Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse

SUMMARYChromosome segregation errors that cause oocyte aneuploidy increase in frequency with maternal age and are considered a major contributing factor of age-related fertility decline in females. A common age-associated chromosome segregation phenomenon in oocytes is the lagging anaphase chromosome, but whether anaphase laggards actually missegregate and cause aneuploidy is unclear. Here we show unexpectedly that lagging chromosomes in mouse oocytes comprise two mechanistically distinct classes of motion that we refer to as ‘Class-I’ and ‘Class-II’. We use imaging approaches and mechanistic interventions to dissociate the two classes, and find that whereas Class-II laggards are benign, Class-I laggards can directly cause aneuploidy. Most notably, a controlled prolongation of meiosis-I specifically lessens Class-I lagging to prevent aneuploidy. Our data thus reveal lagging chromosomes to be a cause of age-related aneuploidy in mouse oocytes and suggest that manipulating the cell cycle could increase the yield of useful oocytes in some contexts.

An unbiased, quantitative and versatile method for determining misaligned and lagging chromosome during mitosis

ABSTRACTAccurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Therefore, tools to sensitively and quantitatively measure chromosome alignment at metaphase will facilitate understanding of how changes in the composition and regulation of the microtubule attachment machinery impinge on this process. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semi-automated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive, unbiased, and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.

Kinetochore protein depletion underlies cytokinesis failure and somatic polyploidization in the moss Physcomitrella patens

Lagging chromosome is a hallmark of aneuploidy arising from errors in the kinetochore–spindle attachment in animal cells. However, kinetochore components and cellular phenotypes associated with kinetochore dysfunction are much less explored in plants. Here, we carried out a comprehensive characterization of conserved kinetochore components in the moss Physcomitrella patens and uncovered a distinct scenario in plant cells regarding both the localization and cellular impact of the kinetochore proteins. Most surprisingly, knock-down of several kinetochore proteins led to polyploidy, not aneuploidy, through cytokinesis failure in >90% of the cells that exhibited lagging chromosomes for several minutes or longer. The resultant cells, containing two or more nuclei, proceeded to the next cell cycle and eventually developed into polyploid plants. As lagging chromosomes have been observed in various plant species in the wild, our observation raised a possibility that they could be one of the natural pathways to polyploidy in plants.

Kinetochore protein depletion underlies cytokinesis failure and somatic polyploidization in the moss Physcomitrella patens

Lagging chromosome is a hallmark of aneuploidy arising from errors in the kinetochore–spindle attachment in animal cells. However, kinetochore components and cellular phenotypes associated with kinetochore dysfunction are much less explored in plants. Here, we carried out a comprehensive characterization of conserved kinetochore components in the moss Physcomitrella patens and uncovered a distinct scenario in plant cells regarding both the localization and cellular impact of the kinetochore proteins. Most surprisingly, knock-down of several kinetochore proteins led to polyploidy, not aneuploidy, through cytokinesis failure in >90% of the cells that exhibited lagging chromosomes for several minutes or longer. The resultant cells, containing two or more nuclei, proceeded to the next cell cycle and eventually developed into polyploid plants. As lagging chromosomes have been observed in various plant species in the wild, our observation raised a possibility that they could be one of the natural pathways to polyploidy in plants.

Molecular and cytogenetic assessment of Dipterygium glaucum genotoxicity

ABSTRACT The aim of the present study is to assess the genotoxicity of Dipterygium glaucum grows widely in Saudi Arabia desert to produce safety herbal products. This work is considered the first and pioneer report so far due to the lack and poor evaluated reports of the plant species for their mutagensity, genotoxicity and cytogenetics effects. Cytogenetic effects of D. glaucum on mitotic in roots of Vicia faba showed reduction in mitotic activity using three extracts; water, ethanol and ethyl acetate. Chromosomal abnormalities were recorded that included stickiness of chromosomes, chromatin bridge, fragments, lagging chromosome and micronuclei. Protein bands and RAPD analyses of V. faba treated with three D. glaucum extracts revealed some newly induced proteins and DNA fragments and other disappeared. Chemical constitution of the plant species should be identified with their biological activities against human and animal cells like HeLa cancer cell line. We are recommending using additional genotoxicity tests and other toxicity tests on animal culture with different concentrations and also utilizing several drought and heat tolerant genes of the plant species in gene cloning to develop and improve other economical crop plants instead of using the species as oral herbal remedy

Chromosomal Instability Induced by Overexpression of Telomere Repeats Binding Factor 1.

Telomere dysfunction has been associated with chromosomal stability in process of oncogenesis. Telomere, a special nucleoprotein complex at the termini of linear eukaryotic chromosomes, functions to prevent chromosomes from degradation by endogenous nucleases, fusion and recombination of chromosome ends, triggering DNA damage response and checkpoint-induced cell cycle arrest or apoptosis. Telomere homoeostasis and length are maintained by a set of telomere binding proteins. Among these, telomere repeats binding factor 1(TRF1) serves as a negative regulator of telomere length since TRF1 overexpression would elicit the shortening of telomere length in telomerase-positive cells. To date, the expression level of TRF1 in human cancers remains controversial and its role and mechanism in tumorigenesis are poorly understood. To answer these questions, we introduced EGFP-tagged TRF1 plasmid into HeLa cells to establish a stable cell line with TRF1 overexpression. Positive clones were selected by G418(1mg/ml) and GFP fusion protein was confirmed by immunoblotting analysis with anti-GFP antibodies. Interestingly, these cells demonstrated a higher percentage of mitotic abnormality including chromosome misalignment, anaphase bridging, lagging chromosome and multipolar spindles than controls after over 10 passages(20.0% vs 1.5%, p<0.01). To further clarify if this phenomenon resulted from chromosomal fusion, metaphase chromosome spreads were prepared. As expected, the TRF1 stable cells showed an elevated frequency of dicentric chromosomes compared with untransfected cells, which implicated chromosomal end-to-end fusion in these cells. Our results suggested high expression of TRF1 would induce chromosome missegregation at mitosis, a feature of chromosomal instability which is proposed to be a critical step in carcinogenesis. These findings bring fourth a new hint for our further understanding of carcinogenesis led by telomere dysfunction.

Merotelic Kinetochore Orientation Is a Major Mechanism of Aneuploidy in Mitotic Mammalian Tissue Cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 μM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2–5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.

Live analysis of lagging chromosomes during anaphase and their effect on spindle elongation rate in fission yeast

The fission yeast Schizosaccharomyces pombe is widely used as a model system for studies of the cell cycle and chromosome biology. To enhance these studies we have fused GFP to the chromodomain protein Swi6p, thus allowing nuclear and chromosome behaviour to be followed in living cells using time-lapse fluorescence microscopy. Like endogenous Swi6p, GFP-Swi6p localises to the nucleus and is concentrated at the heterochromatic centromeres and telomeres. The nucleus is highly dynamic during interphase: the clustered centromeres, in particular, are highly mobile. By expressing GFP-(α)2-tubulin and GFP-Swi6p in the same cells we observe that the clustered centromeres move in concert with the cytoplasmic microtubules, which is likely to reflect their association with the spindle pole body. Drug treatment indicates that this movement is dependent on intact cytoplasmic microtubules. We have also used GFP-Swi6p to investigate the properties of lagging chromosomes observed in mutants with defects in chromosome segregation. Lagging chromosomes display a variety of behaviours on anaphase spindles, most surprisingly, chromosomes appear to initiate microtubule interactions and move to the poles late in anaphase B. Interestingly, in cells displaying lagging chromosomes, the rate of spindle elongation is slowed by a factor of two. This suggests that cells are able to sense the presence of a lagging chromosome and slow anaphase B in order to allow it extra time to reach the pole. However, this mechanism is not dependent on the spindle checkpoint proteins Bub1p or Dma1p, raising the possibility that a novel checkpoint mechanism operates to retard spindle elongation if lagging chromosomes are detected. An alternative model is also discussed in which single defective kinetochores on lagging chromatids are able to interact simultaneously with microtubules emanating from both poles and affect spindle dynamics by counteracting the spindle elongation force.