ndc80 associated with CENP-C forms the KMN (Knl1, Mis12, Ndc80) network and may be the microtubule-binding “workhorse” of the kinetochore. Our data also suggest that CENP-O and CENP-R may regulate the size of the inner kinetochore without influencing the assembly of the outer kinetochore.

scholarly journals Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ivana Primorac ◽  
John R Weir ◽  
Elena Chiroli ◽  
Fridolin Gross ◽  
Ingrid Hoffmann ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Binding Mode ◽  
Signaling Networks ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Downstream Signaling ◽  
Signaling Components ◽  
Insight Into

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.

scholarly journals SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans

2008 ◽  
Vol 183 (2) ◽  
pp. 187-194 ◽  
Author(s):  
Takaharu G. Yamamoto ◽  
Sonoko Watanabe ◽  
Anthony Essex ◽  
Risa Kitagawa
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Pole ◽  
Cell Stage ◽  
Anaphase Onset ◽  
Mitotic Delay ◽  
Bipolar Spindles

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD–ZW10–ZWILCH complex. In two-cell–stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.

scholarly journals MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 406
Author(s):  
María Arroyo ◽  
Antonio Sánchez ◽  
Ana Cañuelo ◽  
Rosalía F. Heredia-Molina ◽  
Eduardo Martínez-Molina ◽  
...  
Keyword(s):  
Cell Death ◽  
Chromosome Segregation ◽  
Topoisomerase Ii ◽  
Mitotic Cell ◽  
Primary Microcephaly ◽  
Checkpoint Response ◽  
Checkpoint Pathway ◽  
Chromosome Alignment ◽  
Topo Ii ◽  
A Minor

The capacity of Topoisomerase II (Topo II) to remove DNA catenations that arise after replication is essential to ensure faithful chromosome segregation. Topo II activity is monitored during G2 by a specific checkpoint pathway that delays entry into mitosis until the chromosomes are properly decatenated. Recently, we demonstrated that the mitotic defects that are characteristic of cells depleted of MCPH1 function, a protein mutated in primary microcephaly, are not a consequence of a weakened G2 decatenation checkpoint response. However, the mitotic defects could be accounted for by a minor defect in the activity of Topo II during G2/M. To test this hypothesis, we have tracked at live single cell resolution the dynamics of mitosis in MCPH1 depleted HeLa cells upon catalytic inhibition of Topo II. Our analyses demonstrate that neither chromosome alignment nor segregation are more susceptible to minor perturbation in decatenation in MCPH1 deficient cells, as compared with control cells. Interestingly, MCPH1 depleted cells were more prone to mitotic cell death when decatenation was perturbed. Furthermore, when the G2 arrest that was induced by catalytic inhibition of Topo II was abrogated by Chk1 inhibition, the incidence of mitotic cell death was also increased. Taken together, our data suggest that the MCPH1 lack of function increases mitotic cell hypersensitivity to the catalytic inhibition of Topo II.

scholarly journals Mps1 dimerization and multisite interactions with Ndc80 complex enable responsive spindle assembly checkpoint signaling

2020 ◽  
Vol 12 (7) ◽  
pp. 486-498 ◽  
Author(s):  
Ping Gui ◽  
Divine M Sedzro ◽  
Xiao Yuan ◽  
Sikai Liu ◽  
Mohan Hei ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Direct Interaction ◽  
Spindle Assembly ◽  
Kinase Domain ◽  
Checkpoint Signaling ◽  
Terminal Fragment ◽  
Repeat Domain ◽  
Ndc80 Complex ◽  
Chromosome Attachment ◽  
Tetratricopeptide Repeat Domain

Abstract Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, which is monitored by the spindle assembly checkpoint (SAC) signaling. As an upstream factor of SAC, the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling. However, the underlying molecular mechanism remains elusive. Here, we demonstrated that the multisite interactions between Mps1 and Ndc80 complex (Ndc80C) govern Mps1 kinetochore targeting. Importantly, we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C. We further identified that Mps1 C-terminal fragment, which contains the protein kinase domain and C-tail, enhances Mps1 kinetochore localization. Mechanistically, Mps1 C-terminal fragment mediates its dimerization. Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1, leading to aberrant mitosis due to compromised SAC function. Taken together, our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling.

scholarly journals Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

2010 ◽  
Vol 190 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Laura Hewitt ◽  
Anthony Tighe ◽  
Stefano Santaguida ◽  
Anne M. White ◽  
Clifford D. Jones ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Aurora B ◽  
Core Complex ◽  
Obvious Effect ◽  
Checkpoint Signaling ◽  
Mitotic Entry ◽  
The Core ◽  
Chromosome Congression ◽  
Chromosome Alignment

Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.

scholarly journals Structure of the RZZ complex and molecular basis of its interaction with Spindly

2017 ◽  
Vol 216 (4) ◽  
pp. 961-981 ◽  
Author(s):  
Shyamal Mosalaganti ◽  
Jenny Keller ◽  
Anika Altenfeld ◽  
Michael Winzker ◽  
Pascaline Rombaut ◽  
...  
Keyword(s):  
Chemical Biology ◽  
Spindle Assembly Checkpoint ◽  
Complete Model ◽  
Electron Cryomicroscopy ◽  
Independent Manner ◽  
Checkpoint Signaling ◽  
Macromolecular Assemblies ◽  
Self Assembling ◽  
Chromosome Alignment ◽  
Spindle Microtubules

Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD–Zwilch–ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13–Sec31, and αβ’ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein’s cargo at human kinetochores.

Sign in / Sign up

Export Citation Format

Share Document