FLCCR is a fluorescent reporter system that quantifies the duration of different cell cycle phases at the single-cell level in fission yeast

Traditionally, assessing the timing of cell duplication in single-celled organisms has relied on monitoring the optical density of a culture over a restricted timeframe. However, this method overlooks variations in cell size across different strains. Alternatively, the use of a Neubauer chamber provides snapshots at various culture times but fails to differentiate between live and dead cells. Given the limitations of these traditional methods, we developed a reporter system, FLCCR, capable of not only measuring the duration of the entire cell cycle but also discerning the duration of each phase in time-lapse experiments. This reporter system comprises 4 distinct fluorescent markers: SynCut3 (which localizes to the nuclei during mitosis) [16], Pcn1 (which exhibits a punctate pattern during S phase), Sid2 (marking the spindle pole body and the cell division site), and RitC (labeling the cellular membrane) [17]. The combination of these markers enables precise identification of each cell cycle phase and determination of the total cycle duration (Fig 1A). Importantly, the inclusion of these markers does not compromise cell viability or growth compared to an isogenic strain lacking the markers (S1AB Fig).

Next, we quantified the different cell cycle phases in asynchronous cultures through time-lapse experiments (Fig 1B and S1 Movie) or snapshots from the same cultures (S1C Fig). In our time-lapse experiments, individual cells were tracked, with the peak nuclear fluorescence of SynCut3 indicating mitosis and the maximum standard deviation of Pcn1 fluorescence signifying the peak of S phase (Fig 1C). While the extremely short G1 phase in fission yeast precludes resolution in our 3-min interval experiments, we determined a wild-type strain’s cell cycle length to be 165.26 ± 26.58 min, with M+G1 accounting for 16% and S phase for 14% of the cycle duration (Fig 1D and 1G). When cells were grown in rich media (YE5S), we observed a shortening of the cell cycle duration, mainly because the G2 phase was reduced (Fig 1E–1G). To differentiate M from G1, we repeated the time-lapse experiments, capturing images every minute within a 3-h window to avoid reporter signal photobleaching (S1D Fig). Under these conditions, we estimated G1 duration (time between the maximum separation of nuclei and entry into S phase) in a wild-type strain as 3.5 ± 1.65 min (S1E and S1F Fig).

Fig 1. Cell cycle reporter strain, FLCCR.

(A) Scheme depicting all the fluorescent markers and their localization along the cycle: Pcn1 (magenta, nuclear), RITC (magenta, membrane), Sid2 (cyan, spindle pole body and septum), SynCut3 (green, nuclear in mitosis, cytoplasmic in S-G2). (B) Airyscan microscopy of FLCCR strain. Arrows in merged image indicate cells in G1 (cyan), S (magenta), G2 (salmon), or mitosis (green). Scale bar = 5 μm. (C, E) Nuclear SynCut3 mean fluorescence and Pcn1 Std fluorescence of cells grown in EMM (C) or YE5S (E). Individual cycles were synchronized to peak SynCut3 nuclear intensity. Green box marks M-G1 phases, magenta box marks S phase. (D, F) Quantification of the different cell cycle phases of cells grown in EMM (D) or YE5S (F). Boxplot represents quartile distribution. Outliers are depicted as dots. (G) Table shows mean ± standard deviation. EMM n = 224 cells; YE5S n = 37 cells.

https://doi.org/10.1371/journal.pbio.3002969.g001

Next, we decided to test FLCCR in conditions in which either G1/S or G2 phases were extended. For extended G1/S, we utilized a rep2Δ strain known for slow S phase progression and G1 arrest at low temperatures [18] (Fig 2A). Conversely, to induce an extended G2 phase, we employed a strain harboring a cdc25 allele (cdc25-22) that exhibits delayed G2/M transition at the semi-permissive temperature of 30°C [19] (Fig 2A). These mutations were introduced into the FLCCR strains bearing the 4 fluorescent markers, which did not compromise survival in spot assays or growth in liquid cultures (S2AB Fig).

Fig 2. FLCCR validation.

(A) Scheme of the cell cycle indicating the main point of action of Rep2 and Cdc25. Rep2 promotes START by MBF activation and Cdc25 promotes mitosis entry by activating the CDK Cdc2. (B, C) Nuclear SynCut3 mean fluorescence and Pcn1 Std fluorescence in a rep2Δ strain (B, n = 51) and in a cdc25-22 strain grown at 30°C (C, n = 34). (D, E) Quantification of the different cell cycle phases. Boxplot represents quartile distribution. Outliers are depicted as dots. Table shows mean ± standard deviation. WT n = 224 cells, rep2Δ n = 51 cells, and cdc25-22 n = 34 cells. *: p p p

https://doi.org/10.1371/journal.pbio.3002969.g002

Following the same experimental setup employed with the wild-type strain, subsequent analysis of asynchronous cultures through time-lapse experiments, with images captured every 3 min over a span of 10 h (S2C Fig), unveiled notable alterations in the cell cycle dynamics. In the rep2Δ strain (Fig 2B and S2 Movie), while the overall cell cycle length exhibited a marginal increase from 159.34 min in the wild type to 170.50 min, there was a notable extension of the M/G1 phase (72.93 min versus 26.67 min in the wild type) alongside a concomitant shortening of the G2 phase (62.16 min versus 104.21 min in the wild type). This reduction in G2 phase duration sufficiently compensated for the extended G1 phase, maintaining a normal cycle length (Fig 2D and 2E). Conversely, analysis of the cdc25-22 strain showed a cell cycle length prolonged by 43.57 min exclusively attributable to an extended G2 phase (156.87 min versus 107.23 min in the wild type) (Fig 2C and S3 Movie). Given the inherently brief durations of G1 and S phases in fission yeast, no compensatory mechanism was feasible to counteract the lengthening of the cell cycle in the cdc25-22 background if there was any kind of cell size homeostasis (Fig 2D and 2E).

We assessed the performance of the FLCCR system in genetic backgrounds known to potentially influence the duration of the G2 phase, resulting in cells longer than wild-type cells. Our selection of mutants included cdr1Δ and cdr2Δ, implicated in the mitotic G2 cell size control checkpoint signaling [20], alongside a strain carrying 5 copies of the wee1 gene (5xwee1), inducing a G2/M transition delay [21]. While these 3 strains exhibited larger cell sizes compared to the wild-type strain, the cell cycle remained unaffected in cdr1Δ and cdr2Δ strains, with only minor impact observed in the 5xwee1 strain (S2D Fig).

We postulated that the distinct growth rates among the tested strains might account for the observed differences in the final cell size. To explore this hypothesis, we determined the growth rates in these strains and the wild type by measuring size differences across 10 time points (equivalent to 30 min) after septation and before entry into mitosis. Remarkably, the growth rates obtained under these conditions in a wild-type background closely mirrored those previously obtained using 2 different fluorescent markers [10]. Notably, while rep2Δ exhibited a growth rate similar to the wild type, the remaining strains displayed heightened growth rates (ranging from 30% to 45% increase; see below), which in itself could justify their larger size without impacting the length of the cell cycle.

However, a consideration arose in this calculation: the growth rate is not uniform during the cell cycle. During septation, for instance, cells cease growth at their tips. This phenomenon is readily observable using an active growth marker like CRIB [10]. To precisely quantify the duration of halted growth, we incorporated GFP-CRIB into the FLCCR system, omitting Sid2-GFP, which also labels the septum (Fig 3A). The strain with this modification enabled us to gauge CRIB intensity at cell tips across the different cell cycle phases (Fig 3B and 3C and S4 Movie) and ascertain that the period that cells spend in septation corresponds with the cessation of cell growth (Figs 3D and S3). Then, we integrated this information into our cell size prediction model as follows:

With this straightforward calculation, we were able to determine that the expected size of a wild-type cell was 15.17 μm, which is remarkably close to the actual measurement of 15.68 μm (Fig 3E). To validate our approach, we applied the same calculation to other mutant strains used in our study and found that our method reliably predicts cell size, with a maximum error of less than 3% (wild type and 5xwee1).

Fig 3. Measurement of different growth parameters.

(A) Scheme depicting all the fluorescent markers and their status along the cell cycle. Pcn1 (magenta, nuclear), RITC (magenta, membrane), CRIB (gray, growing tips and septum), SynCut3 (green, nuclear in mitosis, cytoplasmic in S-G2). (B) Epifluorescence microscopy of the CRIB and FLCCR strain. Arrows in merged image signal S phase (magenta), G2 (salmon) and mitosis (green). Scale bar = 5 μm. (C) Signal profile of nuclear SynCut3-mTagBFP2 (green), nuclear mCherry-Pcn1 (magenta), CRIB-3GFP at old-growth tips (gray), and cell length (blue) during the cell cycle. Individual cycles were synchronized to peak SynCut3 nuclear intensity. n = 17 cells. (D) Quantification of the septation time (time from Sid2 appearing at the septum until cell separation by RitC) using Sid2 or CRIB localization at the septum as septation initiation mark. Cytokinesis end was considered when a parental cell splits into 2 daughter cells. Sid2 n = 49 cells, CRIB n = 54 cells. (E) Table showing the mean ± SD of all the growth parameters that can be measured with the cell cycle reporter strain. Different cell cycle mutants were used to determine the measured and the calculated cell size (rep2Δ for G1/S; cdr1Δ, cdr2Δ, 5xwee1 and cdc25-22 for G2/M).

https://doi.org/10.1371/journal.pbio.3002969.g003

The MAP kinase Sty1, on top of being essential for the cells to survive stress situations, has been believed for more than 30 years to be involved in the regulation of cells cycle under unstressed conditions [13,14]. To elucidate the precise role of Sty1 in cell cycle regulation, extending beyond the established genetic interactions with the Cdc25 phosphatase coding gene (Fig 4A), we deleted sty1 in the FLCCR strain. Additionally, as a control, we deleted sty1 in a strain expressing a constitutively active Atf1 (atf1.10D), which mimics the Sty1-dependent phosphorylations, thereby generating an Atf1 variant whose activity remains independent of Sty1 [22]. When either of these 2 mutations was introduced into the FLCCR strain containing the 4 fluorescent markers, the resulting strains behaved similarly to the original strains indicating the fluorescent labeled proteins in the FLCCR strain did not alter the phenotype of sty1Δ or sty1Δatf1.10D (S4A and S4B Fig). Using the same experimental setup as with the wild-type strain, analysis of asynchronous cultures through time-lapse experiments, capturing images every 3 min over a period of 10 h (Figs 4B–4D and S4C and S5 and S6 Movies), revealed no major changes in the length of the cell cycle or in the distribution of the different phases. In any case, the strain that exhibited the most significant deviation from the wild-type strain was the sty1Δ atf1.10D mutant, which is supposed not to have affected its cell cycle progression (Figs 4E and S4D). Although sty1Δ cells display an almost unchanged cell cycle length and an unperturbed distribution of the phases, they exhibit a significantly larger size compared to wild-type cells (21.41 μm versus 15.68 μm of wild type). This observed increase in size aligns with our predicted size calculation of 20.25 μm (Fig 4F).

Fig 4. Cell cycle measurement in Sty1 mutants.

(A) Scheme depicting all the fluorescent markers and their localization along the cycle: Pcn1 (magenta, nuclear), RITC (magenta, membrane), Sid2 (cyan, spindle pole body and septum), SynCut3 (green, nuclear in mitosis, cytoplasmic in S-G2). The arrow indicates the putative point of regulation of the cell cycle by Sty1. (B, C) Nuclear SynCut3 mean fluorescence and Pcn1 Std fluorescence of a sty1Δ (B) and sty1Δ atf1.10D (C) strains. (D) Boxplot represents quartile distribution. Outliers are depicted as dots. (E) Mean and SD of the quantification of all growth parameters measured with the FLCCR. WT n = 224 cells, sty1Δ n = 43 cells, and sty1Δ atf1.10D n = 65 cells. *: p p p

https://doi.org/10.1371/journal.pbio.3002969.g004

However, there was a caveat using a constitutively Sty1-deficient strain, given the pleiotropic phenotypes of this strain, ranging from heightened sensitivity to various extracellular and intracellular stresses to a shortened lifespan [23,24]. To circumvent potential issues arising from long-term adaptation in a Sty1-deficient strain, we decided to use a conditional allele of sty1. We used a sty1-as allele that allowed the inhibition of Sty1 with a bulky analog of ATP [24,25]. The growth of all these strains remained unaffected under unstressed conditions, whether in a wild-type background or when incorporated into the FLCCR background (S5A and S5B Fig). We validated the efficacy of 3-MB-PP1 treatment by confirming the lack of Atf1 phosphorylation upon H₂O₂ stress (S5C Fig). To assess the impact of conditional Sty1 inactivation, we analyzed asynchronous cultures of wild-type and sty1-as strains treated with 3-MB-PP1. In one set of experiments, 3-MB-PP1 was administered just before initiating image capture every 3 min (Fig 5A and S7–S10 Movies), while in another set, cells were pre-treated with 3-MB-PP1 for 12 h before imaging (Fig 5B and S11–S14 Movies). Surprisingly, as depicted in Fig 5C and 5D, no significant alterations were observed in the cell cycle length or phase distribution upon conditional Sty1 inactivation compared to wild-type cells. However, we noted a proportional increase in cell size corresponding to the duration of Sty1 inactivation with 3-MB-PP1 (Fig 5E). This observed increase in cell size appears to be solely sustained by an elevated cell growth rate (Fig 5E).

Fig 5. Sty1 chemical inhibition does not alter cell cycle progression.

(A, C) Quantification of the different cell cycle phases of WT and sty1-as without pretreatment. Boxplots (A) represent quartile distribution with outliers depicted as dots and table (C) shows mean ± standard deviation. WT DMSO n = 66 cells, WT 3MB-PP1 n = 51 cells, sty1-as DMSO n = 64 cells, sty1-as 3MB-PP1 n = 51 cells. *: p p p sty1-as after an overnight pretreatment with 10 μm 3MB-PP1. Boxplots (B) represent quartile distribution with outliers depicted as dots and table (D) shows mean ± standard deviation. WT DMSO n = 53 cells, WT 3MB-PP1 n = 40 cells, sty1-as DMSO n = 55 cells, sty1-as 3MB-PP1 n = 40 cells. *: p p p

https://doi.org/10.1371/journal.pbio.3002969.g005

Another approach to discern the possible effect of Sty1 on cell cycle regulation was to investigate previously described genetic interactions. Since the first one described was with cdc25, we generated a double mutant, cdc25-22 sty1-as. Upon Sty1 inhibition with the ATP analog, cells acquired the elongated phenotype typical of sty1Δ cells, which was even more pronounced in the cdc25-22 sty1-as double mutant (S5D Fig). Despite this effect, we observed no significant changes in the distribution or duration of the various cell cycle phases (S5E Fig). Interestingly, when cultures were left to reach saturation and cells entered the stationary phase, sty1-as cells became smaller, while cdc25-22 sty1-as cells grew even larger. A second genetic interaction reported in the literature involves the phosphorylation of Polo kinase Plo1 at Ser402 by Sty1, either directly or indirectly. It was previously shown that a phosphomimetic mutation plo1.S402E can partially rescue the cdc phenotype in sty1Δ cells. However, when we introduced this same mutation into the sty1-as strain, we did not observe the expected effect on cell size (S5F Fig), suggesting that the genetic interaction between Sty1 and Plo1 does not depend on Sty1 kinase activity.

All S. pombe strains are listed in S1 Table and were grown in EMM or YE5S as described previously [27]. The cell cycle reporter strain was constructed in the following way: Sid2-GFP strain was obtained from elsewhere [28]. All the plasmids used in this work are listed in S2 Table. pAV plasmids [17] were obtained from the National Biosource Project (NBRP). In order to stably integrate the mCherry-Pcn1 tagging into the lys3 locus, we isolated a 5.7 kb region from pAV0785 [25] containing the pcn1 promoter, followed by mCherry-Pcn1, the nmt1 terminator and part of the TEV promoter using KpnI. The insert was cloned into pAV0357 [17], yielding pAY1035. synCut3 plasmid was kindly given by Paul Nurse [9]. mTagBFP2 was extracted from pAV0816 and used to replace the original mCherry from SynCut3-mCherry plasmid and generate SynCut3-mTagBFP2 plasmid pAY1193. Pcn1, RitC, and SynCut3 were generated by stable integration of plasmids pAV0607, pAY1035 and pAY1193. Gene deletions were performed using a polymerase chain reaction (PCR)-based method and were confirmed by PCR on genomic DNA. To generate the cdc25-22 (cdc25-C532Y) and the sty1-as (sty1-T97A) and plo1-S402E reporter strains, SpEDIT CRISPR/Cas9 method was used [29] using the primers listed in S3 Table.

Growth curves were performed as previously described [30]. Briefly, exponentially growing cultures were diluted to an initial OD₆₀₀ = 0.1 and were inoculated per duplicate in a 96-well non-coated polystyrene microplates with an adhesive seal. Plates were incubated in a Power Wave microplate scanning spectrophotometer (Bio-Tek) at 30°C with continuous shaking. OD₆₀₀ was automatically recorded every 10 min for 48 h using Gen5 software. Graphs were made using Pandas, Pyplot, and Seaborn packages from Python.

Assays were performed as previously described [31]. Cells were grown at 30°C in EMM until they reached logarithmic phase to an OD₆₀₀ = 0.5. The same number of cells (10⁵ and 1/10 serial dilutions) in 3 μl were spotted on plates that also contained 10 μm 3MB-PP1, H₂O₂, or HU where indicated. The spots were allowed to dry and the plates were incubated at 30°C (or at the indicated temperature) for 2 to 4 days.

To analyze Atf1 phosphorylation, cells were grown in EMM to log phase (OD₆₀₀ = 0.5). Sty1-as inhibition was done using 10 μm 3MB-PP1 (A602960, Toronto Research Chemicals) 15 min prior 1 mM H₂O₂ treatment for 5 min. Protein extraction was performed as previously described [31]. Trichloroacetic acid (TCA) was added to a final concentration of 10%. Cells were pelleted and washed in 20% TCA. The pellets were resuspended in 100 μl of 12.5% TCA and lysed by vortexing in the presence of glass beads. Cell lysates were pelleted, washed in 1 ml ice-cold acetone, and dried. Pellets were resuspended in Tris buffer (0.1 M Tris—HCl (pH 8.0), 1 mM EDTA, 1% SDS), loading buffer was added and samples were boiled for 5 min at 100°C. Samples were separated by SDS-PAGE and detected by immunoblotting with polyclonal anti-Atf1 [31].

Image analysis was performed using the TrackMate plugin [32] from Image J [33]. A Laplacian of Gaussian (LoG) particle detector was used on red channel and particle diameter was set at 2.2 μm or 3.0 μm depending on nuclei size. An automatic first quality threshold was applied without a second filtering. To follow nuclei during the time-lapse, a simple LAP tracker algorithm was used with a maximum linking distance of 5 μm, a maximum gap-closing distance of 5 μm and a maximum frame gap-closing of 10 frames. Then, the numerical values were extracted as a.csv file and manually analyzed using excel. Mean intensity values were used to follow SynCut3-mTagBFP2 and standard deviation of intensity values were used to follow DNA synthesis in mCherry-Pcn1. Individual cell cycles were synchronized to maximum SynCut3-mTagBFP2 values. To distinguish between the different cell cycle phases, an increase of 100% SynCut3 nuclear signal from baseline was used as threshold determination for mitosis entry, and an increase of 50% of mCherry-Pcn1 Standard Deviation from baseline was used as threshold for S-phase delimitation. Graphs were made using Pandas, Pyplot, and Seaborn packages from Python.

Cell size at septation was measured from cell pole to cell pole using the straight-line tool from Fiji when Sid2-GFP appeared at the septum. Septating time was measured as the time lapse between Sid2-GFP or GFP-CRIB fluorescence appeared at the septum and the 2 daughter cells were clearly separated. Growth rate was measured using the straight-line tool from Fiji when cells were under growth time (considered as non-septating time). CRIB signal at cell tips was measured as described in [17]. Briefly, the rectangle square from Fiji was used to measure the maximum CRIB signal, comprising only the cell tip from one cell.