Changes in nuclear morphology and size in mammalian temperature-sensitive mutants
2026 年 51 巻 1 号 p. 191-201
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2026 年 51 巻 1 号 p. 191-201
Nuclear morphology alters during development and disease. Nuclear size and shape are regulated through several mechanisms. In this study, details of phenotypes of temperature-sensitive mutants, which were isolated from Chinese hamster CHO-K1 cells to identify genes responsible for the maintenance of chromosome integrity, were characterized with particular focus on changes in their nuclear size and shape. DNA replication has been implicated in a mutant exhibiting elongation of the nucleus with an increase in its ellipticity during incubation at the nonpermissive temperature of 39°C. Incubation at this temperature also resulted in nuclear enlargement in other mutants accompanied by increased DNA damage and led to a remarkable increase in cells harboring an abnormal nucleus, particularly multiple nuclei or segmented nuclei. These findings may lead to the discovery of a novel mechanism that regulates nuclear size and shape. Identification of genes responsible for these defects is highly desirable.
Key words: 53BP1, γH2AX, mammalian, replication, temperature-sensitive mutant
Graphical Abstract
Nuclear morphology alters during development and disease. Nuclear size and shape vary across cell types and developmental stages (Lele et al., 2018; Kalukula et al., 2022), and nuclear volume changes during cell cycle progression from G1 to G2 phase (Iida et al., 2022). Enlarged nuclei are a hallmark of malignancy in cancer cells (Fischer, 2020). Diseases such as Hutchinson-Gilford progeria syndrome result from mutations in lamin genes, which cause nuclear deformation and premature aging (Schibler et al., 2023). Nuclear size and shape are regulated through several mechanisms. The nuclear envelope is composed of inner and outer membranes, embedded with nuclear pore complexes, and supported by the nuclear lamina (lamins A/C, B1, B2). Linker of nucleoskeleton and cytoskeleton complex connects the cytoskeleton to the nucleus, transmitting mechanical forces and influencing nuclear positioning and shape (Jahed et al., 2021). Euchromatin and heterochromatin distribution affects nuclear rigidity and shape (Schibler et al., 2023). Nucleocytoplasmic transport regulates nuclear size by controlling protein and RNA flux, which refers to the continuous and regulated movement of RNA molecules, especially messenger RNA (Mukherjee et al., 2016). Lamin A interacts with histone H3, and mutations in lamin genes disrupt nuclear morphology (Schibler et al., 2023). Histone modifications (e.g., H3K27 methylation) also influence nuclear size and shape (Mukherjee et al., 2016).
To identify genes responsible for the maintenance of chromosome integrity, 25 temperature-sensitive (ts) mutants were isolated from mutagenized CHO-K1 cells, and 13 complementation groups were identified. Nine of these groups exhibited chromosomal instability at a nonpermissive temperature. Based on the induction patterns of sister chromatid exchanges and chromosomal aberrations, the mutants were classified into three categories. Flow cytometric analysis revealed that many chromosomally unstable mutants were arrested in the S or S/G2 phases with reduced DNA synthesis at the nonpermissive temperature, suggesting that defects in DNA replication contribute to the induction of chromosomal abnormalities (Tsuji et al., 1990). The present author isolated and characterized responsible genes from three of these ts mutant cells: tsTM4, tsTM18, and tsTM3. The results revealed that chromosome instabilities were caused by ts defects in the following three molecules: Rpb1, the largest subunit of RNA polymerase II; Smu1, a protein involved in splicing; and Uba1, the ubiquitin-activating enzyme (see reviews by Sugaya, 2018, 2019). The author then tried to isolate the responsible gene from other mutants using genetical complementarity. However, numerous analyses with these other mutants did not progress, and a responsible gene remains unknown. In the course of examining the association between chromosomal instability and epigenetics in these ts mutants, the author found mutants showing characteristic changes in nuclear size and morphology (Fig. 1A). In the present study, the author therefore reconsidered the research direction to characterize details of phenotypes of the mutant cells and performed experiments that focus particularly on changes in their nuclear size and shape.
Changes in size and shape of nuclei of temperature-sensitive (ts) mutants
(A) Images of fluorescently labeled nuclei of CHO-K1, tsTM19, and tsTM20 at 34°C and at 39°C. DNA was stained with Hoechst 33342. After 10-h incubation at 39°C, changes in the size and shape of the nuclei were found in the ts mutants. (B) Effect of incubation at 39°C on the size of the nucleus. Pixel size in the equatorial section of the nuclei, like those in the images in (A), was analyzed and is expressed as a box-and-whisker plot. Asterisks indicate statistically significant differences from 34°C (*P<0.01, **P<0.001) by Mann-Whitney U test. Increases in the size of the nuclei were found in all cell lines, especially in tsTM20. (C) Effect of incubation at 39°C on the shape of the nucleus. Major and minor axes in the equatorial section of the nuclei, like those in the images in (A), were calculated using ellipse fitting and are represented as a box-and-whisker plot of ellipticity (major/minor axis) in each cell line. Asterisks indicate statistically significant differences from 34°C (**P<0.001) by Mann-Whitney U test. Significant elongation in the shape of the nuclei was found in CHO-K1, and especially in tsTM19. In contrast, the ellipticity of tsTM20 decreased to become more rounded in shape. The numbers of images and nuclei analyzed for this figure are listed as sample sizes in Table S1. (D) Summary of the changes in size and shape of nuclei of the ts mutants. Degrees of changes in size and ellipticity of each cell line in (B) and (C) are represented by the averages in each cell line with standard deviations. Asterisks indicate statistically significant differences from CHO-K1 (the ** in red font: P<0.001) by Mann-Whitney U test. Blue and orange ellipses represent schematic and relative views of the nucleus of each cell line at 34°C and after 10-h incubation at 39°C, respectively, in comparison to that of CHO-K1 at 34°C.
The Chinese hamster cell line CHO-K1 and its ts mutant cell lines, tsTM19, and tsTM20 (Tsuji et al., 1990), were grown on glass coverslips placed in culture dishes in Ham’s F-12 medium (Wako, Osaka, Japan) containing 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (Gibco/Invitrogen, Carlsbad, CA, USA) at 34°C. For analysis of the ts phenotype, cells were shifted up to the nonpermissive temperature (39°C) by transferring culture dishes between incubators. Five frozen stocks in each cell line, whose passage number was less than five after receiving those cells from Dr. Tsuji, were used in present study. Temperature sensitivity was validated by checking viability at 39°C in every stock.
Indirect immunolabeling and microscopyProcedures for the indirect immunolabeling and microscopy of immunolabeled cells were described previously (Hongo et al., 2008). The incorporation of the halogenated nucleoside analogue was carried out by changing medium pre-warmed at the target temperature. Primary antibodies used in this study were mouse anti-BrdU (clone MD5110, 1:800; Caltag, Burlingame, CA, USA), rat anti-BrdU (clone BU1/75, 1:200; Serotec, Puchheim, Germany), mouse anti-α-tubulin (clone DM1A, 1:500; Sigma-Aldrich), mouse anti-gamma H2AX (γH2AX) antibody (clone 3F2, 1:1000; Novus Biologicals, Littleton, CO, USA), and mouse anti-53BP1 antibody (clone 6B3E10, 1:1000; Novus Biologicals). Most images were collected with an Olympus DP73 digital charge-coupled device camera fitted on an Olympus IX73 microscope (Olympus, Tokyo, Japan), except for images of CHO-K1, tsTM19, and tsTM20 in Fig. 1, which were collected with an Olympus DP30BW digital charge-coupled device camera fitted on an Olympus IX71 microscope. Analysis of the size and shape of fluorescently labeled nuclei and the number thereof was performed with ImageJ 1.42q (http://rsb.info.nih.gov/ij/).
Statistical analysisAll values are shown as the mean ± standard deviation (SD). The statistical significance of the differences in size and shape of the nuclei and the formation of foci of BrdU, γH2AX, and 53BP1 was determined with a Mann-Whitney U test. A P value <0.01 was considered to indicate significance.
As described in the Introduction, this study stemmed from comparative nuclear observations of 13 ts mutants and their parental CHO-K1 cells, which revealed mutants with distinctive nuclear size and morphological changes at restrictive temperatures. The effect of 10-h incubation at the restrictive temperature, 39°C, on the nuclear size and shape of mutant cells was investigated with the use of Hoechst 33342 staining (Fig. 1A). In this study, the equatorial plane image of the stained nucleus in interphase obtained by microscopy was evaluated as representing nuclear size and shape, even though the nucleus was three-dimensional. Incubation at 39°C led to changes in nuclear size and shape even in the parental CHO-K1 cells, which became bigger (Fig. 1B) and slightly longer (Fig. 1C). The nuclear size and nuclear ellipticity of CHO-K1 at 34°C and at 39°C were 116 ± 10 μm2 and 133 ± 15 μm2 and 1.44 ± 0.04 and 1.54 ± 0.07, respectively. Remarkable changes were also found in tsTM19 and tsTM20 cells. Using the nuclear size and ellipticity of the parental CHO-K1 cells cultured at 34°C as a reference, changes in nuclear size and ellipticity in each cell line after 10 h of incubation at 39°C were calculated and are schematically illustrated in Fig. 1D. Incubation of tsTM19 cells at 39°C resulted in the formation of an elongated nucleus, which was 1.17 times longer than that at 34°C (Fig. 1C, D). The nuclear size and nuclear ellipticity of tsTM19 at 34°C and at 39°C were 133 ± 12 μm2 and 144 ± 18 μm2 and 1.60 ± 0.06 and 1.88 ± 0.12, respectively. In contrast, the ellipticity of the nucleus of tsTM20 cells decreased to close to a circular shape, and its size became significantly bigger than that at 34°C (Fig. 1B–D). The nuclear size and nuclear ellipticity of tsTM20 at 34°C and at 39°C were 133 ± 10 μm2 and 186 ± 21 μm2 and 1.38 ± 0.06 and 1.34 ± 0.04, respectively. Successfully quantifying and validating these dramatic microscopic changes provided a strong sense of accomplishment and served as motivation to explore their underlying causes. The present study thus focused on understanding the mechanism underlying the changes in nuclear size and shape of these two ts mutants, tsTM19 and tsTM20. Their characteristic nuclei were designated as “elongated ellipse” for tsTM19 and “big and round” for tsTM20.
Effect of 24-hour incubation at the nonpermissive temperatureNext, the effect of much longer incubation at 39°C was examined for the two mutants. At 39°C for 10 h, tsTM19 and tsTM20 cells and other ts mutants showed no notable nuclear changes, except for the alterations in nuclear size and ellipticity in tsTM19 and tsTM20. After 24 h of incubation at the non-permissive temperature, it became difficult to assess nuclear size and morphology, as shown in Fig. 1, due to the frequent appearance of cells with abnormal nuclei such as multinucleated cells in tsTM20 cells. Therefore, we evaluated the appearance of cells with abnormal nuclei, and their frequency is shown in Fig. 2. The CHO-K1 parental cells showed existence of cells harboring an abnormal nucleus, which appeared as cells having a bump or micronucleus, even at 34°C (Fig. 2B). The frequency of cells having a bump or micronucleus in CHO-K1 cells was 6.3 ± 3.5% at 34°C and 9.3 ± 5.6% at 39°C, respectively. The frequency of CHO-K1 cells displaying aberrant nuclear morphologies, multiple nuclei or segmented nuclei, was exceedingly low, remaining below 1% at both 34°C and 39°C (Fig. 2C). In the case of tsTM19 cells, the frequency of cells having a bump or micronucleus was lower than that observed in the parental CHO-K1 cells, being 2.0 ± 2.5% at 34°C and increasing to 5.0 ± 3.9% after 24 h at 39°C (Fig. 2B). The frequency of tsTM19 cells exhibiting abnormal nuclear morphologies, including multiple nuclei or segmented nuclei, was likewise very low, remaining at approximately 1% at both 34°C and 39°C (Fig. 2C), though cells harboring an elongated nucleus increased (Fig. 2A). The most significant changes were found in tsTM20 cells (Fig. 2A). The frequency of tsTM20 cells having a bump or micronucleus was comparable to that of the parental CHO-K1 cells at 34°C (6.9 ± 4.0%), but it markedly increased to 22.1 ± 7.9% after 24 h at 39°C (Fig. 2B). The frequency of tsTM20 cells exhibiting abnormal nuclear morphologies, including multiple nuclei or segmented nuclei, increased even more substantially, remaining similar to the parental CHO-K1 cells at 34°C (1.2 ± 1.4%) but reaching 28.8 ± 7.9% after 24 h at 39°C (Fig. 2C).
Effect of 24-h incubation at 39°C
(A) Cells were fixed, and α-tubulin was indirectly immunolabeled with Alexa 594. DNA was counterstained with Hoechst 33342. The merged views, which are composed of α-tubulin (red channel) and fluorescently labeled nuclei (blue channel), represent upper rows of 34°C and 24-h incubation at 39°C (39°C*24 h), respectively. Bottom rows represent only fluorescently labeled nuclei of each cell line. Incubation at 39°C for 24 h led to the appearance of abnormal nuclei, such as multiple nuclei (blue arrowheads), micronuclei (green arrowheads), and nuclei with a bump (green arrowheads). (B, C) Frequencies of the appearance of abnormal (B: e.g., bump, micro; C: e.g., multiple) nuclei. The numbers of several kinds of abnormal nuclei like those in the images in (A) were counted and are represented as box-and-whisker plots. Asterisks indicate statistically significant differences from 34°C (*P<0.01, **P<0.001) by Mann-Whitney U test. Significant increases in abnormal nuclei were found especially in tsTM20. The numbers of images and nuclei analyzed for this figure are listed as sample sizes in Table S2.
To investigate the cause of the appearance of the cells harboring an abnormal nucleus, indirect immune fluorescence of γH2AX was conducted in the tsTM19, and tsTM20 cells, and their parental cell line, CHO-K1. In the analysis by Tsuji et al. (1990), tsTM19 and tsTM20 were characterized phenotypically by a high frequency of sister chromatid exchanges and chromosomal aberrations. Analysis of DNA damage is also considered to be effective for investigating the high frequency of sister chromatid exchanges and chromosomal aberrations. Phosphorylation of the Ser-139 residue of the histone variant H2AX, γH2AX, is an early cellular response to DNA damage (Mah et al., 2010). The most significant changes were found in tsTM20 cells (Fig. 3A). To characterize foci of γH2AX in detail, they were classified into four types: dark or few (Fig. 3B), not bright and a few (Fig. 3C), bright or many (Fig. 3D), and bright and many (Fig. 3E), and as a combination of two types: negative (–: Fig. 3B, C) and positive (+: Fig. 3D, E). Both the ts mutants and parental cells showed some endogenous damage at 34°C (Fig. 3F). These relatively high values, around 50%, may be attributable to the classification criteria used in this study. Nevertheless, incubation at 39°C resulted in a remarkable increase in positive cells of tsTM20 in focus formation of γH2AX (Fig. 3F). The frequency of γH2AX-positive tsTM20 cells reached 91.4 ± 6.2% and 98.5 ± 8.1% after 8 h and 24 h at 39°C, respectively. The composition of the foci of γH2AX of the CHO-K1 and tsTM19 cells appeared to be similar at 34°C and after incubation at 39°C (Fig. 3F). Although the analysis was performed at 8 h rather than 10 h under the restrictive temperature, the 8-h incubation is considered to be sufficiently appropriate for examining the processes underlying changes in nuclear size and morphology.
Effect of incubation at 39°C on the focus formation of γH2AX
Indirect immune fluorescence of γH2AX. (A) An image of γH2AX-labelled nuclei of tsTM20 after 24-h incubation at 39°C. Scale bar, 20 μm. The majority of γH2AX labelling appeared to be classified as many bright foci in an abnormal nucleus. (B–E) γH2AX-labelled nuclei were classified into four types: (B) dark or few, (C) not bright and a few, (D) bright or many, and (E) bright and many. Scale bar, 10 μm. (F) Effect of incubation at 39°C on the focus formation of γH2AX. γH2AX-labelled nuclei were classified as “positive (+)”, like those in (D) and (E), and are expressed as box-and-whisker plots. Asterisks indicate statistically significant differences from 34°C (**P<0.001) by Mann-Whitney U test. Significant changes in focus formation of γH2AX were found especially in tsTM20 at 39°C*8 h and at 39°C*24 h. The numbers of images and nuclei analyzed for this figure are listed as sample sizes in Table S3.
Following the result of indirect immunostaining of γH2AX, the focus formation of 53BP1 (P53 binding protein 1) was investigated to check the DNA damage-response mechanism. 53BP1 is a key protein involved in the DNA damage response, particularly in the repair of double-strand breaks (Zimmermann and de Lange, 2014). Again, the most significant changes were found in tsTM20 cells (Fig. 4A). As with γH2AX, the focus formation of 53BP1 was classified into two types: more than 10 foci (+10: Fig. 4B, C) or a few foci (+1: Fig. 4D, E). Both the ts mutants and parental cells included a few cells indicating a focus of 53BP1 even at 34°C (Fig. 3F). The frequency of 53BP1-positive cells for CHO-K1, tsTM19, and tsTM20 was 0.8 ± 1.2%, 2.1 ± 6.0%, and 1.3 ± 2.0%, respectively (Fig. 4F). Incubation at 39°C led to a slight increase in cells having a few foci of 53BP1 in CHO-K1 cells after 8 h (3.7 ± 4.6%), which was restored after 24 h (0.2 ± 0.6%), presumably due to the existence of a functional repair system (Fig. 4F). In tsTM19 cells, gradual and significant increases in cells having a few foci of 53BP1 were found (Fig. 4F). During incubation at 39°C, the proportion of 53BP1-positive tsTM19 cells was 3.0 ± 3.3% after 8 h, but it increased significantly, reaching 6.6 ± 6.1% after 24 h. The 24-h incubation at 39°C resulted in a remarkable increase in 53BP1-positive tsTM20 cells (Fig. 4F). During incubation at 39°C, the frequency of 53BP1-positive tsTM20 cells was 4.3 ± 3.4% after 8 h but increased substantially, reaching 48.4 ± 12.7% after 24 h. This result appears to be in reasonable agreement with the remarkable increase in tsTM20 cells positive for the focus formation of γH2AX (Fig. 3F).
Effect of incubation at 39°C on the focus formation of 53BP1
Indirect immune fluorescence of 53BP1. (A) An image of 53BP1-labelled nuclei of tsTM20 after 24-h incubation at 39°C. Scale bar, 20 μm. The majority of 53BP1 labelling appeared to be classified as many bright foci in an abnormal nucleus. (B–E) 53BP1-labelled nuclei were classified into two types in regard to focus formation: more than 10 foci (+10), e.g., (B) and (C), or a few foci (+1), e.g., (D) and (E). Scale bar, 10 μm. (F) Effect of incubation at 39°C on the focus formation of 53BP1. 53BP1-labelled nuclei were classified as “+10”, like those in (B) and (C) and are expressed as box-and-whisker plots. Asterisks indicate statistically significant differences from 34°C (*P<0.01, **P<0.001) by Mann-Whitney U test. Significant changes in the focus formation of 53BP1 were found especially in tsTM20 at 39°C*24 h. The numbers of images and nuclei analyzed for this figure are listed as sample sizes in Table S4.
The effect of incubation at 39°C on DNA replication was investigated by the incorporation of the halogenated nucleoside analogue bromodeoxyuridine (BrdU), as previous study by Tsuji and colleagues (1990) showed that both the tsTM19 and tsTM20 mutant strains exhibit defects in DNA synthesis under restrictive temperatures and consequently arrest the cell cycle in the S phase. After indirect immune staining with anti-BrdU antibody, cells in the S phase of the cell cycle showed various types of staining, as can be seen in Fig. 5A, B, which is known as the replication pattern (see Cook, 2001 for a book offering a comprehensive review). DNA replication starts from euchromatin regions and moves into heterochromatin regions, which corresponds to the sequential images shown in Fig. 5C–F. That is, the nucleus contains numerous tiny foci at the beginning of replication (Fig. 5C), and the foci spread brightly and homogeneously through the entire nucleus (Fig. 5D). Then, replication sites spread in the heterochromatin regions such as the peripheries of the nuclear membrane and nucleoli (Fig. 5E, F), with the foci becoming bigger heterogeneously. In this study, BrdU-labelled nuclei, like those in the images in Fig. 5A, B, were classified into homogeneous labeling as “early”, like those in panels C and D, and heterogeneous as “late”, like those in panels E and F.
Changes in DNA replication at 39°C
(A) An image of nuclei of tsTM19 at 34°C, which were fluorescently labeled by BrdU indirectly. Scale bar, 20 μm. Many types of BrdU-labelled nuclei were observed as depicted in (C–F). (B) Image of BrdU-labelled nuclei of tsTM19 after a 24-h incubation period at 39°C. Scale bar, 20 μm. The majority of BrdU labelling appeared to be classified as many tiny foci in an elongated nucleus. (C–F) Four typical types of DNA replication pattern are listed in order from early to late. Scale bar, 10 μm. Images of nuclei of CHO-K1 (C, D, F) and tsTM19 (E) at 34°C, which were fluorescently labeled by BrdU indirectly. (G) Effect of incubation at 39°C on replication. BrdU-labelled nuclei are counted and expressed as box-and-whisker plots. (H, I) Nuclei like those in the images in (A) were classified into homogeneous labeling as “early”, like those in (C) and (D), and heterogeneous labeling as “late”, like those in (E) and (F), and are expressed as box-and-whisker plots: H, early and I, late, respectively. Black and red asterisks indicate statistically significant differences by Mann-Whitney U test from 34°C (*P<0.01, **P<0.001) and from CHO-K1 (the * in red font: P<0.01, the ** in red font: P<0.001), respectively. The numbers of images and nuclei analyzed for this figure are listed as sample sizes in Table S5.
In the case of parental CHO-K1 cells, incubation at 39°C led to an increase in the composition of S-phase cells after 8 h and a decrease after 24 h (Fig. 5G), especially in early fractions of the S phase (Fig. 5H). After 8 h of incubation at 39°C, the proportion of CHO-K1 cells in S phase and especially those in the early fraction increased to over 60% (62.6 ± 15.2) and 40% (42.5 ± 13.0), respectively. This may be associated with the increase in nuclear size and the higher nuclear ellipticity observed in the CHO-K1 cells (Fig. 1). As a result of 24 h of incubation at 39°C, the proportion of CHO K1 cells in S phase, particularly those in the early fraction, decreased to 20% (23.4 ± 8.6) and 10% (8.8 ± 3.3), respectively. This decrease is likely attributable to progression through the cell cycle.
At the permissive temperature of 34°C, tsTM19 cells displayed a significantly increased S-phase population compared with the parental CHO-K1 line, with both the early and late S-phase subfractions being markedly enriched. The proportions of tsTM19 cells in the S phase at 34°C and those in the early fraction and late fraction were 67.7 ± 11.5%, 39.4 ± 9.5%, and 28.4 ± 8.2%, respectively (Fig. 5G–I). After 8 h of incubation at 39°C, the proportions of tsTM19 cells in S phase and in the early and late S-phase fractions showed no change from those at 34°C, remaining high at 68.1 ± 13.3%, 42.4 ± 11.1%, and 25.7 ± 11.7%, respectively (Fig. 5G–I). The tsTM19 cells exhibited higher ratios of cells in the S phase than in the CHO-K1 cells, particularly in the early fraction of the S phase after 24-h incubation, though those in the late fraction decreased (Fig. 5B, G–I). The proportions of tsTM19 cells in the S phase after 24 h of incubation at 39°C and those in the early fraction and late fraction were 54.5 ± 12.4%, 41.3 ± 14.1%, and 13.3 ± 7.3%, respectively (Fig. 5G–I). The large number of S-phase cells, especially in the early fraction, was remarkable and suggested an increased number of cells entering into and stopping at the S phase (Fig. 5G, H).
In the case of tsTM20 cells, after an 8-h incubation at 39°C, the proportion of cells in the early S-phase fraction was significantly lower than that of the CHO-K1 cells, whereas the late S-phase fraction was significantly increased compared with that at 34°C (Fig. 5H, I). The proportions of tsTM20 cells after 8 h of incubation at 39°C in the early and late fractions of S phase were 27.3 ± 10.9% and 26.0 ± 9.2%, respectively (Fig. 5H, I).
Tsuji et al. (1990) reported that tsTM19 cells fail to properly regulate the cell cycle, arresting at the S phase with a decrease in DNA synthesis activity. The relatively high frequency of the early fraction of the S phase in the tsTM19 cells at 39°C remains at a high level of around 40%. Although the present study did not detect a reduction in DNA synthesis activity, these results are consistent with the findings reported by Tsuji et al., reinforcing the notion that alterations in S-phase dynamics underlie the observed cellular phenotypes. In particular, our data indicate that an expansion of the early S-phase fraction contributes materially to the overall increase in S-phase cells. This shift in S-phase distribution is compatible with scenarios involving accelerated entry into the S phase and/or delayed progression within the early S, either of which would increase the representation of S-phase cells at the population level. Though our current dataset does not disentangle these possibilities, the concordance with prior work strengthens the interpretation that modulation of early S-phase dynamics is a key driver of the phenotype observed here. Cdt1 is a chromatin licensing factor, and its overexpression loads excess amounts of minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication to allow origin over-licensing, which results in re-replication (Li and Blow, 2005). To test the possibility of re-replication underlying the nuclear elongation of the tsTM19 cells, the expression and localization of Cdt1 were investigated by indirect immunostaining using anti-Cdt1 antibody. However, no temperature effect was found in the tsTM19 cells or the other ts mutants. Similar and negative results were obtained from the investigation of MCM7, which is a core component of the hexameric MCM helicase complex. It appears to be obvious that DNA replication is likely closely related to elongation of the nucleus of tsTM19 cells during incubation at 39°C, but details of the mechanism remain unidentified. Further investigation, especially isolation of the gene responsible for the ts defect of tsTM19 cells, is needed.
Increase of DNA damage in tsTM20 cellsIn tsTM20 cells, incubation at 39°C resulted in increased numbers of cells showing enlargement of the nucleus, which became almost circular in shape, after 10 h and in an increase of cells harboring multiple or segmented nuclei after 24 h. It is reasonable that the nuclei of tsTM20 cells become enlarged at 39°C, as tsTM20 cells fail to properly regulate cell-cycle progression and consequently arrest in the S phase (Tsuji et al., 1990). Moreover, it is known that nuclear size increases during the transition from S phase to G2 phase (Iida et al., 2022). The pronounced increase in γH2AX and 53BP1 focus formation observed in tsTM20 cells strongly suggests a substantial rise in DNA damage levels. However, interpreting the absolute number of DNA damage foci requires careful consideration of cell-cycle dynamics. Because genomic DNA content increases progressively from S phase through G2 phase, variations in cell-cycle distribution can considerably influence the apparent burden of DNA lesions. This is particularly relevant in contexts in which cell-cycle progression is perturbed, as even identical levels of damage per unit DNA may manifest as different focus counts depending on DNA content. Thus, the interplay between DNA damage signaling and cell-cycle–dependent changes in chromatin organization must be taken into account when evaluating the degree of genomic instability. Moreover, DNA damage—especially double-strand breaks—is known to induce chromatin decondensation, which in turn alters nuclear mechanics. Such chromatin relaxation reduces nuclear stiffness and promotes nuclear swelling, as demonstrated by Dos Santos et al. (2021). These biophysical changes may further exacerbate the phenotypic consequences of accumulated DNA lesions, potentially contributing to abnormal nuclear morphology and impaired cellular homeostasis. Collectively, our findings underscore the importance of integrating DNA damage markers, cell-cycle status, and chromatin dynamics when assessing the molecular basis of nuclear abnormalities in tsTM20 cells. Decondensed chromatin facilitates access for repair proteins. The results obtained from the tsTM20 cells appeared to be compatible with this previous report. DNA damage also causes nuclear blebbing and rupture, especially when chromatin-based stiffness is compromised (Eskndir et al., 2025). This nuclear blebbing and rupture appear to be similar to the abnormal nuclei with bumps and multiple or segmented nuclei found in the present study. Live-cell imaging-based analysis is considered to be an effective approach for elucidating the processes of abnormal nuclear formation in tsTM20 cells cultured under restrictive temperature conditions, including the transition from nuclear enlargement to multiple or segmented nuclei.
Molecules involved in the regulation of nuclear size and shapeTo investigate the chromatin state of the ts mutants, indirect immunostaining with antibodies raised against 13 histone modifications was carried out (e.g., anti-H3K9ac, anti-H3K27ac, anti-H3K9me3, anti-H4K20me1, anti-H4K20me2, and anti-H4K20me3). However, incubation at 39°C yielded no effect on the expression and distribution of any histone modification in the ts mutants or parental cells. As described in the Introduction, factors associated with the nuclear envelope are known to play critical roles in regulating nuclear size and morphology. Similar negative results were obtained by indirect immunostaining with antibodies raised against Lamin B1, Lamin A/C, and NUP98. Regarding the ts mutants analyzed in this study, the mechanisms and molecules governing nuclear size and morphology cannot be thoroughly investigated with our current experimental equipment and available antibody repertoire. Thus, the gene-identification approaches outlined in the subsequent section are likely to be the most effective means of elucidating the underlying responsible genes.
Isolation of responsible genesAs mentioned in my previous paper (Sugaya, 2018), one big issue in the study of ts mutants that requires resolution is the isolation of the genes responsible for ts defects. Again, target capture sequencing such as whole exome sequencing is an efficient method of directly identifying the causative mutations of genetic disorders. Whole exome sequencing is primarily being applied to human genetic disorders (Hodges et al., 2007; Ng et al., 2010; Rabbani et al., 2014). The whole-genome sequence of CHO cells has already been determined, and the feasibility of designing probes for exome analysis has been established (Lewis et al., 2013). By referring to this genomic information, it should be possible to identify mutations by RNA sequencing analysis, albeit limited to genes that are actively expressed. Of course, if sufficient research funding and analytical resources are available, mutations could be identified by determining the whole-genome sequence of the mutant cells. At present, considering both reliability and a realistically achievable research scale, exome analysis is regarded as the most promising approach. To apply this strategy of isolating the responsible gene from ts mutants of CHO-K1 cells, the author plans to develop a novel set of target capture probes that can identify mutations in exonic regions. Further study, as well as obtaining funding to design a customized capture kit for the desired target sequences in CHO-K1 cells, is necessary and is presently still under way.
This work was supported by a donation from QST Mirai Kikin [zi82].
Conflict of Interest StatementThe author declares no competing interests.
Data Availability StatementAll data generated or analyzed during this study are included in this published article and its supplementary information. All data are available from the corresponding author upon reasonable request.
Author Contribution StatementConceptualization, Funding acquisition, Investigation, Supervision, Visualization, Writing – original draft, Writing – review & editing: KS
Ethics Approval and Consent to ParticipateEthics approval is not required in this study.
Patient Consent for PublicationNot applicable.
The author acknowledges Mrs. Kaomi Higuchi for her technical assistance.
