Mitosis is a crucial stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe demonstrates catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from, for example, mutations in the acetyl-CoA/biotin carboxylase (cut6), fatty acid synthase (fas2, also known as lsd1) or transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It has been previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride supplementation. In this study, we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity.

Mitosis is a crucial stage in the cell cycle of any eukaryotic cell. Irregularities within this process can have far-reaching consequences, such as aneuploidy, mutations and/or cell death. These, in turn, can result in uncontrolled proliferation and tumour formation in metazoans (Saeki et al., 2009), or cause decreased fitness in populations of unicellular organisms, such as yeasts (Hayles et al., 2013). Because of its significance, the entry into mitosis and progression through mitotic phases are tightly regulated by a network of redundant regulatory pathways, creating a fail-safe mechanism of control (Bähler, 2005). This network also integrates numerous internal and external factors that influence the decisions to proceed to the next phase. In yeasts, cell cycle regulation is strongly affected by the availability of nutrients, such as carbon and nitrogen (Fantes and Nurse, 1977).

In contrast to the open mitosis of higher eukaryotes, many unicellular species undergo a closed mitosis. The nuclear envelope (NE) remains intact during the whole process of a closed mitosis, even though substantial NE remodelling is required (Boettcher and Barral, 2013). For example, in the fission yeast Schizosaccharomyces pombe, the NE surface area needs to expand by 33% during mitosis to properly accommodate the elongating spindle and segregating chromosomes (Yam et al., 2011). Notably, a number of abnormal mitotic phenotypes associated with closed mitosis have been described. These include ‘cut’ (for ‘cell untimely torn’), a form of mitotic catastrophe where cytokinesis takes place before nuclear division has been properly resolved and where the mother nucleus gets transected by and trapped in the forming septum (Fig. 1A) (Uemura and Yanagida, 1984; Hirano et al., 1986), ‘lsd’ (for ‘large and small daughters’), a segregation error where the resulting daughter nuclei are of unequal sizes (Saitoh et al., 1996); and nucleus displacement, which is similar to ‘cut’, but is where the forming septum misses the nucleus, resulting in one of the daughter cell being anucleate and the other one containing a diploid nucleus (Yukawa et al., 2021). Typically, such events are lethal for at least one of the daughter cells. The ‘cut’ phenotype has been described for mutations in a range of mitosis-related genes, including the separase (cut1) and securin (cut2) (Uzawa et al., 1990), condensin (cut3) (Saka et al., 1994), anaphase-promoting complex (APC/C; cut4, cut9) (Yanagida et al., 1999) and others (Yanagida, 1998).

Fig. 1.

Addition of ammonium or glutamate suppresses mitotic defects associated with perturbed lipid metabolism. Cells were grown to an exponential phase in the indicated media, fixed, stained with DAPI and subjected to microscopy. (A) Examples of WT and Δcbf11 cell populations. An overlay of DAPI and DIC channels is shown; catastrophic mitoses are marked with stars. (B,C) Mitotic defects in Δcbf11 cells are partially suppressed by ammonium supplementation at all concentrations tested. (D,E) Glutamate can also partially suppress mitotic defects in Δcbf11 cells, but it is less potent than ammonium. Poor nitrogen sources, proline and uracil, do not suppress mitotic defects (uracil was used in 2× lower concentration because the molecule contains two atoms of nitrogen). (F) Ammonium can also partially suppress mitotic defects triggered by cerulenin treatment. Mean±s.d from three or four independent experiments are shown in B–F (exact n indicated by individual data points). To determine statistical significance a one-way ANOVA followed by a Dunnett's statistical test was applied for the multiple comparison with a control (B–E), or a one-sided unpaired Student's t-test was applied for the two-sample comparison (F).

Fig. 1.

Addition of ammonium or glutamate suppresses mitotic defects associated with perturbed lipid metabolism. Cells were grown to an exponential phase in the indicated media, fixed, stained with DAPI and subjected to microscopy. (A) Examples of WT and Δcbf11 cell populations. An overlay of DAPI and DIC channels is shown; catastrophic mitoses are marked with stars. (B,C) Mitotic defects in Δcbf11 cells are partially suppressed by ammonium supplementation at all concentrations tested. (D,E) Glutamate can also partially suppress mitotic defects in Δcbf11 cells, but it is less potent than ammonium. Poor nitrogen sources, proline and uracil, do not suppress mitotic defects (uracil was used in 2× lower concentration because the molecule contains two atoms of nitrogen). (F) Ammonium can also partially suppress mitotic defects triggered by cerulenin treatment. Mean±s.d from three or four independent experiments are shown in B–F (exact n indicated by individual data points). To determine statistical significance a one-way ANOVA followed by a Dunnett's statistical test was applied for the multiple comparison with a control (B–E), or a one-sided unpaired Student's t-test was applied for the two-sample comparison (F).

Interestingly, perturbations of lipid metabolism can also cause mitotic catastrophe in S. pombe (Zach and Převorovský, 2018). It has been shown that mutations in acetyl-CoA/biotin carboxylase (cut6), fatty acid (FA) synthase (fas2, also known as lsd1) (Saitoh et al., 1996) and the CSL (an acronym for CBF-1 and RBPJ-κ in Homo sapiens and Mus musculus, respectively, Suppressor of Hairless in Drosophila melanogaster, Lag-1 in Caenorhabditis elegans) transcriptional regulator of lipid metabolism (cbf11) genes (Převorovský et al., 2015), as well as treatment with the FA synthesis inhibitors cerulenin (Saitoh et al., 1996) or Cutin-1 (Takemoto et al., 2016), lead to the ‘cut’ and/or ‘lsd’ phenotypes. It was assumed that decreased supply of precursors of membrane phospholipids (PL) leads to insufficient NE expansion during anaphase and mitotic failure (Takemoto et al., 2016). However, we recently found additional factors contributing to decreased mitotic fidelity in cells with perturbed lipid metabolism, as cbf11 and cut6 mutants show altered cohesin dynamics and H3K9 modifications at the centromeric regions (Vishwanatha et al., 2023).

Nitrogen is an important macronutrient required for the synthesis of amino acids, nucleotides and many other biomolecules, and the availability of nitrogen has a profound effect on the timing of entry into mitosis (Petersen and Nurse, 2007). To coordinate growth and division with available resources, the cell employs several nutrient-responsive regulatory factors. In S. pombe, these include protein kinase A (PKA), the stress-response MAP kinase Sty1, and the target of rapamycin (TOR) kinases (Yanagida et al., 2011). Notably, similar mechanisms operate in mammalian cells as well (Davie et al., 2015). There are two TOR complexes (TORCs) in S. pombe, each containing a different TOR kinase paralog. TORC1 (containing Tor2 kinase) is a major regulator of nitrogen metabolism and stimulates growth and proliferation. By contrast, TORC2 (containing Tor1 kinase) is involved in stress responses and maintenance of genome integrity. Importantly, there is crosstalk within the TOR network and the two TORCs operate in an antagonistic manner (Ikai et al., 2011; Schonbrun et al., 2009). Moreover, apart from the quantity of available nitrogen, the exact chemical nature (quality) of a nitrogenous substance must be taken into account. Some, like ammonium and glutamate, are classified as ‘good’ sources that can be utilised easily and efficiently by the cells, whereas others, like proline, are ranked as ‘poor’ sources, despite of them all containing equal amounts of nitrogen atoms per molecule. However, this binary classification is somewhat arbitrary as in reality there is likely a ‘goodness’ continuum of nitrogen sources (Petersen and Russell, 2016; Carlson et al., 1999).

Intriguingly, we have previously demonstrated that the incidence of mitotic catastrophe in the cbf11 and cut6 lipid metabolism mutants is suppressed when cells are grown in the minimal defined Edinburgh minimal medium (EMM), which contains abundant ammonium chloride as the nitrogen source (Převorovský et al., 2015). A similar suppression effect was achieved by growing mutant cells in the complex (and relatively nitrogen-poor) YES medium supplemented with ammonium chloride (Zach et al., 2018). We now show that this improvement of mitotic fidelity is indeed nitrogen dependent and is related to the TOR network function. Unexpectedly, we found that nitrogen supplementation does not restore the expression of lipid metabolism genes or lipid composition in Δcbf11 cells, nor does it restore NE expansion during mitosis, indicating that the nature of the suppression is indirect. Moreover, we demonstrate that nitrogen supplementation can also suppress the effects of a range of other, non-lipid, ‘cut’ mutations, suggesting a more general effect of nitrogen on mitotic fidelity. Our results highlight a previously unappreciated role of nitrogen availability in successful progression through mitosis.

The mitotic defects of Δcbf11 cells can be suppressed by nitrogen in the form of ammonium and glutamate

We have shown previously that mitotic defects in fission yeast lipid metabolism ‘cut’ mutants (Fig. 1A) can be suppressed by the addition of ammonium chloride to culture media (Zach et al., 2018). As the first step towards understanding this phenomenon, we compared the frequency of mitotic catastrophe in Δcbf11 cells in the standard complex YES medium, and in minimal defined EMM supplemented with ammonium chloride or ammonium sulphate. We found that the magnitude of the suppressive effect was similar between the two ammonium salts (Fig. 1B), indicating that the previously observed suppression had been caused by the ammonium, not the chloride.

Different nitrogen-containing compounds can be utilised by the cell with varying ease and efficiency (‘good’ versus ‘poor’ nitrogen sources) (Petersen and Russell, 2016; Carlson et al., 1999). Therefore, we next tested several nitrogen-containing compounds for their ability to suppress mitotic defects in Δcbf11 cells. We found that ammonium, a prototypical good nitrogen source, had the most profound effect, suppressing mitotic defects even at reduced concentrations (Fig. 1C). Glutamate, another good nitrogen source, could also improve mitotic fidelity, although only at higher concentration (Fig. 1D). By contrast, the poor nitrogen sources proline and uracil did not suppress the mitotic defects of Δcbf11 cells at the concentrations tested (Fig. 1E). Thus, within the limited set of nitrogen sources we tested, the magnitude of the suppressive effect correlates with the preferability of a given compound as a nitrogen source to fission yeast cells (Petersen and Russell, 2016; Carlson et al., 1999).

Mitotic catastrophe can also be triggered by chemical inhibition of the fatty acid synthase (FAS). This is thought to be caused by insufficient supply of the membrane building blocks for NE expansion during the anaphase of closed mitosis (Yam et al., 2011; Takemoto et al., 2016). We found that the frequency of mitotic catastrophe in wild type (WT) cells treated with the FAS inhibitor cerulenin (Saitoh et al., 1996) could also be partially rescued by ammonium supplementation (Fig. 1F). This finding thus seems to be compatible with the hypothesis of membrane building block shortage being responsible for mitotic defects upon lipid metabolism disturbances (Yam et al., 2011; Takemoto et al., 2016).

Ammonium does not suppress perturbations of lipid metabolism

To investigate whether ammonium supplementation suppresses mitotic defects via stimulating production of new membranes, we performed measurements of NE expansion during mitosis using live-cell microscopy. We used a fluorescently tagged nuclear pore protein, Cut11–GFP, as an NE marker (Fig. 2A) (West et al., 1998). When comparing Δcbf11 and WT cells, we found that the mutant cells had a smaller NE cross-section, both at the start of mitosis (Fig. 2B) and at the moment of maximum NE expansion (Fig. 2C). Importantly, we did not observe any differences between Δcbf11 cultures grown with or without ammonium supplementation. This indicates that the nitrogen-dependent improvement of mitotic fidelity is not achieved by enhancing the anaphase NE expansion. Indeed, we have previously shown that ammonium supplementation does not correct the aberrant lipid droplet (LD) content (i.e. fewer or even no LDs) of Δcbf11 cells (Zach et al., 2018). Also, we demonstrated that the defects in Δcbf11 mitosis have already manifested before anaphase (Vishwanatha et al., 2023). Here, we also note that the addition of ammonium had an effect on NE cross-section in WT cells, making it significantly smaller at the end of anaphase (tmax) compared to that for WT cells grown in plain YES medium (Fig. 2C). However, the impact of this observation is unclear.

Fig. 2.

Small nuclear size in mitotic Δcbf11 cells is not suppressed by ammonium. WT and Δcbf11 cells expressing the Cut11–GFP marker of the NE were grown to exponential phase in the indicated medium and subjected to time-lapse microscopy. (A) Example of a dividing WT cell and the threshold mask used for area measurements is shown. The moment of mitotic spindle appearance as a bright dot (arrow) was set as the t0 point; the latest moment when the nucleus can still be seen as a single compartment was set as tmax. (B,C) The area of nuclear cross-section at the beginning (B) and end (C) of anaphase for 41–45 cells per condition is shown. Boxplots display medians (thick line), Q1 and Q3 quartiles (the box), and the smallest and largest values within a 1.5× interquartile range from the Q1 and Q3 boundaries, respectively (whiskers). To determine statistical significance one-way ANOVA was applied followed by simultaneous tests for general linear hypotheses.

Fig. 2.

Small nuclear size in mitotic Δcbf11 cells is not suppressed by ammonium. WT and Δcbf11 cells expressing the Cut11–GFP marker of the NE were grown to exponential phase in the indicated medium and subjected to time-lapse microscopy. (A) Example of a dividing WT cell and the threshold mask used for area measurements is shown. The moment of mitotic spindle appearance as a bright dot (arrow) was set as the t0 point; the latest moment when the nucleus can still be seen as a single compartment was set as tmax. (B,C) The area of nuclear cross-section at the beginning (B) and end (C) of anaphase for 41–45 cells per condition is shown. Boxplots display medians (thick line), Q1 and Q3 quartiles (the box), and the smallest and largest values within a 1.5× interquartile range from the Q1 and Q3 boundaries, respectively (whiskers). To determine statistical significance one-way ANOVA was applied followed by simultaneous tests for general linear hypotheses.

To corroborate our new findings, we performed an RNA-seq analysis of a panel of lipid-metabolism mutants to assess the effect of nitrogen on the transcriptome. Our panel included strains with mutations in the transcriptional regulators Cbf11 (Δcbf11 and cbf11DBM, a mutant with abolished binding to DNA; Princová et al., 2023) and Mga2 (Δmga2; Burr et al., 2016), and the FA synthesis rate-limiting enzyme Cut6 (Pcut6MUT, which shows a 50% reduction of cut6 transcript levels; Převorovský et al., 2016). As a control, we treated WT cells with the FA synthesis inhibitor cerulenin. Fig. 3 shows the expression profiles of a number of lipid metabolism genes regulated by Cbf11 and/or Mga2, which provide a useful readout on whether the absence of the two main regulators of non-sterol lipid metabolism genes has been compensated for or not. Genes measured include cut6, fas1 and fas2, the acyl-coA desaturase ole1, and the long-chain-fatty-acid-CoA ligases lcf1 and lcf2, all of which were downregulated in the Δcbf11, Δmga2 and Δmga2 Δcbf11 double mutant, as well as in the cbf11DBM strain (Fig. 3). This is in agreement with the previously published studies of Δcbf11 and Δmga2 transcriptomes (Převorovský et al., 2015; Burr et al., 2016). For the sake of completeness, we also note that the downregulation of fas2 in Δcbf11 and cbf11DBM cells observed in the current study was not deemed statistically significant in our recent work when measured by RT-qPCR (Marešová et al., 2024). In contrast to the regulator mutants, targeted inhibition of FA synthesis (Pcut6MUT, cerulenin treatment) resulted in modestly increased expression of lipid metabolism genes, likely as a feedback reaction to FA shortage (Fig. 3). Importantly, lipid gene expression was not restored in regulator mutants grown in a medium supplemented with ammonium chloride, except for fas1 and fas2 in cbf11DBM and, to a lesser extent, in Δmga2 cells (Fig. 3). This shows that the improvement of mitotic fidelity by ammonium is unlikely to be achieved by boosting the expression of lipid metabolism genes. Finally, we also note that the addition of ammonium to WT cells resulted in the downregulation of nitrogen compound transporters. This includes the amino acid transporters per1 and put4, which have been previously shown to be downregulated by ammonium (Kaufmann et al., 2010), as well as a number of other nitrogen compound transport genes, such as aat1, isp5 and others (GO:0071705; https://www.pombase.org/term/GO:0071705) (Fig. 3; Table S1). These results are in line with the known phenomenon of amino acid transport being repressed in the presence of abundant ammonium (Karagiannis et al., 1999), and they confirm that under our conditions the addition of ammonium has a noticeable effect on gene expression. Given that lipid metabolism genes were not affected in WT, it also demonstrates the specificity of the impact of ammonium. Curiously, the expression profiles of nitrogen-responsive genes (especially per1 and isp5) in the cbf11 or mga2 mutants were quite different from those in WT (Fig. 3), which might suggest perturbed nitrogen-related signalling in these strains.

Fig. 3.

Decreased expression of lipid metabolism genes in cbf11 and mga2 mutants is not restored by ammonium. A set of known target genes regulated by Cbf11 and/or Mga2 is shown (‘Lipid metabolism’, Převorovský et al., 2015; Burr et al., 2016), together with a group of nitrogen-regulated amino acid transporter genes (‘AA transport’). RNA-seq results are shown as log2 of expression fold change values normalised to WT grown in plain YES. Gene expression in the Pcut6MUT mutant and in WT treated with cerulenin are shown for comparison. Three biological replicates were analysed, and mean values are shown.

Fig. 3.

Decreased expression of lipid metabolism genes in cbf11 and mga2 mutants is not restored by ammonium. A set of known target genes regulated by Cbf11 and/or Mga2 is shown (‘Lipid metabolism’, Převorovský et al., 2015; Burr et al., 2016), together with a group of nitrogen-regulated amino acid transporter genes (‘AA transport’). RNA-seq results are shown as log2 of expression fold change values normalised to WT grown in plain YES. Gene expression in the Pcut6MUT mutant and in WT treated with cerulenin are shown for comparison. Three biological replicates were analysed, and mean values are shown.

To further test for any potential effects of ammonium on the lipid metabolism in mitotic mutants, we analysed the total lipid content of Δcbf11 and WT cells using thin layer chromatography (TLC) and their FA composition by gas chromatography. The results showed that the total amount of FA is decreased in Δcbf11 cells (Fig. 4A), which correlates well with our previous data on the decreased number of storage LDs in this strain (Převorovský et al., 2015). Also, total FA saturation level is higher in Δcbf11 cells compared to in WT cells (Fig. 4B), which might be caused by the decreased expression of the ole1 desaturase gene (Fig. 3). This tendency towards higher FA saturation is also visible at the level of individual FA species (Fig. 4C, compare C18:1 with C18:0 and C16:0). Finally, the TLC analysis revealed that the content of squalene and lanosterol esters is markedly increased in Δcbf11 cells (Fig. 4D,E; Fig. S1). Notably, none of the detected changes in the Δcbf11 FA and lipid composition were reversed by ammonium supplementation (Fig. 4; Figs S1, S2). Taken together, our results strongly suggest that ammonium does not rectify the disturbed lipid metabolism in Δcbf11 cells, and therefore it likely brings about the partial mitotic rescue in a different, indirect way. Fig. S1 also shows that ammonium triggered an increase in TAG content in WT cells. This is in agreement with our previous findings that ammonium boosts the lipid droplet content in WT cells (Zach et al., 2018).

Fig. 4.

Lack of cbf11 leads to pronounced changes in FA and lipid composition which are not affected by ammonium supplementation. WT and Δcbf11 cells were grown to exponential phase in the indicated medium, and their lipid composition was analysed. (A) Δcbf11 cells have lower FA content per unit of dry cell weight compared to WT. (B) The degree of FA saturation is higher in Δcbf11 cells. (C) Abundance of selected FA species in total lipid samples. Two different y-axis scales are shown to better visualise the full range of FA abundances. (D) Thin layer chromatography (TLC) analysis of neutral lipids. Only a section of the TLC plate is shown (see Fig. S1 for the full TLC plate and additional replicates). (E) Quantification of neutral lipids TLC spots intensity. SE-1, SE-2, sterylester species (ergosterol-based and lanosterol-based, respectively); SQ, squalene. Groups are colour-coded as in C. Mean±s.d. from three independent experiments are shown in A–C and E. To determine statistical significance one-way ANOVA followed by simultaneous tests for general linear hypotheses (A,B) or two-sided unpaired Student's t-test with Holm correction (C,E) were applied. AU, arbitrary units.

Fig. 4.

Lack of cbf11 leads to pronounced changes in FA and lipid composition which are not affected by ammonium supplementation. WT and Δcbf11 cells were grown to exponential phase in the indicated medium, and their lipid composition was analysed. (A) Δcbf11 cells have lower FA content per unit of dry cell weight compared to WT. (B) The degree of FA saturation is higher in Δcbf11 cells. (C) Abundance of selected FA species in total lipid samples. Two different y-axis scales are shown to better visualise the full range of FA abundances. (D) Thin layer chromatography (TLC) analysis of neutral lipids. Only a section of the TLC plate is shown (see Fig. S1 for the full TLC plate and additional replicates). (E) Quantification of neutral lipids TLC spots intensity. SE-1, SE-2, sterylester species (ergosterol-based and lanosterol-based, respectively); SQ, squalene. Groups are colour-coded as in C. Mean±s.d. from three independent experiments are shown in A–C and E. To determine statistical significance one-way ANOVA followed by simultaneous tests for general linear hypotheses (A,B) or two-sided unpaired Student's t-test with Holm correction (C,E) were applied. AU, arbitrary units.

Ammonium has a general positive effect on mitotic fidelity

We have previously shown that the duration of mitotic phases in Δcbf11 cells is typically longer and more variable compared to that in WT cells. The delays first manifest well before anaphase and they increase during the whole mitotic duration (Vishwanatha et al., 2023). Therefore, we investigated whether mitotic timing is affected by ammonium. We performed live-cell microscopy of strains with fluorescently tagged histone H3 (Hht2–GFP) and α-tubulin (mCherry–Atb2) to visualise the chromatin and mitotic spindle, respectively (Fig. 5A). We found that ammonium reduced the overall length of mitosis (Fig. 5B), and this effect manifested both during the prophase and metaphase (Fig. 5C), as well as in the anaphase stages (Fig. 5D) in Δcbf11 cells, bringing them close to WT values. Also, mitotic timing became more uniform among individual treated Δcbf11 cells (Fig. 5). Note that telophase duration was not included in the overall mitosis length due to the technical limitations of our experimental setup.

Fig. 5.

Ammonium suppresses prolonged mitotic duration in Δcbf11 cells. (A) Examples of mitotic phases as they are observed under the microscope. Green, chromatin (Hht2–GFP); magenta, microtubules (mCherry–Atb2). (B–D) The duration of the whole mitosis (prophase, metaphase and anaphase combined) (B), as well as prophase+metaphase (C) or anaphase (D) separately are all prolonged in cells lacking cbf11. This aberrant timing does not occur in the presence of ammonium. Data for 31–41 cells per condition are shown in panels B–D. Barplot (B) displays mean±s.d. together with individual data points. Boxplots (C,D) display medians (thick line), Q1 and Q3 quartiles (the box), and the smallest and largest values within a 1.5× interquartile range from the Q1 and Q3 boundaries, respectively (whiskers), together with individual data points. To determine statistical significance one-way ANOVA was applied followed by simultaneous tests for general linear hypotheses. Only cells which successfully completed mitosis were analysed.

Fig. 5.

Ammonium suppresses prolonged mitotic duration in Δcbf11 cells. (A) Examples of mitotic phases as they are observed under the microscope. Green, chromatin (Hht2–GFP); magenta, microtubules (mCherry–Atb2). (B–D) The duration of the whole mitosis (prophase, metaphase and anaphase combined) (B), as well as prophase+metaphase (C) or anaphase (D) separately are all prolonged in cells lacking cbf11. This aberrant timing does not occur in the presence of ammonium. Data for 31–41 cells per condition are shown in panels B–D. Barplot (B) displays mean±s.d. together with individual data points. Boxplots (C,D) display medians (thick line), Q1 and Q3 quartiles (the box), and the smallest and largest values within a 1.5× interquartile range from the Q1 and Q3 boundaries, respectively (whiskers), together with individual data points. To determine statistical significance one-way ANOVA was applied followed by simultaneous tests for general linear hypotheses. Only cells which successfully completed mitosis were analysed.

Our results so far indicate that nitrogen might have a more general effect on mitotic fidelity. To test this hypothesis, we determined the effect of ammonium on a diverse panel of mutants, none of them being related to lipid metabolism, which are prone to develop the ‘cut’ phenotype with varying severity. Remarkably, ammonium supplementation significantly decreased the frequency of catastrophic mitotic events in many, but not all, of those mutants (Fig. 6A–C). The group of suppressed ‘cut’ mutants consisted of condensin (cut3), cohesin (psm3), members of the SMC5/6 complex [smc6 (Lehmann et al., 1995) and nse3 (Zabrady et al., 2016)] and a nuclear proteasome tethering factor (cut8). By contrast, mutants without significant suppression included separase (cut1), securin (cut2), APC/C subunits (cut4 and cut9) and a nuclear import factor (cut15) (Fig. 6D).

Fig. 6.

Ammonium supplementation suppresses a range of cut mutants. (A–C) Cells were grown to exponential phase in the indicated medium, fixed, stained with DAPI and subjected to microscopy. Cultivation temperatures were chosen to provide semi-restrictive conditions for temperature-sensitive strains. Mean±s.d. from two or three independent experiments are shown (exact n indicated by individual data points). To determine statistical significance one-sided unpaired Student's t-test with Holm correction was applied. (D) Mutants responsive to ammonium supplementation tend to be associated with pre-anaphase (‘pre-A’) rather than anaphase (‘A’). Grey background, no suppression.

Fig. 6.

Ammonium supplementation suppresses a range of cut mutants. (A–C) Cells were grown to exponential phase in the indicated medium, fixed, stained with DAPI and subjected to microscopy. Cultivation temperatures were chosen to provide semi-restrictive conditions for temperature-sensitive strains. Mean±s.d. from two or three independent experiments are shown (exact n indicated by individual data points). To determine statistical significance one-sided unpaired Student's t-test with Holm correction was applied. (D) Mutants responsive to ammonium supplementation tend to be associated with pre-anaphase (‘pre-A’) rather than anaphase (‘A’). Grey background, no suppression.

It should be noted though that the magnitude of the suppression varied between the ammonium-responsive mutants, and we suggest possible explanations for this behaviour in the Discussion. Taken together, the ammonium-mediated suppression of mitotic defects is not limited to cells with perturbed lipid metabolism, and seems to operate early in the cell cycle, prior to anaphase.

The nitrogen-dependent improvement of mitotic fidelity is related to the TOR network

The evolutionarily conserved TOR signalling network is a major hub for controlling nitrogen sensing and utilisation. Therefore, we tested whether and how TOR was involved in the nitrogen-dependent improvement of mitotic fidelity. First, we suppressed the activity of TOR by treating cells with the TOR inhibitor rapamycin (Heitman et al., 1991). Rapamycin is known to target Tor2/TORC1 in many species (Hayashi et al., 2007; Nakashima et al., 2010; Takahara and Maeda, 2012); however, its actions are somewhat divergent in S. pombe. Namely, rapamycin alone does not kill S. pombe cells and must be combined with caffeine to inhibit cell growth (Takahara and Maeda, 2012). Also, rapamycin has been found to inhibit certain functions of Tor1/TORC2 as well (Weisman et al., 2001, 2005). Therefore, rapamycin treatment can provide indications whether the TOR network is implicated in general. Notably, we found that rapamycin improved mitotic fidelity in Δcbf11 cells to an extent similar to that of ammonium. Additionally, a combined treatment showed that the effects of rapamycin and ammonium were non-additive (Fig. 7A). These results strongly indicate that both chemicals affect the same target pathway or process.

Fig. 7.

The TOR network is related to the ammonium-mediated suppression of Δcbf11 mitotic defects. (A) Rapamycin treatment partially suppresses the mitotic defects of Δcbf11 cells and its effect is not additive with ammonium supplementation. The Δcbf11 mitotic defects can also be suppressed, to varying degrees, by the introduction of a tor1-D temperature-sensitive allele, tor1 deletion (B) or deletion of ssp2 (C). Cells were grown to exponential phase in the indicated medium and temperature, fixed, stained with DAPI and subjected to microscopy. Mean±s.d. from three independent experiments are shown. NA, not analysed. To determine statistical significance one-way ANOVA was applied followed by Dunnet's test (A) or simultaneous tests for general linear hypotheses (B), or one-sided unpaired Student's t-test with Holm correction was applied (C).

Fig. 7.

The TOR network is related to the ammonium-mediated suppression of Δcbf11 mitotic defects. (A) Rapamycin treatment partially suppresses the mitotic defects of Δcbf11 cells and its effect is not additive with ammonium supplementation. The Δcbf11 mitotic defects can also be suppressed, to varying degrees, by the introduction of a tor1-D temperature-sensitive allele, tor1 deletion (B) or deletion of ssp2 (C). Cells were grown to exponential phase in the indicated medium and temperature, fixed, stained with DAPI and subjected to microscopy. Mean±s.d. from three independent experiments are shown. NA, not analysed. To determine statistical significance one-way ANOVA was applied followed by Dunnet's test (A) or simultaneous tests for general linear hypotheses (B), or one-sided unpaired Student's t-test with Holm correction was applied (C).

Our next objective thus was to determine the importance of each of the two branches of the TOR network for the suppression. To address this, we first employed mutants of the non-essential tor1 gene (TORC2). We found that the temperature-sensitive tor1-D allele (Ikai et al., 2011) increased mitotic fidelity of the Δcbf11 background when cells were grown at the semi-restrictive temperature of 34°C. Moreover, an even stronger suppressive effect was achieved by introducing a complete deletion of tor1 into Δcbf11 cells (Fig. 7B). It is important to note that the two TOR complexes are antagonistic and the inhibition or depletion of Tor1/TORC2 boosts the activity of Tor2/TORC1 (Ikai et al., 2011; Schonbrun et al., 2009).

Next, we focused on the role of the essential Tor2 kinase. First, we decreased Tor2/TORC1 kinase activity by combining the Δcbf11 mutation with the temperature sensitive tor2-S allele (Hayashi et al., 2007). We did not observe any significant change in the mitotic fidelity at semi-restrictive temperature compared to those in Δcbf11 alone (Fig. S3). Then, to increase Tor2 activity we took advantage of the fact that the activity of Tor2/TORC1 is negatively regulated by the AMP-activated protein kinase (AMPK) complex (Davie et al., 2015). Therefore, we deleted the ssp2 gene, which encodes the AMPK catalytic subunit (Matsuzawa et al., 2012; Valbuena and Moreno, 2012). Indeed, the deletion of ssp2 in Δcbf11 cells led to a decrease in catastrophic mitotic events (Fig. 7C). The frequency of catastrophic mitosis in Δtor1 and Δssp2 single mutants was also analysed (one separate experiment, n>350 cells per sample), showing near-zero values: WT, 0.58%; Δtor1, 0.55%; and Δssp2, 0.27% (data not shown). Collectively, these results strongly suggest that the nitrogen-dependent suppression of mitotic defects is related to the functions of the TOR network and indicate a role for TOR in ensuring successful progression through (closed) mitosis.

The general availability and the particular quality of a nitrogen source have long been recognised as important factors regulating progression through the cell cycle. The absence of a nitrogen source (nitrogen starvation) or a shift to a less preferable nitrogen source (nitrogen stress) inhibit cell growth, and trigger quiescence or sexual differentiation, or accelerate the entry into mitosis, respectively (Fantes and Nurse, 1977; Egel and Egel-Mitani, 1974). However, we recently demonstrated that nitrogen also plays a role in mitotic fidelity in cells with perturbed lipid metabolism, with ammonium chloride supplementation being able to partially rescue catastrophic mitosis phenotypes (Převorovský et al., 2015; Zach et al., 2018). Intriguingly, our current data revealed that this nitrogen-dependent improvement of mitosis is not accompanied by corrections of the aberrant FA and lipid composition, hinting at an indirect suppressor mechanism. Indeed, we found that the suppressive effect is only partial and also of a more general nature, not limited to mutants in lipid metabolism. Moreover, we showed that nitrogen availability also affects the progression through mitosis, not just the G2/M transition (mitotic entry). While the mechanistic details remain to be elucidated, we demonstrated that the effect of nitrogen is related to the functions of the TOR regulatory network.

It was previously suggested that the mitotic defects of fission yeast cells with chemically or genetically perturbed lipid metabolism are caused by insufficient NE expansion due to a shortage of membrane precursors (Yam et al., 2011; Takemoto et al., 2016). It is also possible that these mitotic defects could be related to altered mechanical properties of cell membranes that are important for spindle pole body integration and other crucial steps of the closed mitosis (West et al., 1998). We have indeed found a number of aberrations in the composition of lipids in Δcbf11 cells grown in the complex YES medium compared to WT. These aberrations included an overall lower content of FA, which are required for the production of new membranes. However, we did not observe any notable corrections in the mRNA levels of lipid metabolism genes (Fig. 3) or in composition of FA and lipids (Fig. 4; Figs S1, S2) in Δcbf11 cultures grown in an ammonium-supplemented YES medium, where mitotic defects are suppressed. Neither did we observe any significant improvement in NE expansion during mitosis in YES+ammonium (Fig. 2). These results are consistent with our recent findings that, in addition to any issues with the production of new membranes, mitotic fidelity in lipid metabolism mutants is affected by changes in centromeric chromatin structure and cohesin dynamics (Vishwanatha et al., 2023).

While we did not detect any nitrogen-dependent improvement in mitotic NE dynamics and FA and lipid composition composition, we found that ammonium supplementation clearly normalised the timing of mitotic progression, restoring the duration of individual mitotic phases close to their WT state (Fig. 5). Strikingly, we also found that the phenomenon of nitrogen-mediated improvement of mitotic fidelity is not limited to lipid metabolism-related problems, as mitotic defects in multiple unrelated ‘cut’ mutants could also be suppressed by ammonium supplementation (Fig. 6). Importantly, not all tested ‘cut’ mutants showed responsiveness to nitrogen availability, and among those which did, the magnitude of the suppressive effect varied. Notably, the rescuable strains are typically mutants in genes involved in pre-anaphase processes, while all ammonium-insensitive ‘cut’ mutants are related to anaphase (securin/separase, APC/C) (Fig. 6). Thus, this specificity of the suppressive effect might be dictated by the particular time during which the respective ‘cut’ genes perform their mitosis-related functions. Taken together, the ammonium-mediated suppression of mitotic defects is not limited to cells with perturbed lipid metabolism, and it seems to operate in cell cycle phase(s) prior to anaphase. This also suggests that our findings might be relevant even for organisms that do not employ closed mitosis. Interestingly, we have recently shown that cell cycle arrest in the presence of genotoxic stress is aberrant in Δcbf11 cells, and these cells are sensitive to the topoisomerase I poison camptothecin (Marešová et al., 2024).

The TOR network is known to be one of the key cell-cycle regulators, promoting or inhibiting mitotic onset according to nutrient availability (Petersen and Nurse, 2007; Uritani et al., 2006). Interestingly, previous reports hinted that TOR might also have a role later in mitosis. First, the viability of several temperature-sensitive separase and securin mutants (including those used in our study) was improved by treating cells with the TOR inhibitor rapamycin or by introducing the tor2-S mutation that impairs TORC1 kinase activity. However, the authors did not specifically test whether mitotic fidelity was also improved (Ikai et al., 2011; Vijayakumari et al., 2022). Second, one of these studies also found that TORC1 inactivation could actually suppress the mitotic defects of the cdc48-353 mutant (and the effect was related to separase function), but it had no effect on other mitotic mutants such as a cut3 condensin mutant (Vijayakumari et al., 2022), for which we did see suppression (Fig. 6). In our hands, the occurrence of mitotic defects in the cut1-206 and cut2-447 mutants was not suppressed by ammonium supplementation, a treatment we showed to have an impact similar to rapamycin treatment (Fig. 6). Neither did we observe any differences in mitotic fidelity between Δcbf11 and Δcbf11 tor2-S cells (Fig. S3). It is therefore possible that any impact of TOR on the fidelity of anaphase events is regulated separately, by mechanism(s) different from those related to the nitrogen-dependent suppression that we report here. Indeed, the authors themselves reported that the phenotype of the tor2-S mutant manifested clearly only in the peptone-containing YPD medium and not in the ammonium-containing EMM2, and they observed the cut1/cut2 suppression under suboptimal nitrogen availability in YPD (Ikai et al., 2011). Notably, some mitotic proteins, such as the Cut1/separase, seem to exert translational sensitivity related to the TOR network (Vijayakumari et al., 2022). However, when we analysed the proteins annotated as mitotic in PomBase by label-free quantitative mass spectrometry (mitotic nuclear division; GO:0140014; n=136, of those 90 were detected) we did not see any significant differences in their levels in Δcbf11 cells grown in plain YES compared to YES with ammonium.

It was also reported that TOR is linked to growth phase-related changes in lipid metabolism. The switch between membrane phospholipid and storage triacylglycerol production is regulated by the lipin phosphatidic acid phosphatase (Santos-Rosa et al., 2005). In the budding yeast Saccharomyces cerevisiae, TOR controls lipin activity to ensure sufficient supply of phospholipids for new membrane production during active proliferation (Dubots et al., 2014). On the other hand, TOR/lipin-dependent overproduction of FA and endoplasmic reticulum membrane leads to mitotic defects and formation of micronuclei in mammals (Merta et al., 2021). Nevertheless, we did not observe any major changes in phospholipid composition in S. pombe cells grown in ammonium supplemented YES medium compared to plain YES (Fig. S2). Even though ammonium addition led to increased TAG content in WT cells (Fig. S1).

We showed increased mitotic fidelity in Δcbf11 cells when the stress-response branch of the TOR network was suppressed, either by ablation of Tor1/TORC2 or by de-repressing the pro-growth Tor2/TORC1 branch (Fig. 7). These data are in agreement with previous findings that Tor2/TORC1 inhibition mimics nitrogen starvation (Matsuo et al., 2007; Weisman et al., 2007). And vice versa, Tor2/TORC1 hyperactivation delays the response to nitrogen starvation (Urano et al., 2007), highlighting that the two branches of the TOR network are antagonistic and act in a negative feedback loop. Our results indicate that interventions providing cells with more (well-utilisable) nitrogenous compounds (e.g. ammonium supplementation), or merely triggering internal signals mimicking the state of such nitrogen availability (i.e. de-repressing Tor2/TORC1 activity by ablating TORC2 or AMPK), make the mitotic defects in cells prone to catastrophic mitosis less severe. The results of the rapamycin treatment are, however, controversial. While usually perceived as a TORC1 inhibitor and stress inducer mimicking nitrogen shortage, in our case rapamycin had a positive effect on mitotic fidelity, similar to ammonium. That can be explained by the previously reported fact that in S. pombe rapamycin can be suppressing certain cell functions associated with the nitrogen stress, such as sexual development (Weisman et al., 2001). Additionally, it should be noted that ammonium-dependent improvements in mitotic dynamics manifested very early on during the mitosis of Δcbf11 cells (Fig. 5). It is therefore also possible that the TOR network does not act on mitosis directly during the process of nuclear division, but rather earlier in the cell cycle helps establish conditions more favourable for smooth mitotic progression and increased fidelity further down the road.

In any case, the exact mechanism of the nitrogen-mediated improvement of mitotic fidelity remains to be characterised in detail, including the regulatory level(s) on which the observed effect is achieved. Given that the TOR proteins are kinases, it is likely that the suppression is mediated, at least in part, at the post-translational level by phosphorylation of downstream effector proteins. These could be identified, for example, by screening of a knock-out library (Kim et al., 2010) combined with the Δcbf11 mutation, as removal of these effectors should abolish the positive effects of nitrogen on mitotic fidelity and/or overall viability. Such studies could also explain the general character of the suppression, which occurs in a functionally diverse group of ‘cut’ mutants (Fig. 6). Finally, the effects of nitrogen availability on mitotic fidelity are worth testing in organisms featuring open mitosis, such as human cells.

Strains, media and cultivation

Standard methods and media were used for the cultivation of Schizosaccharomyces pombe strains (Sabatinos and Forsburg, 2010). YES medium was prepared using Bacto Yeast Extract (BD Biosciences) and SP Supplements (Formedium). EMM was prepared using EMM Broth without nitrogen (EMM-N; Formedium). Ammonium chloride (Sigma) or ammonium sulphate (Lachema or Chempol) were added to EMM-N to the final concentration of 93 mM, unless indicated otherwise. L-glutamic acid monosodium hydrate, L-proline or uracil (Sigma) were added to the final concentrations from 20 to 100 mM, as required. S. pombe cell cultures were pre-grown for 8 h at 32°C (or 25°C for temperature-sensitive strains) in 5 ml YES medium, then transferred to the medium with the appropriate supplements or stressors, diluted to an optical density at 600 nm (OD600)=0.005 (WT) or ∼0.03–0.05 (mutants) and incubated overnight to the early exponential phase (OD600=0.5) at 32°C or at appropriate semi-restrictive temperature. Cerulenin (Abcam) was added 2 h before harvesting to the final concentration of 10 μg/ml. Rapamycin (Merck) was added at the time of starting the final culture to the final concentration of 0.3 μg/ml. Routine OD measurements of liquid cell cultures were taken using the WPA CO 8000 Cell Density Meter (Biochrom). The strains used in the study are listed in Table S2 and are available upon request. Deletion of cbf11 was carried out using the pMP91 or pMP92 targeting plasmid, based on pCloneNAT1 or pCloneHYG1 respectively, as described previously (Gregan et al., 2006) and confirmed by PCR. All other strains used in this study were constructed by standard genetic crosses. Plasmids and oligonucleotides used in this study are listed in Tables S3 and S4, respectively.

Microscopy

For nuclear staining, exponentially growing S. pombe cells were pelleted by centrifugation (1000 g, 3 min), fixed in 70% ethanol and stored at 4°C prior to imaging. Then cells were rehydrated in water and stained with DAPI at a 0.1 μg/ml final concentration. Samples were analysed using a Leica DM750 microscope with a HC FL PLAN 100×/1.25 oil objective. For each sample at least ten images (∼500–1500 cells total) were acquired. The frequency of catastrophic mitosis was counted manually using our standard scoring criteria (Vishwanatha et al., 2023). Briefly, at least 400 cells from the acquired images of asynchronous populations were analysed per sample, and mitotic defects were scored based on nuclear morphology and the position of the nucleus relative to the septum or cell wall. If medial cell wall invagination was already apparent, a dividing cell was counted as two separate daughters.

For live-cell microscopy 1 μl of a slightly resuspended cell pellet was applied on 2% agarose-YES medium solidified in a 2 mm polydimethylsiloxane (PDMS) spacer (Costa et al., 2013), and covered with a coverslip. Slides were placed into an OKOlab environmental chamber set to 32°C. Time-lapses were acquired using a Nikon Ti2 microscope with Plan Apo Lambda 60× oil objective coupled with a Hamamatsu ORCA-Flash4.0 camera in 16-bit, 2×2 binning mode. Snapshots were taken at 2 min intervals. Images were acquired as Z-stacks, as five to nine slices with a 0.3 μm step. Fluorophores were excited using CoolLED pE-4000 device. The GFP fluorophore was filmed using 460 nm excitation at 21% light power, for a 100 (Cut11–GFP) or 50 ms (Hht2–GFP) exposure time and with a 510 nm emission filter. The mCherry fluorophore was filmed using 580 nm excitation at 20% power, for a 200 nm exposure time and with a 590 nm emission filter.

Image analysis was performed using ImageJ software (Schindelin et al., 2012). Z-stacks were processed as maximum intensity projections. Time-lapses were corrected for photobleaching (Miura, 2020) and sample movement (‘Linear Stack Alignment with SIFT’) (Lowe, 2004). The nucleus cross-section was determined as an area enclosed by high-intensity pixels (i.e. by the Cut11-decorated nuclear envelope) in the GFP channel. Threshold levels for image segmentation were set according to local signal-to-noise ratio to fully enclose any given nucleus at every time point, and the void space in the middle of the resulting masks were filled.

RNA-seq

Samples were prepared from three biological replicates. Cells were cultured to exponential phase, 10 ml was harvested (600 g, 2 min) and the cell pellet was flash-frozen with liquid nitrogen. Total RNA was isolated using a hot acidic phenol method followed by phenol-chloroform extractions and precipitation (Lyne et al., 2003). Extracted RNA was treated with TURBO DNase (Thermo Fisher Scientific) and purified using RNeasy columns (Qiagen). RNA quality was assessed on Bioanalyzer 2100 (Agilent). A detailed sample preparation protocol is available at ArrayExpress database (https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-13302).

WT and Δcbf11 samples were processed at the Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Germany. Libraries were prepared using the Illumina TruSeq stranded mRNA Library (poly-A) and sequenced in the pair-end mode on an Illumina NovaSeq 6000 instrument with the NovaSeq 6000 SP Reagent Kit with 100 cycles.

Samples of WT, cbf11DBM, Δmga2, Δmga2Δcbf11, Pcut6MUT and cerulenin treatment were processed at the Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czechia. The sequencing libraries were synthesised using KAPA mRNA HyperPrep Kit (Illumina platform) (Roche, KK8581) and analysed on an Illumina Nextseq 500 instrument using the NextSeq 500/550 High Output Kit v2.5 (75 Cycles) (Genetica, 20024906) with single-end, 75 bp, dual index 2×8 bp setup.

RNA-seq data are available at ArrayExpress database (https://www.ebi.ac.uk/arrayexpress/) under the accession numbers: E-MTAB-13302 (WT, Δcbf11), E-MTAB-13303 (Δmga2, Δmga2 Δcbf11) and E-MTAB-13305 (WT, cbf11DBM, Pcut6MUT, and cerulenin treatment).

RNA-seq data analysis

The reference S. pombe genome and annotation were downloaded from PomBase (2022-05-30; https://pombase.org; Wood et al., 2002; Lock et al., 2019). Read quality was checked using FastQC version 0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapters and low-quality sequences were removed using Trimmomatic 0.39 (Bolger et al., 2014). Clean reads were aligned to the S. pombe genome using HISAT2 2.2.1 (Kim et al., 2015) and SAMtools 1.13 (Li et al., 2009). Read coverage tracks were then computed and normalised to the respective mapped library sizes using deepTools 3.5.1 (Ramírez et al., 2016). Mapped reads and coverage data were inspected visually in the IGV 2.9 browser (Robinson et al., 2011). Gene counts were generated using the GenomicAlignments package (Lawrence et al., 2013) in R/Bioconductor (https://R-project.org; Gentleman et al., 2004). Datasets from different sequencing runs were normalised using RUVseq (Risso et al., 2014). Differentially expressed genes (DEG) were detected using DESeq2 (Love et al, 2014). Gene ontology (GO) term and other gene list enrichment analysis was performed using AnGeLi (Bitton et al., 2015).

Lipid analysis

Samples were prepared from three biological replicates. Cells were cultured to exponential phase in YES medium, 50 ml were harvested (1000 g, 3 min). Cell pellets were flash-frozen with liquid nitrogen and freeze-dried using HyperCOOL 3055 device (Gyrozen).

Total lipids were extracted using a method previously reported with minor modifications (Garaiova et al., 2017). Briefly, dry cell weight (DCW) was determined gravimetrically prior to lipid extraction. Freeze-dried cells (∼10–15 mg) were mixed with 100 µl of ice-cold water followed by the addition of 1 ml chloroform and methanol mixture (2:1, v/v). Cells were disrupted by a FastPrep disintegrator (MP Biomedicals) with glass beads (diameter 0.4 mm, 3×40 s at the highest speed, with 5 min cooling on ice between cycles). Lipids were extracted by incubating in chloroform/methanol/water (1:2:0.8, v/v) and subsequently adjusting the mixture proportion to 2:2:1.8 (v/v) at room temperature. The organic phase containing the lipids was separated by centrifugation (1000 g, 5 min) and dried under a stream of nitrogen. The resulting dry lipids were dissolved in 100 µl of chloroform and methanol mixture (2:1, v/v).

For fatty acid analysis total lipid extracts were transmethylated with 5% Na-OCH3 in methanol. Fatty acid methyl esters (FAMEs) were then extracted using n-hexane as described previously (Iwabuchi et al., 2003). The analysis of FAMEs involved injecting 1 μl aliquots into a GC2010Plus gas chromatography (GC) apparatus (Shimadzu) equipped with a BPX70 capillary column (30 m×0.25 mm×0.25 µm, SGE Analytical Science) as described previously (Garaiova et al., 2017; Mietkiewska et al., 2011). Identification of individual FAMEs was accomplished by comparing them with authentic standards of a C4−C24 FAME mixture (Supelco). The quantification of individual fatty acids was conducted using heptadecanoic acid methyl ester as an internal standard (Sigma Aldrich).

For the thin layer chromatography (TLC), an aliquot of lipid extract corresponding to 8 mg of DCW was applied to silica gel TLC plates (Merck) by a Linomat 5 semiautomatic sample applicator (Camag). Neutral lipids were separated by a two-step TLC solvent system using a method described previously (first step: petroleum ether/diethyl ether/acetic acid, 70:30:2; second step: petroleum ether and diethyl ether, 49:1; Spanova et al., 2010). Individual lipid spots were visualised by charring the plates as previously reported (Garaiová et al., 2014). Individual lipid spots were identified using lipid standards. Intensity of individual lipid spots was quantified by scanning absorption at 475 nm. Phospholipids were separated by the solvent system (chloroform/methanol/acetic acid/water, 75:45:3:1) as described previously (Garner et al., 2012).

Data analysis and visualisation

Data was handled using R software (https://R-project.org). A one-tailed unpaired Student's t-test was applied for statistical comparisons of two groups, if not specified otherwise. One-way ANOVA test was applied for multiple group comparisons, followed by an appropriate post-hoc test. Dunnet's test was applied for multiple comparisons with a single control. Simultaneous tests for general linear hypotheses were applied for pairwise comparisons between multiple groups (glht function in R software). A single-step method was used for P-value adjustment, if not specified otherwise. Multiple comparison statistical tests were performed using the multcomp package (Hothorn et al., 2008). Plots were generated using the gplots (https://CRAN.R-project.org/package=gplots) and ggplot2 (Wickham, 2016) packages. Scripts for generation of the plots are available at https://www.github.com/ViacheslavZemlianski/Cut_paper.

We are very grateful to Phong Tran for his advice and material support for live-cell microscopy; Akshay Vishwanatha and Patrik Hohoš for the help with development of live-cell microscopy protocols; and Adéla Kracíková and Kateřina Svobodová for excellent technical assistance. The cut1-206, cut2-447, cut3-477, cut4-533, cut8-563, cut9-665, cut15-85, psm3-304, tor1-D and tor2-S strains were provided by NBRP, Japan. The smc6-x and nse3-R254E strains were provided by Jan Paleček. Microscopy was performed in the Vinicna Microscopy Core Facility co-financed by the Czech-BioImaging large RI project LM2023050. Computational resources were supplied by the project ‘e-Infrastruktura CZ’ (e-INFRA LM2018140) provided within the program Projects of Large Research, Development and Innovations Infrastructures. The authors acknowledge the Imaging Methods Core Facility at BIOCEV, institution supported by the MEYS CR (LM2023050 Czech-BioImaging), for their support and assistance in this work.

Author contributions

Conceptualization: V.Z., M.P.; Methodology: R. Holič; Formal analysis: V.Z., R. Holič, M.P.; Investigation: V.Z., A.M., J.P., R. Holič, M.J.R.d.R., L.L., M.Z.; Resources: A.M., J.P., R. Häsler; Data curation: V.Z.; Writing - original draft: V.Z., M.P.; Writing - review & editing: V.Z., A.M., J.P., R. Holič, R. Häsler, M.P.; Visualization: V.Z.; Supervision: M.P.; Project administration: V.Z.; Funding acquisition: V.Z., M.P.

Funding

This work was supported by the Grant Agency of Charles University (GA UK grant no. 1311120 to V.Z.). Parts of the sequencing analysis were supported by the Deutsche Forschungsgemeinschaft (DFG) Research Infrastructure NGS CC (project 407495230) as part of the Next Generation Sequencing Competence Network (project 423957469). These parts of the NGS analysis were carried out at the Competence Centre for Genomic Analysis (Kiel, Germany). Work focused on FA and lipid analyses was supported by the Slovak Research and Development Agency grant number APVV-20-0166 and by the Ministry of Education, Science, Research, and Sport of the Slovak Republic, and the Slovak Academy of Sciences grant number VEGA 2/0036/22.

Data availability

RNA-seq data are available at ArrayExpress database (https://www.ebi.ac.uk/arrayexpress/) under the accession numbers: E-MTAB-13302 (WT, Δcbf11), E-MTAB-13303 (Δmga2, Δmga2 Δcbf11) and E-MTAB-13305 (WT, cbf11DBM, Pcut6MUT, and cerulenin treatment). The scripts used for sequencing data processing and analyses are available at https://github.com/mprevorovsky/RNA-seq_ammonium (WT, Δcbf11), https://github.com/mprevorovsky/RNA-seq_CSL-DBM_cerulenin (WT, cbf11DBM, cerulenin treatment), and https://github.com/mprevorovsky/RNA-seq_mga2 (Δmga2, Δmga2 Δcbf11). Raw data and scripts for generation of the plots are available at https://github.com/ViacheslavZemlianski/Cut_paper.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information