Cellular mechanisms of aneuploidy induction in mammalian cells.

2001 EEMS Young Scientist Award Lecture

Dr. Daniela Cimini, Center for Evolutionary Genetics c/o Dept. of Genetics and Molecular Biology, University of Rome "La Sapienza", Via degli Apuli, 4 00185 Rome, Italy

Current address: Department of Biology, 607 Fordham Hall, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Phone: 1-919-962-2354

Fax: 1-919-962-1625

e-mail: ciminid@hotmail.com

 

Introduction

This paper summarizes a study focused on the following three main research objectives: chromosome segregation in mitosis, analysis of different types of malsegregation events, and identification of cellular mechanisms involved in the production of aneuploid cells. Aneuploidy is the condition of a cell that has lost its normal diploid chromosome number, because it has lost or gained one or more chromosomes during cell division. The role of aneuploidy in human meiotic cells is well known for inducing severe pathological genetic syndromes (for review see Nicolaidis and Petersen, 1998). In addition, recent studies have indicated that aneuploidy in somatic mitotic cells plays a significant role in tumorigenesis (reviewed in Sen, 2000). An increasing number of studies have shown that abnormalities like aneuploidy and whole-chromosome loss of heterozygosity are commonly present in tumor cells. This suggests that chromosome segregation defects play a critical role in tumor development and progression (Lengauer et al., 1997; Cahill et al., 1998; Stoler et al., 1999; Saunders et al., 2000). Several studies indicate that aneuploidy is a dynamic mutational event. Aneuploidy amplification can result because, after aneuploidy initially occurs, it induces a destabilization of the karyotype that increases the probability of malsegregation during mitosis (Lengauer et al., 1997; Duesberg et al., 1998). Furthermore, studies of both human and rodent cells in vitro have shown that aneuploidy occurs during the early stages of tumor transformation and can induce cellular immortalization (Namba et al., 1996; Li et al., 1997; Duesberg et al., 2000).

Errors in chromosome segregation during cell division are responsible for alterations in chromosome number. The impairment of each of the structural or regulative proteins involved in the mitotic process can result in an alteration of chromosome segregation. Abnormalities of the mitotic apparatus have been frequently identified in tumor cells. For example, centrosome defects associated with aneuploidy have been identified in prostate, colon, brain, and breast tumors (Pihan et al., 1998; Lingle et al., 1998). Untimely separation of sister chromatids has been suspected as a cause of aneuploidy in human tumors. And this seems to be the case, since a vertebrate securin (v-securin) that seems to be involved in cell transfromation and tumorigenesis has recently been identified (Zou et al., 1999). Furthermore, defects in the mitotic spindle checkpoint may result in chromosome segregation defects. The mitotic spindle checkpoint is a control mechanism that inhibits anaphase onset if a bipolar spindle is not correctly assembled and if chromosomes are not correctly attached by their kinetochores to spindle microtubules. In the presence of such defects the mitotic spindle checkpoint is activated, inducing the cell to arrest in metaphase. This provides time for repair of errors (for review see Rieder and Salmon, 1998; Shah and Cleveland, 2000). Mutations in the mitotic checkpoint genes hBUB1and hBUBR1 have been identified in colorectal tumor cells showing inactivation of the mitotic spindle checkpoint (Cahill et al., 1998). A reduced expression of the human homologue of MAD2 (hMAD2) (another mitotic checkpoint protein) has also been observed in a breast tumor cell line with a defective mitotic checkpoint (Li and Benezra, 1996). These findings provide further evidence of the strong relation between chromosome missegregation and tumorigenesis.

Both the identification of cellular mechanisms responsible for aneuploidy induction and the development of methods to detect aneuploidy are of great interest in order to better understand the crucial role that aneuploidy has in tumorigenesis and tumor progression.

This paper summarizes the results I obtained over the last few years in the study of aneuploidy and aneuploidy-inducing mechanisms, and it is focused on two main topics:

1. development of new methods to detect aneuploidy as a tool to identify aneugenic compounds; 2. identification of cellular mechanisms producing aneuploidy during mitosis.

Development of new methods to detect aneuploidy

Several methods have been developed and used both to identify aneugenic compounds and to measure aneuploidy in biomonitoring studies of exposed populations. One of the most widely used methods for the detection of aneuploidy is the cytokinesis-block assay (Fenech and Morley, 1985). This assay allows identifying the products of a single mitosis, since the two daughter nuclei are maintained into the same cytoplasm by inhibition of cytokinesis using cytochalasin B treatment. The combination of the cytokinesis-block assay with anti-kinetochore staining or fluorescence in situ hybridization with pan-centromeric probes has been used to analyze both chromosome loss and breakage after treatment of mammalian cells with chemical or physical agents (Eastmond and Tucker, 1989; Antoccia et al., 1993; Norppa et al., 1993). Fluorescence in situ hybridization (FISH) with chromosome-specific alphoid probes has also been shown to be a powerful method to detect aneuploidy in interphase human cells, allowing the identification of aneuploid cells by counting the number of signals inside the nucleus (Eastmond and Pinkel, 1990). The combination of the cytokinesis-block assay and FISH staining with chromosome-specific probes detects not only chromosome loss (absence of chromosome migration to spindle poles during anaphase), visualized as FISH-positive micronuclei in binucleate cells (Figure 1, left), but also errors in chromosome segregation like chromosome non-disjunction, deriving from the migration of both sisters to the same pole during anaphase (Figure 1 right), in cells that have accomplished one mitotic division (see diagram in Figure 1) (for review see Kirsch-Volders et al., 1997). The use of this approach has allowed the identification of the aneuploidy-inducing activity of several compounds, including several spindle poisons (Marshall et al., 1996; Zijno et al., 1996; Elhajouji et al., 1997; Sgura et al., 1997), and X-rays (Kirsch-Volders et al., 1996). Furthermore, this approach has been used in several biomonitoring studies in which the monitored population had been exposed to known or suspected aneuploidy-inducing agents (Surralles et al., 1997; Carere et al., 1998; Marcon et al., 1999). We extensively used FISH staining in combination with the cytokinesis-block method, but we also developed an approach in which FISH staining with chromosome-specific probes is performed on anaphases cells. This system allows to visualize the aneuploidy-inducing event when it occurs (see diagram in Figure 1, top), and, in some cases, to identify the mechanism responsible for aneuploidy induction.

In 1997 we performed double in situ hybridization on human primary fibroblasts with a chromosome 1-specific alphoid probe and a probe for the satellite III on the long arm of chromosome 1 (tandem labeling) in combination with the cytokinesis-block assay. We found that topoisomerase II inhibition during mitosis induces both chromosome breakage and chromosome non-disjunction (Cimini et al., 1997). Furthermore, the tandem labeling on anaphase cells revealed that the non-disjunction induced by topoisomerase II inhibitors is due to the inhibition of sister chromatid decatenation, so that the arms of the two sister chromatids remain connected and one of the sisters is pulled by the other toward its same spindle pole (as diagrammed in Figure 2) (Cimini et al., 1997).

While topoisomerase II inhibitors act on the chromatin, many aneugenic compounds interfere with mitotic spindle functions. In a following study we used nocodazole as a model spindle poison compound and we applied in situ hybridization with alphoid probes specific for chromosome 7 and 11 to ana-telophase cells from human primary fibroblasts recovering from a nocodazole-induced mitotic block to visualize the events leading to aneuploidy directly during anaphase and to investigate the induction of chromosome malsegregation during this mitotic stage (Figure 3A-B). We also compared the frequencies of chromosome malsegregation observed in ana-telophase with the estimated malsegregation obtained in binucleate cells (Figure 3 C-D), to compare the analysis of aneuploidy in anaphase with the aneuploidy evaluated in the cytokinesis-block assay, which, as described above, is a widely used method to detect chemically-induced aneuploidy in mammalian cells. This study showed that the aneuploidy detected in binucleate cells is lower than the aneuploidy observed in anaphase (Cimini et al., 1999). This difference seems to be related to the fact that during the cytochalasin B treatment some of the mitotic cells give rise to mononucleate polyploid cells instead of binucleate cells (Figure 3 E-F) (Cimini et al., 1999). We also showed that the formation of mononucleate polyploid cells during cytochalasin B treatment was promoted by a reduced pole-to-pole distance (Cimini et al., 1999) associated with an absence of both contractile ring and spindle midzone in ana-telophase cells (Figure 4) (Cimini et al., 1998). Under these conditions, the two groups of segregating chromosomes could be included into the same nucleus upon nuclear envelope reassembly at the end of mitosis in cells displaying lagging chromosomes at anaphase. Based on this observation, we suggested that the combined FISH analysis of polyploid mononucleate cells and chromosome loss/non-disjunction in binucleate cells could substantially improve the sensitivity of the cytokinesis-block assay in identifying compounds that interfere with mitotic division and chromosome segregation (Cimini et al., 1999).

Taken together, these data showed that, even though the cytokinesis-block assay remains a very sensitive and useful method to detect aneuploidy, the analysis of malsegregation events in anaphase cells is a very powerful tool, as it allows the identification of both potential aneugenic compounds and cellular mechanisms responsible for induction of aneuploidy.

Identification of cellular mechanisms involved in chromosome loss during mitosis

Having established that anaphase analysis is a powerful method to detect malsegregation events, we took advantage of this approach to study the cellular mechanisms involved in the production of aneuploid cells. As described above, the two major events leading to the formation of aneuploid cells during bipolar mitotic division are chromosome loss and chromosome non-disjunction (Figure 1). We focused our attention on the investigation of cellular mechanisms responsible for chromosome loss in mitosis. In particular, we investigated what is the role of the mitotic checkpoint in the origin of aneuploidy and if chromosome loss occurs in cells that enter anaphase with unattached kinetochores and unaligned chromosomes. The mitotic checkpoint is a control mechanism that prevents metaphase to anaphase transition if one or more chromosomes are not correctly attached to microtubules and not aligned at the metaphase plate (for review see Rieder and Salmon, 1998; Shah and Cleveland, 2000). Therefore, the mitotic spindle checkpoint plays a key role in preventing the unbalanced distribution of chromosomes during mitosis and the production of aneuploid cells. Nevertheless, the fidelity of chromosome segregation is not absolute, and chromosomes left near the spindle equator after anaphase onset (lagging chromosomes) have been observed in cultured mammalian cells (Ford et al., 1988; Izzo et al., 1998; Cimini et al., 1999; Catalán et al., 2000). A model suggested by a large number of studies in grasshopper meiosis, proposed that, although initial kinetochore mal-orientations are very frequent, they are efficiently corrected during prometaphase by a process in which kinetochore microtubules can detach and reattach several times, until the correct orientation is achieved (for reviews see: Nicklas, 1971; Bajer and Molč-Bajer, 1972; Nicklas, 1997). This model also proposed that meiotic or mitotic mis-segregation may occur when kinetochore mal-orientations are not corrected before anaphase onset (Nicklas and Koch, 1969; Ault and Rieder, 1992).

To test this hypothesis we performed a set of experiments in cells recovering from a nocodazole-induced mitotic block. Nocodazole was used because of its reversible effect on the mitotic spindle and because of the high frequency of anaphase lagging chromosomes observed in cells recovering from the mitotic block (Cimini et al., 1999). We used in situ hybridization on anaphase cells and we also performed observations of mitotic progression and chromosome dynamics in living cells expressing an H2B histone-GFP chimeric protein.

As previously shown, in situ hybridization with chromosome specific alphoid probes on anaphase cells allows identification of both chromosome loss and chromosome non-disjunction events (Figure 1). Furthermore, when chromosome loss events are seen as chromosomes lagging behind at the spindle equator in anaphase cells, this approach discriminates between single chromatid loss and loss of both sisters or whole chromosomes (Figure 5 and Figure 6 A-B).

The analysis of chromosome loss in anaphases from human primary fibroblasts recovering from a nocodazole-induced mitotic block hybridized with alphoid probes for chromosomes 7 and 11 (Figure 6 A-B) showed that the loss of a single sister is much more frequent than the loss of both sisters, and that, when both sisters are lost, they exhibit kinetochore separation (Figure 6 C), indicating that the loss of whole chromosomes is a very rare event and that chromosome loss and sister chromatid separation are independent events (Cimini et al., in press).

To investigate the state of an important mitotic spindle checkpoint marker on lagging chromosomes, we immunostained cells with 3F3/2 antibody, which recognizes a kinetochore phosphoepitope that localizes on unattached kinetochores. We showed that no 3F3/2 staining is detectable on the kinetochore of lagging chromosomes in anaphase cells from human primary fibroblasts recovering from a nocodazole-induced mitotic block (Cimini et al., in press). This suggests that anaphase lagging chromosomes interact with kinetochore microtubules at some point during mitosis before anaphase onset and this induces the dephosphorylation of the phosphoepitope recognized by the 3F3/2 antibody.

To test this hypothesis in living cells, we transfected both human primary fibroblasts and PtK1 cells with a vector carrying an H2B-GFP fusion gene under the CMV promoter (Kanda et al., 1998). The expression of the H2B-GFP fusion protein provided a clear GFP-labeling of chromosomes, allowing the visualization of chromosome dynamics in living cells. The analysis of mitotic progression during the recovery from a nocodazole-induced mitotic block in H2B-GFP expressing cells provided the following important observations. First, both MRC-5 (Figure 7 A) and PtK1 mitotic cells showing one or a few chromosomes not aligned at the metaphase plate (mono-oriented chromosomes, i.e. chromosomes in which only one of the sister kinetochores is attached to spindle fibers) did not enter anaphase within the period of live observation (2-3 hours). Second, all anaphase lagging chromosomes appeared in cells in which all the chromosomes had correctly aligned at the metaphase plate before anaphase onset (Figure 7 B). Third, all the lagging chromosomes we observed were not incorporated in one of the daughter nuclei during nuclear envelope reformation at the end of mitosis, but formed micronuclei (Figure 7 C, last frame). This result indicates that lagging chromosomes are never rescued by successful later migration, but remain at the cell equator, giving rise to aneuploid daughter cells in 50% of the cases, being included randomly in either one or the other daughter cell when cytokinesis occurs at the end of mitosis (Cimini et al., in press). In conclusion, the observation that in both cell lines lagging chromosomes are never observed in cells entering anaphase in the presence of mono-oriented chromosomes, demonstrated that the mitotic checkpoint is completely efficient in inhibiting the metaphase to anaphase transition in cells with unattached kinetochores. Taken together, these data indicate that in cells recovering from a nocodazole block all kinetochores successfully attach to microtubules and align at the metaphase plate, switching off the 3F3/2 checkpoint-associated signal, and inactivating inhibitory signals for anaphase onset. After anaphase onset lagging chromosomes appear and do not migrate toward spindle poles due to a mechanism not detected by the mitotic spindle checkpoint.

The following part of the study was then aimed to identify the cellular mechanism that produces anaphase lagging chromosomes (i.e. chromosome loss during mitosis). More specifically, we addressed the following questions: 1. why do lagging chromosomes remain near the spindle equator if they are able to congress to the metaphase plate before anaphase-onset? 2. why doesn’t the mitotic spindle checkpoint sense the defect producing anaphase lagging chromosomes?

Possible hypotheses to explain lagging chromosomes after anaphase onset are: loss of kinetochore microtubules during anaphase, inactivation of pulling forces acting on the kinetochore during anaphase, and merotelic kinetochore orientation (attachment of a single kinetochore to microtubules coming from both poles; Ladrach and LaFountain, 1986).

To identify the cellular mechanism responsible for induction of anaphase lagging chromosomes we used PtK1 cells, a model system widely used to study mitosis and chromosome segregation. We showed by CREST staining of PtK1 cells that anaphase lagging chromosomes are single chromatids with only one CREST signal both in untreated cells and in cells recovering from a mitotic block (Figure 8), and that the loss of paired sisters is a rare event (Cimini et al., 2001). These results agree with the results obtained in our studies of human primary fibroblasts.

We next investigated the state of two crucial mitotic spindle checkpoint proteins on lagging chromosomes: Mad2 and the phosphoepitope recognized by 3F3/2 antibody. We found that kinetochores of anaphase lagging chromosomes did not stain for either Mad2 or 3F3/2 (see Figure 9 for 3F3/2). Thus, the spindle checkpoint activity of lagging chromosome kinetochores is inactivated, observation that confirms the result previously obtained in human cells with the 3F3/2 antibody (Cimini et al., 2001). In addition, by anti-Mad2 and 3F3/2 immunostaining we showed that kinetochores in cells depleted of microtubules by nocodazole treatment expand into crescents around the centromere as seen by both Mad2 and 3F3/2 staining (Figure 9 B), as predicted by the work of Hoffman et al. (2001).

To determine the cellular mechanism inducing lagging chromosomes in anaphase, we investigated if and how the kinetochore of a lagging chromosome is connected to kinetochore microtubules. To analyze the connections between mitotic spindle microtubules and the kinetochore of lagging chromosomes, we combined CREST staining with a-tubulin immunostaining to visualize both centromeric proteins and microtubules. We obtained high resolution optical sections through anaphase cells with lagging chromosomes by fluorescence confocal microscopy. By using this approach, we showed clearly that lagging chromosomes, both in untreated cells and in cells recovering from a mitotic-block, are merotelically oriented (Figure 10). This means that the single kinetochore of a lagging chromosome is connected to microtubule bundles coming from opposite poles (merotelic orientation). Kinetochores of these lagging chromosomes also appeared highly stretched laterally, indicating that they are subjected to pulling forces toward opposite poles (Cimini et al., 2001). We were also able to confirm merotelic orientation of lagging chromosomes by electron microscopy (Cimini et al., 2001).

Since lagging chromosomes remain behind at the spindle equator as cells enter anaphase, we expected to find chromosomes in late prometaphase or metaphase cells with merotelically oriented kinetochores. To test this possibility, we fixed cells in prometaphase and metaphase after a brief release from a nocodazole block, and we performed fluorescence imaging of kinetochores and kinetochore microtubule bundles by confocal microscopy and 3-D image deconvolution. We also used a fixation protocol that preferentially preserves kinetochore microtubules, so that connections between kinetochores and kinetochore microtubules could be more easily analyzed. By using this approach we found, as expected, merotelically oriented kinetochores in prometaphase and metaphase cells (Figure 11), indicating that merotelic kinetochore orientation occurs in prometaphase, when kinetochores establish their connections to kinetochore microtubules.

Taking these results together, we proposed a model in which, single kinetochores acquire a merotelic orientation in prometaphase. During anaphase merotelically oriented chromosomes are not able to migrate toward the spindle poles, because forces directed in opposite directions are applied on their kinetochores. We also proposed that the increased frequency of lagging chromosomes in cells recovering from a nocodazole-induced mitotic arrest might be a consequence of the expansion of kinetochores in crescents during the nocodazole treatment. During the recovery from the mitotic block, the persistence of a crescent morphology on kinetochores, would promote the acquisition of a merotelic orientation. This work clearly showed that merotelic kinetochore orientation during prometaphase is a major mechanism of chromosome loss in mammalian tissue cells; that kinetochore curvature, enhanced by a nocodazole block, may promote the formation of bundles of microtubules to opposite poles; and that merotelic orientation of single kinetochores induces the inactivation of the mitotic spindle checkpoint.

Conclusions and perspectives

My work has shown the great advantage offered by developing new approaches to detect aneuploidy. Our new method for aneuploidy detection in anaphase cells has been proven to be extremely useful, as it allows not only the analysis of aneuploidy, but also the identification of possible mechanisms inducing chromosome malsegregation. Furthermore, both the analysis of mitosis in living cells and the use of high resolution microscopy represent powerful tools for the identification of cellular mechanisms involved in chromosome malsegregation.

The identification of both cellular structures and cellular mechanisms involved in chromosome malsegregation is of crucial interest because of the potential role that aneuploidy has been suggested to have in tumor development and progression (reviewed in Sen, 2000). Genetic instability at the chromosome level has been demonstrated to be a common feature of several tumor types. For this reason, the identification of cellular structures and cellular mechanisms involved in the production of aneuploid cells represents an extraordinarily important clue to understand the genesis of chromosome instability in tumor cells and a starting point for the development of new therapeutic strategies.

Future research should be addressed to the identification of other possible cellular mechanisms of malsegregation. Subsequently, the development of methods to prevent malsegregation and aneuploidy amplification will be easier to achieve. At the same time, a different research line should be addressed on the identification of chemicals and systems to selectively hit aneuploid cells. The discovery of cellular mechanisms involved in the occurrence of aneuploid cells and the finding that aneuploidy may be the unifying feature of many different tumor types may stimulate new approaches to treat common forms of cancer.

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Figure legends

Figure 1. Diagram showing chromosome malsegregation events that may occur during a bipolar mitotic division and the products generated by such malsegregation events if cells are treated with cytochalasin B during mitosis. The cytochalasin B treatment during mitosis prevents cytokinesis and induces the formation of binucleate cells, thus allowing the identification of the products of a single mitosis that are retained within the same cytoplasm. The diagram shows the visualization of malsegregation events as detected by FISH staining with two differently labeled chromosome-specific alphoid probes. The left side of the diagram shows a micronucleus (bottom) originating by an anaphase lagging chromosome (top). The right side of the diagram shows a non-disjunctional event (i.e. migration of two sister chromatids toward the same pole), as detected both in anaphase (top) and in a binucleate cell (bottom).

Figure 2. Diagram showing how inhibition of sister chromatid decatenation by topoisomerase II inhibitors can lead to non-disjunction during mitosis. The arms of the two sister chromatids remain connected and one of the sister kinetochores loses the connection to spindle fibers and is pulled by its sister chromatid toward the opposite spindle pole.

Figure 3. FISH staining on human primary fibroblasts using chromosome 7 (FITC, yellow) and chromosome 11 (rhodamine, red) alphoid probes. DNA was counterstained with DAPI (blue). A. Anaphase cell displaying a copy of chromosome 11 lagging behind at the spindle equator. B. Anaphase cell displaying non-disjunction of chromosome 11 (three red signals to one pole and 1 red signal to the other pole). C. Binucleate cell showing a micronucleus containing a copy of chromosome 7 (chromosome loss). D. Binucleate cell showing non-disjunction of chromosome 11 (three red signals in one nucleus, one red signal in the other nucleus). E. Polyploid mononucleate cell, possessing 4 copies of both chromosome 7 and chromosome 11 (4 yellow and 4 red signals). F. Frequencies of polyploid mononucleate cells in a control cell population (-Cyt B) and in a cell population treated with cytochalasin B (+Cyt B). The histogram shows that the cytochalasin B treatment increases the frequency of mononucleate polyploid cells.

Figure 4. A, B. Giemsa-stained anaphase MRC-5 cells in the presence (B) or in the absence (A) of cytochalasin B. A clear reduction of the pole-to-pole distance is visible in cytochalasin B-treated cells (B) as also indicated by the mean values of pole-to-pole distance measured in two samples of anaphase cells in the presence (d2) or in the absence (d1) of cytochalasin B. C-F. Immunostaining of actin and a-tubulin in untreated human primary fibroblasts (C-D) and cytochalasin B-treated ana-telophase cells (E-F). Contractile ring (red) and spindle midzone (green) are visible both in anaphase (C) and telophase (D) in untreated cells. When cells are exposed to cytochalasin B the two structures are absent both in anaphase (E) and telophase (F).

Figure 5. Diagram showing different chromosome loss events that may occur during bipolar mitotic division. The diagram shows how these malsegregation events would be visualized in anaphase cells hybridized in situ with two chromosome-specific alphoid probes.

Figure 6. FISH staining on anaphase human primary fibroblasts (MRC-5 cells) recovering from a nocodazole-induced mitotic arrest. Alphoid specific probes for chromosome 7 (FITC, yellow) and 11 (Rhodamine, red) were used. DNA was stained by DAPI (blue). A. Loss of a single chromatid of chromosome 7. B. Loss of both sister chromatids of chromosome 7. C. Graph showing quantitative results of the in situ hybridization analysis. The data show that single lagging chromatids are the great majority of loss events (green section) and that when both sisters are lost in anaphase cells, their kinetochores are frequently separated (yellow section) and only in a few cases the kinetochores of the two chromatids are still connected (red section).

Figure 7. Chromosome dynamics during recovery from a nocodazole-induced mitotic arrest in MRC-5 (A) and PtK1 (B-C) cells expressing an H2B-GFP fusion protein. Numbers at the bottom right corner indicate the time in minutes. A. MRC-5 cell displaying non-congressed, unaligned chromosomes (arrows). In the presence of non-congressed chromosomes cells activate the mitotic spindle checkpoint and do not enter anaphase. B. PtK1 cell displaying a lagging chromosome in anaphase/telophase (14, 19, 22, 70, 85 minute frames) after chromosome alignment at the metaphase plate had occurred (0 minutes). C. Same cell shown in (B) visualized by phase contrast microscopy during anaphase and telophase. The lagging chromosome (arrows) is visible until the end of mitosis (85 minutes), when it gives rise to a micronucleus (arrow) upon nuclear envelope reassembly.

Figure 8. Fluorescent images of anaphase lagging chromosomes in PtK1 cells. Kinetochores were stained with CREST antibodies (yellow) while chromosomal DNA was stained with DAPI (pseudocolored red). Examples of lagging chromosomes in an untreated cell (A) and in a cell recovering from a nocodazole-induced mitotic arrest (B). Lagging chromosomes are single chromatids with only one CREST signal.

Figure 9. 3F3/2 immunostaining in an untreated cell (A), a nocodazole-treated cell (B) and a cell fixed after 1 hr recovery from a nocodazole-induced mitotic arrest (C). Overlays of DIC and fluorescence images are shown in the Figure. 3F3/2 staining is present on the kinetochores of unattached and unaligned chromosomes in prometaphase (A), is enhanced on the kinetochores of nocodazole-arrested cells (B), but is not present on the kinetochores of lagging chromosomes (or chromosomes normally moved to the spindle poles) in anaphase cells (C).

Figure 10. Fluorescent and phase contrast images of PtK1 cells fixed at anaphase and immunostained for kinetochores with CREST antibodies (green) and for microtubules with anti-a-tubulin antibodies (red). Overlays of phase contrast and CREST images (left column) and a-tubulin and CREST images (right column) are shown. The CREST and microtubule fluorescent images were obtained by projecting into a single image the maximal brightness at each pixel location through a stack of optical sections acquired at 0.2 mm intervals through the immunostained cells by confocal fluorescence microscopy. (A-A’) Example of a single anaphase lagging chromosome. (B-B’) Example of double lagging chromosome in anaphase. The CREST and a-tubulin merged images clearly show that kinetochores of lagging chromosomes in anaphase are connected to microtubules coming from both poles (merotelic orientation) and that the CREST stained region is stretched compared to the kinetochores correctly localized to the spindle poles.

Figure 11. Merotelic kinetochore orientation in a prometaphase cell observed 15 min after release from a nocodazole-induced mitotic block and possessing a merotelically oriented kinetochore (arrow). The figure shows that merotelic kinetochore orientation occurs by prometaphase on chromosomes near the spindle equator.