Proteins of the inner and outer centromere of mitotic chromosomes

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 541-552 ◽  
Author(s):  
William C. Earnshaw ◽  
Carol A. Cooke
Keyword(s):  
Negative Charge ◽  
Dna Binding Protein ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Region I ◽  
Spindle Microtubules ◽  
Region Ii ◽  
Chromosome Scaffold

We have used immunocytochemistry and molecular cloning methods to identify and characterize structural polypeptides of the centromere. These studies permit us to resolve two distinct regions: the inner and outer centromere, (i) Components of the outer centromere: autoantibodies from certain patients with rheumatic disease identify a family of three immunologically related polypeptides that we have designated CENP-A (17 kDa), CENP-B (80 kDa), and CENP-C (140 kDa). CENP-B has been cloned and sequenced. DNA sequence analysis indicates that this polypeptide possesses two large regions with extraordinary concentrations of acidic residues (region I: 61 residues with 79% glu + asp; region II: 31 residues with 87% glu + asp). Despite this concentration of negative charge, immunocytochemical experiments suggest that CENP-B may be a DNA binding protein. In these experiments, the levels of CENP-B are seen to vary reproducibly from chromosome to chromosome. The role of CENP-B in vivo is unknown. However, it is unlikely to bind directly to the spindle microtubules since it is found at an inactive centromere that apparently does not attach to the spindle. (ii) Components of the inner centromere: we have injected mice with the whole chromosome scaffold fraction to elicit production of monoclonal antibodies. One such antibody identifies two structurally related polypeptides (the INCENP antigens, 135 and 155 kDa) that are preferentially located between the sister chromatids at the centromere. The INCENP antigens undergo dramatic movements from the chromosomes to the central spindle during mitosis. They are ultimately sequestered in the midbody and discarded. Several lines of evidence suggest that the INCENP polypeptides may be involved in the regulation of sister chromatid separation at the metaphase–anaphase transition.Key words: mitosis, centromere, CENP antigens, INCENP antigens, kinetochore, disjunction.

Katanin, the microtubule-severing ATPase, is concentrated at centrosomes

1996 ◽  
Vol 109 (3) ◽  
pp. 561-567 ◽  
Author(s):  
F.J. McNally ◽  
K. Okawa ◽  
A. Iwamatsu ◽  
R.D. Vale
Keyword(s):  
Mitotic Spindle ◽  
Dynamic Properties ◽  
Mitotic Spindles ◽  
Microtubule Severing ◽  
Spindle Microtubules ◽  
Gamma Tubulin ◽  
And Function ◽  
Poleward Flux

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.

scholarly journals Dynamic bonds and polar ejection force distribution explain kinetochore oscillations in PtK1 cells

2013 ◽  
Vol 201 (4) ◽  
pp. 577-593 ◽  
Author(s):  
Gul Civelekoglu-Scholey ◽  
Bin He ◽  
Muyao Shen ◽  
Xiaohu Wan ◽  
Emanuele Roscioli ◽  
...  
Keyword(s):  
Metaphase Plate ◽  
Quantitative Model ◽  
Force Distribution ◽  
Mitotic Chromosomes ◽  
Chromosome Behavior ◽  
Sister Chromatids ◽  
Ejection Force ◽  
Sister Kinetochore ◽  
Spindle Microtubules ◽  
Key Features

Duplicated mitotic chromosomes aligned at the metaphase plate maintain dynamic attachments to spindle microtubules via their kinetochores, and multiple motor and nonmotor proteins cooperate to regulate their behavior. Depending on the system, sister chromatids may display either of two distinct behaviors, namely (1) the presence or (2) the absence of oscillations about the metaphase plate. Significantly, in PtK1 cells, in which chromosome behavior appears to be dependent on the position along the metaphase plate, both types of behavior are observed within the same spindle, but how and why these distinct behaviors are manifested is unclear. Here, we developed a new quantitative model to describe metaphase chromosome dynamics via kinetochore–microtubule interactions mediated by nonmotor viscoelastic linkages. Our model reproduces all the key features of metaphase sister kinetochore dynamics in PtK1 cells and suggests that differences in the distribution of polar ejection forces at the periphery and in the middle of PtK1 cell spindles underlie the observed dichotomy of chromosome behavior.

scholarly journals Polyploids require Bik1 for kinetochore–microtubule attachment

2001 ◽  
Vol 155 (7) ◽  
pp. 1173-1184 ◽  
Author(s):  
Haijiang Lin ◽  
Pedro de Carvalho ◽  
David Kho ◽  
Chin-Yin Tai ◽  
Philippe Pierre ◽  
...  
Keyword(s):  
Cancer Therapeutics ◽  
Imaging Data ◽  
Elastic Recoil ◽  
New Approach ◽  
Binding Interface ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
Polyploid Cells ◽  
Spindle Microtubules

The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore–MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

O-171 Altered meiotic spindle morphology and composition in in vitro matured oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Karamtzioti ◽  
G Tiscornia ◽  
D Garcia ◽  
A Rodriguez ◽  
I Vernos ◽  
...  
Keyword(s):  
Metaphase Plate ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Developmental Potential ◽  
Software Analysis ◽  
Spindle Microtubules ◽  
Non Parametric

Abstract Study question How does the meiotic spindle tubulin PTMs of MII oocytes matured in vitro compare to that of MII oocytes matured in vivo? Summary answer MII cultured in vitro present detyrosinated tubulin in the spindle microtubules, while MII oocytes matured in vivo do not. What is known already A functional spindle is required for chromosomal segregation during meiosis, but the role of tubulin post-translational modifications (PTMs) in spindle meiotic dynamics remains poorly characterized. In contrast with GVs matured in vitro within the cumulus oophorous, in vitro maturation of denuded GVs to the MII stage (GV-MII) is associated with spindle abnormalities, chromosome misalignment and compromised developmental potential. Although aneuploidy rates in GV-MII are not higher than in vivo matured MII, disorganized chromosomes may contribute to compromised developmental potential. However, to date, spindle PTMs morphology of GV-MII has not been compared to that of in vivo cultured MII oocytes. Study design, size, duration GV (n = 125), and MII oocytes (n = 24) were retrieved from hormonally stimulated women, aged 20 to 35 years old. GVs were matured to the MII stage in vitro in G-2 PLUS medium for 30h; the maturation rate was 68,2%; the 46 GV-MII oocytes obtained were vitrified, stored, and warmed before fixing and subjecting to immunofluorescent analysis. In vivo matured MII oocytes donated to research were used as controls. Participants/materials, setting, methods Women were stimulated using a GnRH antagonist protocol, with GnRH agonist trigger. Trigger criterion was ≥2 follicles ≥18mm; oocytes were harvested 36h later. Spindle microtubules were incubated with antibodies against alpha tubulin and tubulin PTMs (acetylation, tyrosination, polyglutamylation, Δ2-tubulin, and detyrosination); chromosomes were stained with Hoechst 33342 and samples subjected to confocal immunofluorescence microscopy (ZEISS LSM780), with ImageJ software analysis. Differences in spindle morphometric parameters were assessed by non-parametric Kruskal–Wallis and Fisher’s exact tests. Main results and the role of chance Qualitatively, Δ2-tubulin, tyrosination and polyglutamylation were similar for both groups. Acetylation was also present in both groups, albeit in different patterns: while in vivo matured MII oocytes showed acetylation at the poles, GV-MII showed a symmetrical distribution of signal intensity, but discontinuous signal on individual microtubule tracts, suggesting apparent islands of acetylation. In contrast, detyrosination was detected in in vivo matured MII oocytes but was absent from GV-MII. Regarding spindle pole morphology, of the four possible phenotypes described in the literature (double flattened and double focused; flattened-focused, focused-flattened, with the first word characterizing the cortex side of the spindle), we observed double flat shaped spindle poles in 86% of GV-MII oocytes (25/29) as opposed to 40.5% (15/37) for the in vivo matured MII oocytes (p = 0.0004, Fisher’s exact test). Further morphometric analysis of the spindle size (maximum projection, major and minor axis length) and the metaphase plate position (proximal to distal ratio, angle) revealed decreased spindle size in GV-MII oocytes (p = 0.019, non parametric Kruskal- Wallis test). Limitations, reasons for caution Oocytes retrieved from hyperstimulation cycles could be intrinsically impaired since they failed to mature in vivo. Our conclusions should not be extrapolated to IVM in non-stimulated cycles, as in this model, the cumulus oophorus is a major factor in oocyte maturation and correlation with spindle dynamics has been inferred. Wider implications of the findings The metaphase II spindle stability compared to the mitotic or metaphase I meiotic one justifies the presence of PTMs such as acetylation and glutamylation, which are found in stable, long-lived microtubules. The significance of the absence of detyrosinated microtubules in the MII-GV group remains to be determined Trial registration number not applicable

scholarly journals The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis.

1987 ◽  
Vol 105 (5) ◽  
pp. 2053-2067 ◽  
Author(s):  
C A Cooke ◽  
M M Heck ◽  
W C Earnshaw
Keyword(s):  
Metaphase Plate ◽  
Pericentromeric Region ◽  
Contractile Ring ◽  
Structural Localization ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Centromere Proteins ◽  
Coarse Fibers ◽  
Chromosome Scaffold

We describe a novel set of polypeptide antigens that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, we have provisionally termed them the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100% of the detectable INCENPs remain scaffold associated. We were therefore unprepared for the fate of the INCENPs at anaphase. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase.

Role of Cohesin-Resolving Protease, Separase, In Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1593-1593
Author(s):  
Lanelle V. Nakamura ◽  
Malini Mukherjee ◽  
Margaret A. Goodell ◽  
Debananda Pati
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Protein Complex ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Stem ◽  
Sister Chromatids

Abstract Abstract 1593 Introduction: Cohesin is an evolutionarily conserved protein complex that forms during the replication of sister chromatids. It is a multi-protein complex that consists of four proteins, Smc1, Smc3, Rad21, and Scc3. Resolution of sister chromatid cohesion at the onset of anaphase depends on Separase, an endopeptidase that separates sister chromatids by cleaving cohesion Rad21. A recent study suggests a new role of Cohesin proteins in gene expression and development with implications in hematopoiesis. Our data indicates that cohesin-resolving protease Separase may play a critical role in hematopoiesis. HYPOTHESIS: We hypothesize that Separase plays a role in hematopoiesis by increasing the quantity of hematopoietic stem cells (HSC). METHODS: Our experimental approach was to isolate murine long-term HSC from WT mice and mice with one mutated copy of Separase (i.e. Separase heterozygotes). In addition, in vivo competitive long term repopulation assays were used assess the function of HSC in Separase heterozyotes. RESULTS: Separase heterozygote have increased HSC numbers (p<0.05) as compared to WT mice. In addition, an improved engraftment in a competitive repopulation assay (p < 0.001) was seen in the Separase heterozyotes. Analysis of the engrafted cells demonstrated no difference between the wild type and Separase heterozygote animals, indicating the increased engraftment may be due to unique features in the primitive hematopoietic stem cells. CONCLUSION: Investigation of the mechanism for improved HSC engraftment in Separase heterozygote mice will significantly contribute to our understanding of marrow engraftment and function. Elucidating the mechanisms of hematopoietic dysregulation will provide insights into the development of life-threatening disorders such as leukemia and, in the setting of bone marrow transplant, engraftment failure. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics

2019 ◽  
Vol 12 (6) ◽  
pp. 462-476 ◽  
Author(s):  
Ruoying Yu ◽  
Huihui Wu ◽  
Hazrat Ismail ◽  
Shihao Du ◽  
Jun Cao ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Kinase Activity ◽  
Genomic Stability ◽  
Lysine Methylation ◽  
Mitotic Chromosomes ◽  
Methyltransferase Activity ◽  
Sister Chromatids ◽  
Spindle Microtubules ◽  
Chemical Inhibition ◽  
Early Mitosis

Abstract Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.

scholarly journals The mammalian kinetochore independently regulates its passive and active force-generating interfaces with microtubules

2017 ◽  
Author(s):  
Alexandra F. Long ◽  
Dylan B. Udy ◽  
Sophie Dumont
Keyword(s):  
Mammalian Cells ◽  
Active Force ◽  
Microtubule Depolymerization ◽  
Chromosome Congression ◽  
Sister Kinetochore ◽  
Spindle Microtubules ◽  
Active Force Generation ◽  
Molecular Nature

SummaryThe kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1,2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore slides along microtubules [3,4]. We do not know the physical and molecular nature [5–7] of these interfaces, or how they are regulated to support diverse mitotic functions in mammalian cells. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8–11]. Hec1’s affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12–15], but its role at the passive and active interfaces remains unclear. Here, we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1’s role as it moves on polymerizing versus depolymerizing microtubules. We show that Hec1 phosphorylation tunes passive friction along polymerizing microtubules, modulating both the magnitude and timescale of responses to force. In contrast, we find that Hec1 phosphorylation does not affect the kinetochore’s ability to grip depolymerizing microtubules, or switch to this active force-generating state. Together, the data suggest that different kinetochore interfaces engage with growing and shrinking microtubules, and that passive friction can be regulated without disrupting active force generation. Through this mechanism, the kinetochore can modulate its grip on microtubules as its functional needs change during mitosis, and yet retain its ability to couple to microtubules powering chromosome movement.

Distribution and function of non-histone proteins in human chromosomes

1992 ◽  
Vol 50 (1) ◽  
pp. 556-557
Author(s):  
W.C. Earnshaw ◽  
C.A. Cooke
Keyword(s):  
Mitotic Spindle ◽  
Centromeric Heterochromatin ◽  
Mitotic Chromosomes ◽  
Histone Proteins ◽  
Sister Chromatids ◽  
Structural Specialization ◽  
And Function ◽  
Passenger Proteins

The role of non-histone proteins in the structure and movements of mitotic chromosomes remains poorly understood. We describe here experiments aimed at characterization of the distribution of two very different classes of these proteins. The first is composed of integral components of the centromere (or primary constriction). The second class consists of proteins that we have termed “chromosome passenger proteins”. These proteins are chromosomal during most of the cell cycle, but appear to be associated with the cytoskeleton during anaphase and telophase.The centromere regions of chromosomes perform three essential functions in mitosis. (1) They form the site of attachment of the chromosomes to the mitotic spindle. (2) They contain the mechanochemical motor molecules that are responsible for the movements of the chromosomes along microtubules. (3) They regulate the pairing of sister chromatids during mitosis. The first two of these mitotic functions are properties of a disk-shaped structural specialization, the kinetochore, which is located at the surface of the centromeric heterochromatin.

Cohesin dependent compaction of mitotic chromosomes

2016 ◽  
Author(s):  
Stephanie A Schalbetter ◽  
Anton Goloborodko ◽  
Geoffrey Fudenberg ◽  
Jon M Belton ◽  
Catrina Miles ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Mitotic Chromosome ◽  
Protein Complexes ◽  
Budding Yeast ◽  
Mitotic Chromosomes ◽  
Chromosome Conformation ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Structural Maintenance Of Chromosomes

Structural Maintenance of Chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply-conserved SMC complexes, organize chromosomes in budding yeast. The canonical role of cohesins is to co-align sister chromatids whilst condensins generally compact mitotic chromosomes. We find strikingly different roles in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosomes arms, independent of and in addition to its role in sister-chromatid cohesion. Cohesin dependent mitotic chromosome compaction can be fully accounted for through cis-looping of chromatin by loop extrusion. Second, condensin is dispensable for compaction along chromosomal arms and instead plays a specialized role, structuring rDNA and peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that SMC-dependent looping is readily deployed in a range of contexts to functionally organize chromosomes.

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