scholarly journals Distinct Chromosome Segregation Roles for Spindle Checkpoint Proteins

2002 ◽  
Vol 13 (9) ◽  
pp. 3029-3041 ◽  
Author(s):  
Cheryl D. Warren ◽  
D. Michelle Brady ◽  
Raymond C. Johnston ◽  
Joseph S. Hanna ◽  
Kevin G. Hardwick ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chromosome Segregation ◽  
Chromosome Instability ◽  
Spindle Checkpoint ◽  
Adjacent Segment ◽  
Effect Analysis ◽  
Checkpoint Proteins ◽  
Chromosome Transmission ◽  
Acid Fragment ◽  
Molecular Functions

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage.

scholarly journals Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2206
Author(s):  
Yuto Fujibayashi ◽  
Reiko Isa ◽  
Daichi Nishiyama ◽  
Natsumi Sakamoto-Inada ◽  
Norichika Kawasumi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Disease Progression ◽  
Chromosome Segregation ◽  
Early Stage ◽  
Chromosome Instability ◽  
Underlying Mechanism ◽  
Mitotic Checkpoint ◽  
Checkpoint Proteins ◽  
Segmental Aneuploidy ◽  
Chromosomal Changes

Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

scholarly journals Loading of the 3F3/2 Antigen onto Kinetochores Is Dependent on the Ordered Assembly of the Spindle Checkpoint Proteins

2006 ◽  
Vol 17 (10) ◽  
pp. 4390-4399 ◽  
Author(s):  
Oi Kwan Wong ◽  
Guowei Fang
Keyword(s):  
Chromosome Segregation ◽  
Spindle Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Assembly Pathway ◽  
Checkpoint Pathway ◽  
Unknown Protein ◽  
Ordered Assembly

Accurate chromosome segregation is controlled by the spindle checkpoint, which responds to the lack of microtubule–kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B, and Plk1/Plx1 kinases. The presence of the 3F3/2 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1 but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopusextracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, and BubR1 and Plx1 second, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid in the eventual identification of the 3F3/2 substrate.

scholarly journals Functional Redundancy in the Maize Meiotic Kinetochore

2000 ◽  
Vol 151 (1) ◽  
pp. 131-142 ◽  
Author(s):  
Hong-Guo Yu ◽  
R. Kelly Dawe
Keyword(s):  
Light Microscopy ◽  
Chromosome Segregation ◽  
Spindle Checkpoint ◽  
Functional Redundancy ◽  
Checkpoint Control ◽  
Checkpoint Proteins ◽  
Spindle Poles ◽  
Division 1 ◽  
Sister Kinetochore ◽  
Meiosis I

Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, ∼6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate.

scholarly journals CBIO-16. SUPPRESSION OF HISTONE H3.3 MITOTIC PHOSPHORYLATION DRIVES DEVELOPMENT OF H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii18-ii19
Author(s):  
Charles Day ◽  
Alyssa Langfald ◽  
Florina Grigore ◽  
Leslie Sepaniac ◽  
Jason Stumpff ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Treatment Options ◽  
Chromosome Instability ◽  
Pericentromeric Heterochromatin ◽  
Low Grade ◽  
High Grade ◽  
Chromosome Missegregation ◽  
Mitotic Phosphorylation ◽  
Chk1 Kinase

Abstract Pediatric midline gliomas – including DIPG – are lethal brain tumors in children, with poor prognosis and limited treatment options that provide only short-term benefits. The majority have a lysine-to-methionine substitution at residue 27 (H3K27M) in genes expressing histone H3 – predominantly in the H3.3 variant. This causes a global reduction in H3 Lys27 tri-methylation (H3K27Me3), comprehensive epigenetic reprogramming, and is a key driver in gliomagenesis. We show that the H3.3K27M mutation also induces chromosome segregation defects, which in high-grade tumors, results in extensive copy number alterations (CNAs). Ser31 is one of five amino acid substitutions differentiating H3.3 from canonical H3.1. Mitotic phosphorylation of H3.3 Ser31 by Chk1 kinase is restricted to pericentromeric heterochromatin, where it plays a role in chromosome segregation. We show that the K27M mutation affects neighboring Ser31 phosphorylation and pericentromeric heterochromatin organization. We demonstrate that (i) H3.3 K27M protein is defective for Ser31 phosphorylation by Chk1 kinase in vitro; (ii) DIPG cell lines have significantly decreased mitotic Ser31 phosphorylation, and are chromosomally unstable; and (iii) CRISPR-reversion of H3.3K27M to Lys27 restores phospho-Ser31 (and Lys27 tri-methylation) and significantly decreases chromosome instability. Expression of H3.3K27M or non-phosphorylatable H3.3S31A mutants in WT cells results in chromosome missegregation; this is suppressed by co-expression of phospho-mimetic H3.3K27M/S31E. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. Finally, in a novel mouse model of glioma, mean survival of mice with tumors induced with H3.3K27M and H3.3S31A was 81 and 68 days: 100% of H3.3S31A mice developed high-grade tumors. H3.3 WT controls developed only low-grade tumors and all survived 100 days. H3.3S31A is WT for Lys27 tri-methylation and thus, loss of Ser31 phosphorylation alone is oncogenic.

scholarly journals The wages of CIN

2008 ◽  
Vol 180 (4) ◽  
pp. 661-663 ◽  
Author(s):  
Karen W. Yuen ◽  
Arshad Desai
Keyword(s):  
Cancer Cells ◽  
Solid Tumors ◽  
Chromosomal Instability ◽  
Chromosome Instability ◽  
Spindle Checkpoint ◽  
Human Cells ◽  
Normal Cells ◽  
Chromosome Missegregation ◽  
Cell Biol ◽  
The Relationship

Aneuploidy and chromosome instability (CIN) are hallmarks of the majority of solid tumors, but the relationship between them is not well understood. In this issue, Thompson and Compton (Thompson, S.L., and D.A. Compton. 2008. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell. Biol. 180:665–672) investigate the mechanism of CIN in cancer cells and find that CIN arises primarily from defective kinetochore–spindle attachments that evade detection by the spindle checkpoint and persist into anaphase. They also explore the consequences of artificially elevating chromosome missegregation in otherwise karyotypically normal cells. Their finding that induced aneuploidy is rapidly selected against suggests that the persistence of aneuploid cells in tumors requires not only chromosome missegregation but also additional, as yet poorly defined events.

scholarly journals Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways

2010 ◽  
Vol 84 (19) ◽  
pp. 9897-9906 ◽  
Author(s):  
Florence Larrous ◽  
Alireza Gholami ◽  
Shahul Mouhamad ◽  
Jérôme Estaquier ◽  
Hervé Bourhy
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Matrix Protein ◽  
Full Length ◽  
M Protein ◽  
Genotype 3 ◽  
Cell Death Pathways ◽  
Acid Fragment ◽  
M Proteins ◽  
Cellular Apoptosis

ABSTRACT The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

scholarly journals Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 685-695 ◽  
Author(s):  
Dong Zhang ◽  
Shen Yin ◽  
Man-Xi Jiang ◽  
Wei Ma ◽  
Yi Hou ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Mitotic Arrest ◽  
Checkpoint Proteins ◽  
Meiotic Progression ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Oocyte Meiosis ◽  
And Function

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

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