Correspondence to: E.D. Salmon, Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280. Tel:(919) 962-2265 Fax:(919) 962-1625 E-mail:tsalmon{at}email.unc.edu.
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Abstract |
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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 (1.25.6-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.
Key Words: aneuploidy, kinetochores, mitosis, microtubules, mitotic spindle checkpoint
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Introduction |
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The unequal distribution of sister chromatids during mitosis or meiosis and cell cytokinesis produces aneuploid daughter cells. The role of aneuploidy in human meiotic cells is well known for generating severe congenital syndromes (for review see
A potential source of aneuploidy in mitotic mammalian tissue cells are chromosomes which are left behind at the spindle equator during anaphase as the great majority of sister chromatids move to their spindle poles (
These lagging chromosomes raise important questions about errors in the mechanisms of chromosome segregation and the mitotic spindle checkpoint. The first major issue is how these lagging chromosomes primarily remain near the spindle equator in anaphase if they are able by anaphase onset to congress to near the spindle equator (Cimini, D., and F. Degrassi, unpublished observations). Chromosome congression to the metaphase plate usually means that one sister kinetochore is attached to and pulled toward one pole by formation of kinetochore microtubules to that pole, whereas the other sister is attached to and pulled toward the opposite pole by formation of kinetochore microtubules to that pole (
Possible hypotheses to explain these lagging chromosomes in anaphase include sister chromatid nondisjunction, detachment of kinetochore microtubules at anaphase, inactivation of pulling forces at kinetochores in anaphase, and merotelic kinetochore orientation. The first hypothesis predicts that lagging chromosomes are paired chromatids with properly oriented sister kinetochores, with one sister kinetochore fiber to one pole and the other sister with its kinetochore fiber to the opposite pole. This has been shown for the inhibition of sister chromatid decatenation by topoisomerase II inhibitors (
We have tested the above hypotheses for the origin of lagging chromosomes during anaphase in mammalian tissue cells by analyzing PtK1 cells in culture. Our data are consistent with the merotelic orientation hypothesis and show that merotelic kinetochore orientation is a major mechanism producing chromosome loss during mitosis. A lagging chromosome was found to almost always be a single chromatid with its kinetochore attached to and stretched between bundles of microtubules oriented toward opposite poles. Apparently, antagonistic pulling forces toward opposite poles prevent normal anaphase segregation of the lagging chromosome. Merotelically oriented kinetochores are not detected by the mitotic spindle checkpoint since they exhibit no staining in anaphase for the mitotic checkpoint protein Mad2 or the 3F3/2 phosphoepitope, markers for checkpoint activation at kinetochores. The expansion of kinetochores into "crescents" around centromeres during a nocodazole mitotic block suggests that kinetochore defects in the "search and capture" mechanism of kinetochore microtubule formation contribute to merotelic kinetochore orientation.
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Materials and Methods |
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Cell Culture and Treatment
PtK1 cells (American Type Culture Collection) were maintained in HAM's F-12 medium (Sigma-Aldrich) complemented with 10% fetal bovine serum, penicillin, streptomycin, and amphotericin B (antimycotic) and grown in a 37°C, 5% CO2 humidified incubator. For nocodazole treatment, a 10 mM stock solution in DMSO was diluted into medium to a final concentration of 2 µM nocodazole. Cells were incubated with nocodazole-containing medium and either fixed after 3 h of treatment, or washed four times with medium at 37°C and incubated in fresh medium for 1 h at 37°C to allow for spindle reassembly and entry into anaphase before fixation. Pilot experiments showed that the majority of mitotic-arrested cells accomplished anaphase 1 h after the release from the nocodazole block.
Immunostaining with CREST and Anti-tubulin Antibodies
Cells were rapidly rinsed in PBS, lysed for 5 min with PHEM buffer containing 0.5% Triton X-100 and then fixed in 95% methanol plus 5 mM EGTA. The cells were then rinsed in PBS and subsequently blocked in 5% boiled donkey serum in PBS for 1 h at room temperature. The coverslips were then incubated in primary antibodies (human calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia [CREST] serum and mouse anti-tubulin) diluted in 5% boiled donkey serum overnight at 4°C. CREST serum (a gift from Dr. B.R. Brinkley, Baylor College of Medicine, Houston, TX) was diluted 1:600. Anti
-tubulin (DM1
; Sigma-Aldrich) was diluted 1:300. Cells were then rinsed in PBST (PBS with 0.05% Tween 20), incubated in secondary antibodies (Rhodamine redX antihuman, diluted 1:100, and Alexa 488 antimouse, diluted 1:1,000) for 45 min at room temperature, rinsed again, and mounted in an antifade solution containing 90% glycerol and 0.5% N-propyl gallate. The same fixation procedure was used for experiments in which only CREST staining was performed.
Immunostaining with Anti-Mad2 and -3F3/2 Antibodies
Cells were rinsed rapidly in PHEM buffer and then lysed for 5 min in 0.5% Triton X-100 in PHEM buffer. For the 3F3/2 staining, 100 nM microcystin (Sigma-Aldrich) was included in the lysis buffer to block phosphatase activity. Cells were fixed for 20 min in PHEM plus 4% formaldehyde freshly prepared from paraformaldehyde. Cells were then rinsed in PBST and blocked in 5% boiled donkey serum in PBS for 1 h at room temperature. For Mad2 staining, a rabbit primary anti-Mad2 antibody (
Fluorescence Microscopy and Image Acquisition
For analysis of CREST and -tubulin staining, immunofluorescently stained cells were recorded with a spinning disk confocal fluorescence microscope system equipped with a 100x 1.4 NA Plan-Apochromatic phasecontrast objective lens. The microscope was an inverted microscope (TE3000; Nikon) equipped with phasecontrast transillumination or epifluorescence illumination by a Yokogawa CS10 spinning disk confocal attachment (PerkinElmer) containing filters and filter wheels for illumination at 488 or 568 nm from a 60 mW argon/krypton laser. Digital images were obtained with an Orca ER-cooled CCD camera (Hamamatsu Photonics). Image acquisition, microscope shutters, and z-axis focus were all controlled by MetaMorph (Universal Imaging Corp.) software in a PC computer. Phasecontrast microscopy was used to visualize the chromosomes. Z-series optical sections through each cell analyzed were obtained in 0.2-µm steps. To enhance resolution of merotelic kinetochore orientation in prometaphase and metaphase cells, three dimensional (3-D)1 deconvolution and reconstruction was performed on the z-series confocal optical sections using a Delta Vision image processing workstation (Applied Precision). For part of the analysis of Mad2 and 3F3/2 staining, immunofluorescence-stained cells were viewed by wide-field epifluorescence using a Nikon Microphot FX-A microscope equipped with a 60x 1.4 NA objective lens and a 2.0x optovar projection lens. Both differential interference contrast and fluorescence images were obtained with a cooled CCD digital camera (C4880; Hamamatsu). Z-series optical sections through each cell analyzed were obtained in 0.5-µm steps using MetaMorph image processing software and a Ludl stepping motor (
Kinetochore Maximal Width
MetaMorph tracking software was calibrated with a stage micrometer and used to measure maximal width of kinetochores in normal and lagging chromosomes. In the same anaphase cell, the maximal widths of kinetochores on lagging chromosomes and on chromosomes that had moved normally to their spindle poles were measured. To facilitate accurate measurements, the images were zoomed 200400%. 25 ana-telophase cells containing lagging chromosomes were analyzed.
Electron Microscopy
Cells released for 1 h from a mitotic block induced by 2 µM nocodazole were fixed for electron microscopy as described in
A 3-D model of a merotelically oriented kinetochore was also generated. Stereopairs for two consecutive sections containing the kinetochore were obtained at 12,500x magnification and ±16° tilt. Microtubules, kinetochore, and chromosome contours were traced in 3-D space using the Stereocon image-reconstruction system (
Online Supplemental Material
A rotatable 3-D file of Fig 4 d and the software that allowed us to rotate the image are available at http://www.jcb.org/cgi/content/full/153/3/517/DC1.
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A Quicktime® movie of Fig 5 is also available at the same location. The movie allows the cell to rotate and discriminate microtubule bundles ending on the kinetochores from bundles that are aligned with the kinetochores, but not attached to them.
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Results |
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Lagging Chromosomes Are Individual Sister Chromatids Whose Frequency of Occurrence Is Highly Increased in Cells Recovering from a Nocodazole Block
Lagging chromosomes could be observed in anaphase in both control PtK1 cells (Fig 1 a) and in PtK1 cells fixed after 1 h recovery from a nocodazole (2 µM)-induced mitotic arrest (Fig 1 b). These chromosomes were left near the spindle equator, often with their arms nearly perpendicular to the spindle axis (Fig 2), whereas all other chromosomes correctly moved to their spindle poles. The presence of anaphase-lagging chromosomes was analyzed in 1,631 control anaphases and 758 nocodazole-released anaphases by immunofluorescence microscopy. Kinetochores were stained with CREST antibodies, microtubules with an anti-tubulin antibody, and chromosomes with DAPI. We observed lagging of paired sister chromatids in only 1 of 20 control anaphase cells showing lagging chromosomes (Table 1). In this case, these chromatids did not exhibit any CREST staining for kinetochores, suggesting that inactivation of the kinetochores had occurred in that chromosome or that they were acentric chromosome fragments. Lagging of paired sister chromatids was observed in only 2 out of 135 cells recovering from a nocodazole-induced mitotic block and showing lagging chromosomes (Table 1). These chromatids did show CREST staining for kinetochores. In all the other cases of lagging chromosomes analyzed, the lagging chromosomes were single chromatids with only one CREST signal, indicating that lagging of paired sisters was a rare event both in untreated cells and in cells recovering from a mitotic block (Table 1). The kinetochore of a lagging chromosome was often very stretched (Fig 1 a and below), but we could always detect the weak fluorescence of the thin region between the two thicker ends. Note that CREST antibodies do not stain the centromere region between sister kinetochores (Fig 2 a).
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Counts of CREST-stained kinetochores also verified that lagging chromosomes were single chromatids. Kinetochore count was performed on only 31 anaphase cells, because in these cells the kinetochores near the poles were sufficiently separated from each other to allow accurate counts. The chromosome number in our PtK1 cells in culture is slightly variable above 12 sister pairs (13.5 ± 1.5). In each anaphase cell analyzed with one or more lagging chromosomes (31 total), the counts always resulted in an even number of kinetochores. For all 11 anaphases analyzed with a single lagging chromosome (with 1 CREST-stained kinetochore), the sum of kinetochores at both poles was always an odd number. A similar result was found for three anaphases with three lagging chromosomes. For the 17 anaphases that had 2 lagging chromosomes, the sum of all CREST-stained kinetochores at the poles was an even number, as predicted by each lagging chromosome having only one kinetochore. Although lagging chromosomes were the major source of aneuploidy in the cells analyzed, there was also evidence that sister pairs might missegregate. In 6 of the 31 anaphases with lagging chromosomes, the number of kinetochores segregated to one pole was greater than the sum of the number at the opposite pole plus the number of lagging kinetochores by two and, in one case, by six. This inequality indicates that occasionally both sisters of one or more sister pairs can migrate toward the same pole, as reported for other mammalian cells (
The data summarized in Fig 1 c and Table 1 show that the mean frequency of anaphases with one or more lagging chromosomes (each showing only one CREST signal; i.e., a single chromatid) was 1.16% in untreated cells and 17.55% in cells released from the nocodazole block. The frequencies of lagging chromosomes in six independent experiments, in which only CREST or both CREST and -tubulin immunostaining were performed, did not show statistically significant differences. The frequency of anaphase cells containing a single lagging chromosome was about ninefold higher in cells released from the mitotic arrest than in untreated cells, whereas the frequency of anaphase cells showing multiple (two or more) lagging chromosomes was increased >100 times, since this event was rarely observed in control cells (Fig 1 c and Table 1). A
2 test confirmed that in cells recovering from the nocodazole block, the frequencies of both single and multiple lagging chromosomes were highly increased compared with control cells (P < 0.001 in both cases). For this reason we used the nocodazole treatment as a tool to increase the number of cells containing lagging chromosomes for analysis.
A Lagging Chromosome Is Connected at Its Kinetochore to Bundles of Microtubules from Opposite Spindle Poles (Merotelic Orientation)
To analyze the connections between mitotic spindle microtubules and the kinetochore of lagging chromosomes during mitosis, we performed immunostaining for centromeric proteins (CREST staining) and microtubules (-tubulin immunostaining) and obtained high resolution optical sections through anaphase cells with lagging chromosomes by fluorescence confocal microscopy. Fig 2, a and b show normal metaphase and anaphase, respectively. The overlay of the phasecontrast image with the CREST staining (Fig 2, left column) confirmed that lagging chromosomes in cells recovering from a mitotic block were single chromatids with one CREST-staining kinetochore (stretched, see below) (Fig 2, ce), whereas the merged images of CREST and tubulin staining (Fig 2, right column) revealed that a single kinetochore was connected to bundles of microtubules coming from opposite spindle poles (merotelic attachment) (Fig 2c'e'). In late anaphase and telophase the connection of microtubules to the kinetochore of a lagging chromosome was not detectable because of the presence of a large number of microtubules in the spindle midzone. However, in 26 cells recovering from a mitotic block in which the connection of microtubules to the kinetochore of a lagging chromosome was clearly detectable, microtubule bundles were seen extending toward both poles (i.e., merotelic attachment).
To test if merotelic attachment is a general cellular mechanism responsible for chromosome loss and not only an abnormal behavior induced by nocodazole treatment, we performed the same immunostaining on cells not treated with nocodazole. We could clearly see for all the 11 lagging chromosomes analyzed in untreated cells that the attachment of kinetochore microtubule bundles to kinetochores was merotelic (Fig 2c and Fig c'), except in the case of the two paired sisters with no CREST signals that did not show any microtubule binding. Thus, merotelic attachment appeared to be responsible for lagging chromosomes during mitosis in untreated cells as well as cells recovering from the nocodazole block.
The analysis of -tubulin immunostaining showed a major decrease in microtubule fluorescence at the kinetochore of the lagging chromosome (Fig 3, middle column). This indicates that the majority of microtubules from opposite poles do not run past the kinetochore, but rather terminate on it. However, there was a low level of microtubule fluorescence at the kinetochore region. This fluorescence could be produced by microtubules attached to different positions along the extended kinetochore or by interpolar microtubules clustered in the vicinity of kinetochore microtubule bundles as already shown by electron microscopy in PtK1 cells (
We were also able to show by electron microscopy that the single kinetochore of an anaphase-lagging chromosome has kinetochore microtubules extending towards opposite poles. Nocodazole-treated cells were released from a mitotic block for 1 h and then fixed for electron microscopy. Samples were plastic-embedded and anaphases with lagging chromosomes were localized by phasecontrast microscopy. Selected cells were relocalized on the plastic after the coverslip was removed and the samples were sectioned. For three lagging chromosomes in two cells, the kinetochore on the lagging chromosome had microtubules extending towards opposite spindle poles as shown in Fig 4 (i.e., merotelic attachment). The figure shows three serial-thick sections through a lagging chromosome in an anaphase cell with its kinetochore connected by kinetochore microtubule bundles to both spindle poles. In the two sections shown in Fig 4b and Fig c, the kinetochore can be seen stretched laterally between microtubule bundles extending toward opposite poles (arrows point at opposite sides of the kinetochore). In these thick sections, the sites of microtubule attachment to the kinetochore are difficult to see because of the section thickness, the attachment of plus ends at various positions along the stretched kinetochore, and the presence of interpolar microtubules which extend pass the kinetochore without attachment. To clearly see the distribution of kinetochore microtubules and the attachment position of their plus ends, a 3-D Stereocon reconstruction (Fig 4 d) was generated by stacking the pertinent information contained in the two sections shown in Fig 4b and Fig c. The resulting image (Fig 4 d) clearly shows that 5 kinetochore microtubules extend toward one pole and 11 extend toward the opposite pole. There were 1012 microtubules in the vicinity of the kinetochore that did not terminate in the kinetochore and these microtubules were not included in the reconstruction so that the kinetochore microtubules are clearly visible.
Since lagging chromosomes remain behind at the spindle equator as chromosomes move to the spindle poles during anaphase, we expected to find chromosomes in late prometaphase or metaphase cells with one of the sister kinetochores merotelically oriented. To test this possibility, we fixed cells 15 min after release from a nocodazole-induced mitotic block at a time when most of the cells had already reestablished bipolar spindle assembly and their chromosomes were partially aligned on a metaphase plate. We needed higher resolution procedures to help clearly see the orientation of microtubule attachment to kinetochores near the metaphase plate, where chromosomes are very crowded, as compared with the resolution we needed for the analysis of one or a few lagging chromosomes at mid- to late anaphase. This was achieved by acquiring z-series stacks of confocal images at 0.2-µm intervals through each cell analyzed and processing the image stacks to obtain 3-D projections after digital image deconvolution (
Analysis of the Kinetochore Stretching in Anaphase Induced by Merotelic Attachment
During mitosis, kinetochore microtubules exert a net pulling force on their chromatids (
We evaluated the difference in the size of kinetochores between normal and lagging chromosomes by measuring the maximal width of the CREST signal. In the same anaphase cell, the maximal width of the kinetochores on lagging chromosomes and on chromosomes that had moved normally to the spindle poles was measured. On average, the maximal width of kinetochores of lagging chromosomes was >2-fold greater than normal kinetochores, but could be stretched laterally up to 5.6-fold (an example of a stretched kinetochore, >5-fold wider than the average, is shown in Fig 6 a, right; see also Fig 1 a and 2, cf'). The stretching observed ranged between 1.2- and 5.6-fold, but we also observed a correlation between the mitotic phase and the degree of stretching by comparing the data for different stages of ana-telophase. In mid-anaphase, the kinetochores of lagging chromosomes were on average 2.6-fold wider (1.47 vs. 0.56 µm) than normal kinetochores. In late anaphase the ratio between lagging and normal kinetochores was 2.1 (1.14 vs. 0.55 µm) and it was reduced to 1.7 in telophase (0.96 vs. 0.57 µm; Fig 6 b). This comparison suggests that during anaphase a merotelically oriented kinetochore is stretched laterally by pulling forces generated by kinetochore microtubules extending to opposite spindle poles. During the later stages of mitosis stretching is reduced, indicating that the pulling forces are decreased. Statistical analysis of the data showed that the differences in kinetochore width between normal and lagging chromosomes were statistically significant for all three mitotic phases analyzed (P < 0.001, Student's t test).
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Mad2 and the 3F3/2 Phosphoepitope Are Not Present on the Kinetochore of a Lagging Chromosome at Anaphase
Untreated cells, nocodazole-treated cells, and cells fixed after 1 h recovery from the nocodazole-induced mitotic arrest were examined to investigate the state of the mitotic spindle checkpoint proteins on lagging chromosomes. We immunostained cells with anti-Mad2 or 3F3/2 antibodies. As shown previously (for review see
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Discussion |
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A Major Mechanism of Aneuploidy Is the Formation of Lagging Chromosomes with Merotelically Oriented Kinetochores
The 1% occurrence of lagging chromosomes at the spindle equator of untreated anaphase PtK1 cells and the order of magnitude higher frequency after mitotic arrest with nocodazole treatment are values similar to those reported previously by in situ hybridization analysis for other types of mammalian tissue cells in culture (
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For these merotelically oriented single kinetochores in anaphase, we measured a 1.25.6-fold lateral stretching. This lateral direction of stretching is not typically experienced by kinetochores attached only to microtubules from one pole. Normally oriented kinetochores are pulled by kinetochore microtubule-associated forces along the kinetochore-centromere axis (
Abnormal Kinetochore Expansion and Curvature May Promote Merotelic Kinetochore Orientation
Kinetochore microtubule formation is thought to occur by a "search and capture mechanism," where kinetochores recruit kinetochore microtubules by capturing the plus growing ends of polar spindle microtubules into binding sites in the kinetochore outer plate (for review see
What produces the substantial increase in merotelic kinetochore orientation after release of the mitotic block? One possibility is that the nocodazole-induced mitotic block may somehow inhibit, in cells recovering from the mitotic block, the normal mechanisms that correct merotelic orientation when it naturally occurs during prometaphase. On the other hand, the substantial increase in merotelic orientation is correlated with the previous expansion of the kinetochore outer domain into expanded crescents around the centromere during nocodazole treatment (Fig 7b and Fig e, and Fig 8 a). Kinetochore crescent formation is typical of the response of kinetochores in mammalian tissue cells to depletion of spindle microtubules (
Merotelically Oriented Kinetochores Are Not Detected by the Mitotic Spindle Checkpoint
Mitotic tissue cells containing chromosomes with merotelically oriented kinetochores, induced either by multiple spindle poles (
Our results also extend these studies by providing biochemical evidence that merotelically oriented kinetochores have inactivated checkpoint activity in cells that enter anaphase. Much as occurs for kinetochores of properly aligned metaphase chromosomes (
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviation used in this paper: 3-D, three dimensional.
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Acknowledgements |
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Thanks to G. Cassels for her expert assistance with electron microscopy.
Supported by Nationsl Institutes of Health grants GM24364 to E.D. Salmon and GM59363 to A. Khodjakov. D. Cimini is a Ph.D. fellow of the University of Rome "La Sapienza." Stereocon is a part of National Resource supported by National Center for Research Resources grant RR012019.
Submitted: 5 January 2001
Revised: 15 March 2001
Accepted: 19 March 2001
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References |
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