New Results

Astral Microtubules Regulate Formation of the Cytokinetic Cleavage Furrow in Fission Yeast

Samantha E. R. Dundon, Thomas D. Pollard
doi: https://doi.org/10.1101/843110

Abstract

Microtubules of the mitotic spindle direct cytokinesis in metazoans but this has not been documented in fungi. We report evidence that astral microtubules help coordinate cytokinetic furrow formation in fission yeast. The temperature-sensitive cps1-191 strain (Liu et al., 1999) with a D277N substitution in β-glucan synthase 1 (Cps1/Bgs1) was reported to arrest with an unconstricted contractile ring. We discovered that rings in cps1-191 cells do constrict slowly and that an S338N mutation in the mto2 gene is required with the bgs1D277N mutation to reproduce the cps1-191 phenotype. Complexes of Mto2 and Mto1 with γ-tubulin regulate microtubule assembly. Deletion of Mto1 along with the bgs1D277N mutation also gives the cps1-191 phenotype, which is not observed in mto2S338N or mto1Δ cells expressing bgs1+. Both mto2S338N and mto1Δ cells nucleate fewer astral microtubules than normal and have higher levels of Rho1-GTP at the division site than wild-type cells. We report multiple conditions that sensitize mto1Δ and mto2S338N cells to furrow ingression phenotypes.

Summary Dundon and Pollard show that compromising the Mto1 or Mto2 regulators of the fission yeast γ-tubulin complex reduces or eliminates astral microtubules, exaggerates the effects of a non-lethal D277N substitution in β-glucan synthase 1 (Cps1/Bgs1) on the rate of cytokinetic furrow formation, and increases Rho-GTP at the cleavage site.

Introduction

Animals, fungi, and amoebas use an actomyosin contractile ring to divide. This ring is constructed at the end of the cell cycle in uninucleate cells, and cellular division must only proceed after successful DNA segregation to generate viable daughters. However, the mechanisms communicating DNA status to the contractile ring are still incompletely understood. Failure to correctly orchestrate this process can cause chromosome segregation errors and aneuploidy, which can promote chromosomal instability.

The fission yeast Schizosaccharomyces pombe is an excellent model for understanding mitosis and cytokinesis, including the most comprehensive cytokinetic parts list. The timing of mitotic and cytokinetic milestones is highly reproducible and well characterized; the stereotypical size and shape of S. pombe cells facilitates rigorous quantitative microscopy; and numerous temperature-sensitive alleles and facile genetic methods enable dissection of molecular processes.

S. pombe cells assemble a contractile ring from precursors called nodes over 10-15 min following separation of the spindle pole bodies (SPBs) at the onset of anaphase at 23°C (Vavylonis et al., 2008). During anaphase B, the ring matures by adding proteins such as the myosin-II isoform, Myp2 (Wu et al., 2003), the major motor for constriction (Laplante et al., 2015). The maturation period ends with onset of constriction 35 min after SPB separation, even if mutations slow the assembly of the ring (Tebbs and Pollard, 2013; Coffman et al., 2009; Roberts-Galbraith et al., 2010; Wang et al., 2014; Laplante et al., 2015). If a ring takes longer than 35 min to assemble, it constricts immediately (Chen and Pollard, 2011; Tebbs and Pollard, 2013; Li et al., 2016).

Three conditions appear to be required to begin furrow ingression: an intact contractile ring; a signal originating from the cell cycle clock; and initiation of septum synthesis. The enzyme β-glucan synthase 1 (Bgs1) is recruited with its regulator Rho1 to the equator where it synthesizes the primary septum (Arellano et al., 1996) and helps anchor the ring to generate inwardly directed force (Arasada and Pollard, 2014; Davidson et al., 2015). Furrow ingression initially depends on both tension produced by the contractile ring and septum deposition by Bgs1 (Zhou et al., 2015; Thiyagarajan et al., 2015), but after ~50% ingression, septum synthesis alone suffices to complete division (Proctor et al., 2012). Although the septum can be completed in the absence of the actomyosin ring, tension in the ring promotes septum deposition and maintains the circularity of the pore during septum deposition (Zhou et al., 2015; Thiyagarajan et al., 2015). Mutations compromising the contractility or tension in the ring decrease the constriction rate (Pelham and Chang, 2002; Proctor et al., 2012; Laplante et al., 2015; Tebbs and Pollard, 2013; Li et al., 2016). Ring constriction is faster if the internal pressure against which the furrow ingresses is reduced through mutation or increasing the osmolarity of the growth medium (Morris et al., 2019; Proctor et al., 2012). Bgs1, activated by Rho1, synthesizes the primary septum (Cortés et al., 2007). This is followed by synthesis of the secondary septum by Bgs4 (Rho1) and Ags1 (Rho2) (Cortés et al., 2012; 2005; Arellano et al., 1996; Calonge et al., 2000). While Rho activity is required for cytokinesis, it must decrease after septation to allow cell separation (Nakano et al., 1997).

The fascinating fission yeast strain cps1-191 (Liu et al., 1999) attracted our attention because it seemed to lack some aspect of the signal for initiating ring constriction. Liu et al. created this strain by chemical mutagenesis and reported that the cells arrested at 36°C with two nuclei and an unconstricted cytokinetic ring. They concluded that colonies did not grow at 36°C due to failed cytokinesis. They identified a candidate gene by genetic mapping, complemented temperature sensitivity of the strain with the wild type cps1+/bgs1+ gene, identified the D277N substitution in the gene and named the strain cps1-191 (Liu et al., 1999). Time-lapse microscopy of the cps1-191 strain confirmed that the nuclei separate normally but actomyosin rings remain intact without constricting for an hour after assembly at the restrictive temperature (Arasada and Pollard, 2014). The cps1-191 strain has been used in many studies to generate cells with nonconstricting actomyosin rings (Pardo and Nurse, 2003; Yamashita et al., 2005; Venkatram et al., 2005; Wachtler et al., 2006; Loo and Balasubramanian, 2008; Roberts-Galbraith et al., 2009) and others.

Based on these reports, we selected the cps1-191 strain to investigate the mechanisms that initiate cytokinetic ring constriction. However, we determine that rings in cps1-191 cells do not arrest per se, but constrict very slowly and that cells with the bgs1D227N mutation die from lysis at 36°C rather than arresting the cell cycle as previously thought. Surprisingly, we found that the cps1-191 constriction phenotype depends on a second point mutation in the gene for the γ-tubulin regulator Mto2, implicating microtubules in the process that drives furrow ingression.

S. pombe has multiple microtubule organizing centers (MTOCs) containing γ-tubulin in addition to the SPB (Sawin and Tran, 2006). During interphase, multiple MTOCs localize along microtubule bundles (Janson et al., 2005). During mitosis, interphase microtubules disassemble and the SPBs nucleate spindle and astral microtubules. Finally, during cytokinesis Myp2 recruits MTOCs to the equator (Samejima et al., 2010), where they nucleate the post-anaphase array (PAA) of microtubules. Mto1 and, to a lesser extent, Mto2 are critical proteins for the function and cell-cycle regulated activities of these MTOCs. In the absence of Mto1, no MTOCs form beyond the SPB, which only nucleates microtubules from its inner face (Zimmerman and Chang, 2005). In biochemical reconstitution experiments, the Mto1/Mto2 complex and MOZART1 homolog Mzt1 are required for γ-tubulin to promote microtubule nucleation (Leong et al., 2019). We find that Mto1/Mto2 complex mutants nucleate fewer than normal numbers of astral microtubules, and report one consequence, higher than normal Rho1-GTP activity at the cleavage site. Our findings reveal a new link between microtubules and furrow ingression in fission yeast.

Results

Cytokinetic furrows ingress slower in the cps1-191 strain than in cells with the bgs1D277N mutation

Long time-lapse movies of the cps1-191 strain with genome-encoded Rlc1-tdTomato (regulatory light chain for both isoforms of myosin-II, Myo2 and Myp2) revealed that the actomyosin ring constricted ~30-fold slower (median 0.02 μm/min) at the restrictive temperature of 36°C than in wild-type cells (median 0.62 μm/min) (Fig. 1A, B). We never observed abnormal ring structures such as detachment from the cell wall (Arasada and Pollard, 2014; Laplante et al., 2015; Cheffings et al., 2019), therefore we consider Rlc1-tdTomato constriction to reflect furrow ingression. Thirty percent of these fully-formed contractile rings slid along the membrane of cps1-191 cells at 36°C, as reported (Arasada and Pollard, 2014; Cortés et al., 2015). We shifted cps1-191 cells from the permissive (25°C) to restrictive (36°C) temperature on the microscope to determine the time required for the phenotype to manifest. When the SPBs separated within 30 min of the temperature shift, furrow ingression was normal. If SPB separation occurred >30 min after the shift, furrows did not form normally (Fig S1A).

Figure 1.
Figure 1. Both the bgs1D277N and the mto2S338N mutations are required to cause the cps1-191 constriction phenotype in a wild-type background.

A) Kymographs of inverted contrast, maximum-intensity projected images of contractile rings in strains with Rlc1-tdTomato at 36°C. Wild-type cells were imaged at 1-min intervals, and bgs1D277N and cps1-191 cells were imaged with 5-min intervals. The kymograph of the wild-type cell is displayed (left sub-panel) as acquired and (right sub-panel) rescaled to match the timescale of the kymographs (other panels) of the cps1-191 and six different bgs1D277N strains. Horizontal scale bars = 15 min, vertical scale bar = 1 μm. B) Rates of cytokinetic ring constriction measured from a subset of kymographs in A. The data are not normally distributed, so the median and 1st and 3rd quartiles are indicated by black bars, n ≥ 55 cells. C) Log10-transformed cytokinetic ring constriction rates of cells carrying the bgs1D277N mutation measured from kymographs as in A. The median and 1st and 3rd quartiles are indicated by black bars, n ≥ 57 cells. Significance differences were determined by Welch’s ANOVA followed by Tukey post-hoc test (p < 0.05). D) Cytokinetic ring constriction rates of cells carrying bgs1+ measured from kymographs as in A. The median and 1st and 3rd quartiles are indicated by black bars. No significant differences were detected by Welch’s ANOVA. E) Cumulative distribution plots showing accumulation of cells with rings that have (•) assembled, (■) initiated constriction, and (▲) completed constriction in wild-type and bgs1+ cells with various additional mutations at 36°C. n ≥ 71 cells for C and D.

We made a strain with just the bgs1D277N mutation in a wild-type background and found that furrowing progressed 3-fold faster (median 0.06 μm/min) than in cps1-191 cells. Nevertheless, these bgs1D277N cells had a growth defect at 36°C similar to the cps1-191 strain (Fig. S1B). Both bgs1D277N and cps1-191 cells lysed frequently during imaging (Fig. S1D), which caused the growth defect at 36°C on solid medium. None of the bgs1+ strains we examined lysed during observations. The lysis frequency varied substantially between replicate samples, suggesting that this phenotype is extremely sensitive to minute environmental differences. Consistent with this hypothesis, the osmotic stabilizer sorbitol partially rescued the growth of cps1-191 and bgs1D277N cells at 36°C (Fig. S1C).

The cps1-191 strain carries a large number of mutations

We sequenced the complete genome of the cps1-191 strain. Comparison to the S. pombe reference genome obtained from PomBase (Wood et al., 2002; Lock et al., 2019) revealed 384 unique mutations, 271 of which were transition mutations consistent with the nitrosoguanidine mutagenesis used to generate the strain (Balasubramanian et al., 1998) (Fig. 2A). Ten of these transition mutations substituted amino acids in eight genes, including the reported bgs1D277N mutation, located across all three chromosomes (Fig. 2B, Table 1).

Figure 2.
Figure 2. The cps1-191 strain carries many mutations.

A) Categorization of mutation types in cps1-191. B) Locations of cps1-191 transition mutations.

View this table:
Table 1. Mutations in ORFs in the cps1-191 strain.

Mutations in the Mto1/Mto2 complex synthetically interact with the bgs1D277N mutation

To determine which mutations contribute to the cps1-191 phenotype, we examined the PomBase annotations for each protein-coding gene with a substitution mutation (Table 1), looking for roles in cell cycle regulation or cytokinesis (Lock et al., 2019). Two of the eight affected genes, Mto2 and Nup40, met this criterion. Next, we took advantage of the fact that we saved multiple progeny from crosses generating cps1-191 strains with labeled rings and SPBs. We measured the furrowing rates at 36°C and determined the mto2 and nup40 mutation status for each of these strains. Only presence of the mto2S338N mutation correlated with the slowest furrowing rate observed for cps1-191 (Table S1). The mto2S338N mutation was also particularly interesting due to previous work linking Mto2 to the cytokinetic ring (Samejima et al., 2010).

Combining the bgs1D277N and mto2S338N mutations in a wild-type background reproduced the slow constriction rate of cps1-191 (Fig. 1A, C). The Mto2 C terminus is highly phosphorylated (Borek et al., 2015), so we tested if the absence of phosphorylation at S338 caused the synthetic interaction with bgs1D277N. Neither S338C, which precludes phosphorylation of S338, nor the [24A] strain with 24 phosphorylated serines mutated to alanines (Borek et al., 2015), constricted as slowly as the bgs1D277N mto2S338N or cps1-191 mutants, but instead resembled the bgs1D277N strain with the single mutation (Fig. 1A, C).

Mto2 contributes to the formation and regulation of non-SPB MTOCs in S. pombe through interactions with its binding partner Mto1 (Sawin and Tran, 2006). Neither mto1+ nor mto2+ are essential genes, and mutations of mto1+ are more severe than mutations of mto2+ (Sawin et al., 2004; Venkatram et al., 2005). In a bgs1+ background with Rlc1-tdTomato to label contractile rings and Pcp1-mEGFP to label SPBs, all mutant strains of mto1 and mto2 formed furrows at the wild-type rate with cytokinesis timelines similar to wild-type cells (Fig. 1D, E). Deletion of Mto1, but not Mto2, slowed furrowing in a bgs1D277N strain to the rate observed in cps1-191 cells, while mto2Δ cells exhibited an intermediate phenotype (Fig. 1C). Therefore, the bgs1D277N mutation sensitizes cells with a compromised Mto1/Mto2 complex.

Mitotic microtubules are disrupted in mto1Δ and mto2S338N cells

We investigated the microtubule cytoskeleton in mto1Δ and mto2S338N cells to determine common phenotypes that may affect furrow ingression. Over the course of spindle elongation in wild-type cells each SPB nucleated multiple, short-lived astral microtubules with a median duration of 20 s (n = 175 microtubules) (Fig. 3A, S2A). The mto2S338N strain produced fewer astral microtubules than normal, and most SPBs nucleated no astral microtubules in mto1Δ, as reported (Sawin et al., 2004)(Fig. 3B, C). We measured the numbers of microtubules nucleated by SPBs only during anaphase B, to avoid intra-nuclear “astral” microtubules nucleated prior to this stage, which may not be affected by mto1 loss (Zimmerman et al., 2004). As reported (Sawin et al., 2004), we did not observe PAA formation in mto1Δ cells, but the PAA formed at the normal time in mto2S228N cells.

Figure 3.
Figure 3. The mto2S338N and mto1Δ mutations perturb astral microtubules and the time of spindle breakage.

A) Time series of inverted contrast maximum-intensity projected fluorescence micrographs of wild type cells at 36°C with astral microtubules labeled with GFP-Atb2 (α tubulin). Arrows of the same color mark the same astral microtubule in consecutive frames. Scale bar 2 μm. B) Representative fluorescence micrographs of three strains (wild type, mto2S338N, and mto1Δ) at 36°C with spindle microtubules labeled with GFP-Atb2. Scale bar 2 μm. C) The number of astral microtubules nucleated from a single SPB over the course of mitosis at 36°C in wild-type, mto2S338N, and mto1Δ cells, n ≥ 21 spindle poles. Significance was determined using pairwise K-S tests with Bonferroni correction. D & E) Cumulative distribution plots comparing mitotic and cytokinetic outcomes in wild type, mto2S338N, and mto1Δ cells with GFP-Atb2 to mark microtubules, Rlc1-tdTomato to mark the cytokinetic ring, and Sfi1-mCherry to mark SPBs at 36°C, n ≥ 21 cells. D) Time course after time zero of the accumulation of cells with broken mitotic spindles. E) Time course after time zero of the accumulation of cells with rings that have (•) assembled, (■) initiated constriction, and (▲) completed constriction.

The time at which the spindle began to disassemble after reaching its maximum length, referred to as spindle breakage, occurred later than normal in both the mto2S338N and mto1Δ strains (Fig. 3D, p < 0.05 by pairwise K-S tests with Bonferroni correction). Both spindle breakage and initial formation of the PAA occur during the ring maturation stage (Fig. S2C).

Although the cytokinetic timelines of mto2S338N and mto1Δ cells were indistinguishable from wild-type cells at 36°C (Fig. 2E), GFP-Atb2 expression caused synthetic phenotypes with these mutations. Ring assembly was slightly delayed in mto2S338N and mto1Δ cells carrying GFP-Atb2 compared with wild-type cells bearing GFP-Atb2 (Fig. 3E, p < 0.05 by pairwise K-S tests with Bonferroni correction, n ≥ 21 cells). Completion of ring constriction was also delayed in mto1Δ cells in this experiment. This substantiates that the mto1Δ and mto2S338N mutations perturb cytokinesis when combined with additional stressors. We believe that the effects we observe on astral microtubule nucleation are not an artifact of GFP-Atb2 expression, as they are consistent with mto1Δ and mto2Δ microtubule-nucleation defects shown by immunofluorescence (Sawin et al., 2004; Janson et al., 2005; Samejima et al., 2005).

We considered whether PAA microtubules nucleated by the Mto1/Mto2 complex might promote trafficking of vesicles with Bgs1 to the division site and cause the synthetic effects of combining a Bgs1 mutation with compromised microtubule-nucleation machinery. However, the number of Bgs1 molecules recruited to the equator during cytokinesis of bgs1+ mto1Δ and bgs1+ mto2S338N cells did not differ from wild-type cells (Fig. S2D), refuting this trafficking hypothesis.

mto1Δ and mto2S338N cells do not exhibit SIN phenotypes

We wondered whether the septation initiation network (SIN) signaling pathway operates normally in the mto1Δ and mto2S338N mutants. The SIN (Hippo in humans and MEN in budding yeast) drives formation of the contractile ring and cytokinesis (Simanis, 2015). Hyperactivation promotes septum formation during interphase (Minet et al., 1979), while low SIN activity results in unstable contractile rings and unreliable cytokinesis, promoting formation of multinucleate cells (Mitchison and Nurse, 1985; Dey and Pollard, 2018). We rarely observed the formation of binucleate interphase cells in the mto1Δ mutant cells and never in mto2S338N cells. The mto1Δ cells have defects in nuclear positioning (Zimmerman and Chang, 2005), so both nuclei occasionally migrate into one daughter compartment after the contractile ring forms, resulting in one binucleate and one anucleate daughter cell. Contractile rings in these cells formed at the correct time in mitosis and did not disassemble. Early in mitosis, the SIN kinase Cdc7 first localizes to both SPBs and then to only one SPB during anaphase B (Sohrmann et al., 1998). Cdc7 localizes to one SPB in cps1-191 cells with an unconstricted ring, which suggests normal SIN activity (Liu et al., 1998).

Cytokinetic rings in cps1-191, bgs1D277N, and mto2S338N cells are so stable that we never observed one disassemble, suggesting that the SIN activity is normal. In 3 of 189 mto1Δ cells examined, a ring formed without SPB separation. One of these cells carried GFP-Atb2 and the microtubules emanated from the SPB in an aster-like array, so the ring likely formed at the normal time during a failed mitosis, rather than during interphase (Fig S2D). One of these three rings was subsequently disassembled, so SIN activity may be abnormal in a very small fraction of mto1Δ cells. The extremely low penetrance of ring formation without SPB separation and subsequent ring disassembly in mto1Δ cells (<0.5% of cells) prevented us from undertaking a quantitative examination of SIN activity in this population. The absence of this phenotype in any of the strains bearing mto1+ (n = 844 cells) suggests that SIN defects are not what sensitizes mto1Δ or mto2S338N cells to the loss of Bgs1 function.

mto1Δ modulates the effects of salt stress on cytokinesis

Acute salt stress produced by addition of 0.6 M KCl to rich medium (YE5S) causes a delay in ring constriction particularly in cells lacking myp2, which encodes one of two type-II myosins in fission yeast (Bezanilla et al., 1997; Okada et al., 2019). Myp2 recruits Mto1 to the ring and is required to generate the PAA (Samejima et al., 2010). We hypothesized that if loss of Mto1 at the equator causes sensitization to salt stress in myp2Δ cells, we would observe the same effect of salt stress in mto1Δ and mto2S338N cells. We found that 0.6 M KCl also decreases the rate of ring constriction in minimal medium (EMM5S) in wild-type, mto2S338N, and mto1Δ cells (Fig. 4A). Surprisingly, rings in mto1Δ cells constricted faster than in wild-type cells in the presence of this stress, though still more slowly than in the absence of KCl, so rings completed constriction earlier than in the other two strains in the presence of KCl (Fig. 4C). However, the mto1Δ and mto2S338N phenotypes are distinct from the myp2Δ phenotype under salt stress (Okada et al., 2019), so the loss-of-function phenotypes differ for the Mto1/Mto2 complex and Myp2.

Figure 4.
Figure 4. The mto2S338N and mto1Δ mutations sensitize cells to stresses.

A-C) Effects of 0.6 M KCl on cytokinesis in wild type, mto2S338N, and mto1Δ cells at 25°C. A) Rates of cytokinetic ring constriction. The median and 1st and 3rd quartiles are indicated by black bars and significance (p < 0.05) was determined using Welch’s ANOVA followed by Tukey post-hoc test. B & C) Time courses of cytokinetic events in cells expressing Rlc1-tdTomato to mark the cytokinetic ring and Pcp1-mEGFP to mark SPBs. Cumulative distribution plots show the accumulation of cells with rings that have (•) assembled, (■) initiated constriction, and (▲) completed constriction in medium with (B) no added KCl or (C) 0.6 M KCl. Dashed gray lines in (B) and (C) indicate the time when 50% of cells in the (B) reach each milestone to facilitate comparison. n ≥ 20 cells for A-C. D-F) Effects of the Rho-GTP biosensor Pkc1(HR1-C2) on cytokinesis at 36°C. D) Rates of cytokinetic ring constriction. The median and 1st and 3rd quartiles are indicated by black bars and significance (p < 0.05) was determined using Welch’s ANOVA followed by Tukey post-hoc test. E & F) Cumulative distribution plots comparing mitotic and cytokinetic outcomes in cells (E) without and (F) with the Pkc1(HR1-C2) Rho1 biosensor. Plots show the accumulation of cells with rings that have with (•) assembled, (■) initiated constriction, and (▲) completed constriction. Dashed gray lines in (E) and (F) indicate the time when 50% of cells in the (F) reach each milestone to facilitate comparison. n ≥ 21 cells for D-F.

The mto1Δ and mto2S338N cells have high Rho-GTP at the cleavage site

To explore one of many possible pathways influenced by the defects in astral microtubules, we localized S. pombe Rho1-GTP using the Pkc1(HR1-C2)-mEGFP biosensor (Davidson et al., 2015). This probe, derived from Saccharomyces cerevisiae Pkc1, contains Rho-GTP binding HR1 domains and a plasma-membrane targeting C2 domain fused to mEGFP (Kono et al., 2012). The fluorescence of this probe is the same when free or bound to Rho-GTP, so it can measure the local accumulation but not the total active Rho-GTP. Binding of biosensors can compromise functions of the target protein, so expression of the biosensor may perturb cytokinesis if Rho1-GTP regulates furrow formation. To limit perturbation, the thiamine-repressible 3nmt1 promoter controlled the expression of Pkc1(HR1-C2)-mEGFP and cells were grown medium lacking thiamine (i.e. inducing conditions) for only ~15 h prior to imaging, the minimum time for maximum expression (Maundrell, 1990). Conveniently, expression from the P3nmt1 promoter was highly variable, giving cells with a range of concentrations of Pkc1(HR1-C2)-mEGFP.

Expression of the biosensor consistently decreased the ring constriction rate of wild-type, mto2S338N, and mto1Δ cells (Fig. 4F), delaying completion of constriction for all genotypes. On the other hand, the constriction rates were not strongly correlated with Pkc1(HR1-C2) expression level (by Spearman correlation test, which was used because Pkc1(HR1-C2) expression levels were not normally distributed)(Fig. S2B). The cytokinetic timelines of wild-type and mto2S338N cells were statistically indistinguishable with and without Pkc1(HR1-C2), but expression of the biosensor delayed further both the duration of maturation and completion of constriction in mto1Δ cells (p < 0.05 by K-S test with Bonferroni correction)(Fig. 4D, E).

Fluorescence microscopy of Pkc1(HR1-C2)-mEGFP confirmed that Rho1-GTP concentrates at cell tips during interphase and at the cytokinetic furrow in wild-type cells (Fig. 5A, Movie S1) as reported (Davidson et al., 2015). Fig. 5A shows a representative mto1+ mto2+ cell with the same pattern of Pkc1(HR1-C2)-mEGFP localization observed in mto1Δ and mto2S338N cells. Pkc1(HR1-C2)-mEGFP also concentrated at other cortical locations; this ectopic localization was much more frequent in mto2S338N (49%) and mto1Δ (46%) mutants than in wildtype (23%) cells (Fig. 5B, Movies S2, S3.). The bgs1D277N cells expressing Pkc1(HR1-C2)-mEGFP grew very poorly at 36°C, so we were unable to quantify Rho activity. This genetic interaction suggests that cps1-191 cells are more sensitive to the effects of the Rho biosensor on Rho1 function.

Figure 5.
Figure 5. The mto2S338N and mto1Δ mutations disrupt Rho signaling.

A & B) Time series (min) at 36°C of inverted contrast maximum-intensity projected fluorescence micrographs of a wild-type cell expressing the Rho biosensor Pkc1(HR1-C2)-mEGFP and Pcp1-mEGFP to mark spindle pole bodies. Scale bars 2 μm. A) Wild-type cells. Active Rho concentrates at poles of interphase cells (arrowheads) and at the equator of cells during cytokinesis and septation. Times are minutes after SPB separation. B) Representative images of mto1Δ and mto2S338N cells during interphase showing fluctuating, ectopic concentrations of the biosensor (arrows) at times denoted in minutes after the start of image acquisition. Ectopic accumulations were observed in 23% of wild-type, 49% of mto2S338N, and 46% of mto1Δ cells; n ≥ 72 cells, p < 0.05 by Chi-Squared test with Bonferroni correction for multiple comparisons. C) Time course of the integrated density of the Rho biosensor at equator of the cell in (A) during cytokinesis normalized to interphase value. The main peak (dark blue) is during furrow formation and the short second peak (light blue) is at cell separation. D & E) Rho biosensor signals at the equators of populations of ≥ 21 cells. Significance (p < 0.05) was determined using Welch’s ANOVA and Tukey’s post-hoc analysis. D) Peak Rho biosensor signal at the equator during cytokinesis and separation. E) Duration of Rho biosensor peak during cytokinesis.

The Pkc1(HR1-C2)-mEGFP signal peaked at the cleavage site during furrow ingression (Fig. 5C), followed by a brief decrease and second peak during cell separation (single light blue data point in Fig. 5C). The normalized peaks of Pkc1(HR1-C2)-mEGFP fluorescence during cytokinesis and at separation were more intense in mto2S338N and mto1Δ cells than wild-type cells (Fig. 5D). Furthermore, the cytokinesis peak of Pkc1(HR1-C2)-mEGFP fluorescence was prolonged in mto1Δ cells (Fig. 5E).

Discussion

Since its description in 1999, the cps1-191 strain has been the gold standard to generate S. pombe cells arrested with stable, unconstricting actomyosin rings. We find a more nuanced phenotype that depends on both the originally-identified bgs1D277N mutation and an additional mutation, mto2S338N. Caution is advised when interpreting published results from cps1-191 experiments examining the “arrest” phenotype, particularly those that required crossing the cps1-191 strain with other strains, as the mto2S338N mutation may no longer be present with the bgs1D277N mutation. The bgs1 and mto2 loci are both on Chromosome II but are ~2 Mb apart. Isolating progeny that undergo a recombination event between these genes is common in our hands. A cps1-191 mto2Δ double mutant was previously examined, though the authors were presumably unaware of the mto2S338N mutation in the cps1-191 strain (Venkatram et al., 2005). However, we found that bgs1D277N mto2S338N had a more severe phenotype than bgs1D277N mto2Δ. The molecular basis of this difference is currently unknown.

Microtubule depolymerization in metazoan cells increases Rho-GTP levels and has profound effects on the actin cytoskeleton (Zhang et al., 1997; Danowski, 1989; Liu et al., 1998; Enomoto, 1996). Astral microtubule plus ends carry the Aurora A activator TPXL-1 to the cortex, where it inactivates Rho in C. elegans embryos (Mangal et al., 2018). Binding to microtubules inactivates Rho GEF-H1 in mammalian cells, so depolymerizing microtubules releases this GEF and thereby increases Rho activity (Krendel et al., 2002). Cells lacking Mto1 produce no PAA microtubules. The loss of the PAA microtubules in addition to fewer astral microtubules may explain why mto1Δ cells recruit more Rho1-GTP to the division site than mto2S338N cells, which form a normal PAA and have an intermediate astral microtubule number. While S. pombe has no clear homologs for either TPXL-1 or GEF-H1, the high and prolonged Rho activity at the cell equator in mto2S338N and mto1Δ cells suggests mitotic microtubules may also inhibit cortical Rho in fission yeast.

When in the active, GTP-bound state, RhoA in metazoan cells localizes to the cell cortex and GDP-dissociation inhibitors (GDIs) sequester GDP-bound GTPases in the cytoplasm (Michaelson et al., 2001). Membrane-localization through the C-terminal CAAX motif is thought to increase Rho activity by increasing the likelihood of activation by GEFs. This hypothesis is supported by the observation that specific localization of the p63RhoGEF to the plasma membrane is sufficient to drive RhoA activation and its downstream effects (van Unen et al., 2015). Therefore, we expect that the pool of Rho1-GTP we detect with the plasma-membrane localized Pkc1(HR1-C2)-mEGFP biosensor represents the relevant pool of active Rho1.

The mto1Δ and mto2S338N cells recruit excess Rho-GTP to the division site, but an additional insult (e.g. Bgs1 mutation, salt stress, Rho biosensor or GFP-Atb2 expression) is required to sensitize these cells and produce a furrow ingression phenotype. Why cells tolerate excess Rho-GTP so well, and how increased Rho-GTP modulates furrow ingression under multiple stressors are unclear. Much more work is required to assess which of the Rho1 regulators contribute to the observed hyperactivation and exactly how this influences furrow ingression. Many Rho1 regulators are known to be involved in septation, but how microtubules might regulate any of these proteins is currently unclear.

An additional mystery is why combination of Mto1/Mto2 mutations with the bgs1D227N mutation decreases the furrowing rate while salt stress increases the furrowing rate, particularly because these mutants recruit normal levels of Bgs1 to the division site. Cells depleted of Bgs1 using the nmt1 promoter and cps1-191 cells exhibit abnormally-thick cell walls and secondary septa, suggesting that other glucan synthases attempt to compensate for the loss of Bgs1 activity (Cortés et al., 2007; 2015; Sethi et al., 2016). Excess Rho1-GTP at the division site may compromise the activity of these enzymes.

Changing Rho expression perturbs the levels of other GTPases (e.g. Rac, Cdc42) through competition for shared GDIs in mammalian cells, and it has been proposed that changes in the activity of Rho could also affect other GTPases through this mechanism (Boulter et al., 2010). S. pombe has a single Rho GDI, Gdi1, so an altered Rho1-GDP/GTP balance may change the amount of Gdi1 available to interact with other GTPases involved in cytokinesis such as Cdc42, Rho2, and Rho4. Rho1 activates Bgs1 (primary septum) and Bgs4 (secondary septum), but Rho2 regulates Ags1 (secondary septum). In bgs1D277N mto2S338N or bgs1D277N mto1Δ cells, increased Rho1 at the division site may perturb Rho2 and Ags1 activity, compromising the ability of cells to compensate for lack of Bgs1 and causing even slower furrow ingression than the bgs1D277N substitution alone.

Our data suggest that mitotic microtubules, and astral microtubules in particular, participate in signaling to the division machinery and regulate furrow ingression. The SPB is known to serve as a hub for cell cycle signals—e.g. the SIN—and proteins localizing to the SPB throughout mitosis serve as markers for progression of the cell cycle (Tatebe et al., 2001; Grallert et al., 2004). Two, non-mutually-exclusive options for astral microtubule involvement in this signaling are facilitating recruitment of known cell-cycle regulators to the SPB and serving as signaling platforms in their own right. Much more research is required to determine exactly how microtubules interact with the three requirements for furrow formation: an intact contractile ring; a signal originating from the cell cycle clock; and initiation of septum synthesis.

Our work demonstrates that crosstalk between microtubules and the furrow machinery mediated by Rho is conserved more broadly than previously thought and raises many intriguing questions regarding the molecular details of this communication in fungal cells. During cytokinesis of metazoan cells, RhoA regulates the location of the cytokinetic ring, activates formins for cytokinetic ring assembly, promotes myosin activity through Rho-associated protein kinase (ROCK), and promotes midbody ring maturation through Citron kinase (Pollard, 2017; El-Amine et al., 2019). Ring positioning is achieved through other mechanisms in fission yeast (Pollard, 2017), but Rho1 contributes to full activation of the SIN to drive ring formation (Alcaide-Gavilán et al., 2014) and has essential roles in septum synthesis through its interaction with glucan synthases (Arellano et al., 1996). This suggests that Rho signaling is an ancient mechanism for coordinating cytoskeletal elements and that during the divergence from the LECA (the last eukaryote common ancestor) Rho signaling has been adapted for a range of functions to regulate cytokinesis.

Methods and Materials

Growth conditions and strain construction

Table S2 lists the S. pombe strains used in this work. Oligonucleotide primers were from Millipore Sigma. Restriction enzymes were from New England BioLabs. Plasmid purification was performed using an E.Z.N.A Plasmid Mini Kit (Omega Bio-tek) with Miraprep modifications (Pronobis et al., 2016). The Keck DNA Sequencing Lab of Yale University performed routine DNA sequencing. All cloning was done with high-efficiency NEB 5-alpha Competent E. coli from New England BioLabs. Standard methods were used for genetic crosses (Moreno et al., 1991).

mto2S338N:KanMX6 and mto2S338C:KanMX6 strains of S. pombe were generated by homologous integration (Bähler et al., 1998). First, pFA6a-mto2S338N-C-KanMX6 was generated. A 245-base fragment of the end of the mto2 coding sequence was amplified from cps1-191 (containing the mto2S338N mutation) with flanking homology to the pFA6a vector using the Expand™ High Fidelity PCR system (Millipore Sigma). The vector backbone was amplified from pFA6a-GFP-KanMX6 using Phusion polymerase (New England BioLabs). Both PCR reactions were digested with DpnI and purified using an E.Z.N.A Cycle Pure Kit (Omega Biotek). pFA6a-mto2S338N-C-KanMX6 was generated using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs) in an assembly containing 5 fmol of each fragment. The mto2S338N segment was verified by sequencing. pFA6a-mto2S338C-C-KanMX6 was generated by site-directed mutagenesis. Mutagenic primers were used to amplify pFA6a-mto2S338N-C-KanMX6 using the Expand High Fidelity PCR system (Millipore Sigma). The PCR product was digested with DpnI and transformed into NEB 5-alpha Competent E. coli. The mto2S338C segment was verified by sequencing.

PCR-based homologous integration cassettes were amplified using the Expand™ High Fidelity PCR system (Millipore Sigma) from the appropriate plasmid with 80 bp flanking homology to replace the 3’ end of mto2 (primers designed with PPPP at www.bahlerlab.info/resources/)(Bähler et al., 1998). Lithium acetate transformation was used to introduce 5-10 μg of integration cassette DNA (Murray et al., 2016). Integration at the correct locus was verified by colony PCR using LongAmp Taq 2X Master Mix (New England BioLabs), and the mto2 gene was sequenced in its entirety to verify the S338N or S338C point mutations.

The bgs1D277N strain was generated by CRISPR (Fernandez and Berro, 2016). A two-step PCR was used to assemble pUR19-adh1-Cas9-rrk1-sgRNA:Fex1+ with a guide sequence targeting bgs1 using Q5 High-Fidelity polymerase (New England BioLabs). pUR19-adh1-Cas9-rrk1-sgRNA:Fex1+ and a 400-bp repair guide fragment containing the bgs1D277N mutation were introduced into fex1Δ fex2Δ S. pombe cells by lithium acetate transformation (Murray et al., 2016). The bgs1 gene was sequenced in its entirety to verify the bgs1D277N mutation.

Whole-genome sequencing

S. pombe genomic DNA was isolated using a Quick-DNA Fungal/Bacterial Miniprep kit and purified by a Genomic DNA Clean & Concentrator-25 kit (both from Zymo Research). Genomic DNA was sequenced on a NovaSeq 6000 (Illumina) with 150 bp paired reads at the Yale Center for Genome Analysis. Sequence data quality was examined using FastQC v 0.11.7 (Babraham Bioinformatics). The S. pombe reference genome was downloaded from PomBase (Lock et al., 2019) and reads were aligned using MagicBLAST (Zhang et al., 2015). Aligned reads were sorted, duplicates removed, and indexed using SAMtools (Li et al., 2009). Deviations from the reference genome were identified using Pilon (Walker et al., 2014), and alignments were viewed using IGV 2.4.10 (Robinson et al., 2017). Identified mutations were cross-referenced with PomBase to determine location, eliminate previously-reported variants, and identify proteincoding mutations (Lock et al., 2019).

Microscopy

Cells were grown in 50 mL baffled flasks in exponential phase at 25°C in YE5S (rich) medium for 24 h, then washed into EMM5S (low-fluorescence) medium for an additional 12 h. For experiments at 36°C, the culture was shifted to 36°C 2 h prior to mounting and imaging. To image, cells were isolated by centrifugation at 2300 x g for 30 s, washed with fresh EMM5S, and resuspended at ~20-fold higher concentration than in culture. Then 1.5-2.0 μL of concentrated cells were mounted on an EMM5S + 2% agarose pad freshly poured on a Therminator block (Davies et al., 2014) and a 24 x 50 mm no 1.5 coverslip was applied (Globe Scientific). Then 150 μL of EMM5S was injected beneath the coverslip in contact with the agarose pad and the edges of the chamber were sealed using VALAP to reduce shrinkage of the pad. For salt stress experiments, cells at 25°C were washed and resuspended in EMM5S + 0.6 M KCl, then immediately mounted on an EMM5S + 0.6 M KCl + 2% agarose pad on a Therminator block and imaged at 25°C.

Cells were visualized using an Olympus IX-71 microscope with a 100×/numerical aperture (NA) 1.4 Plan Apo lens (Olympus) and a CSU-X1 (Andor Technology) confocal spinning-disk confocal system equipped with an iXON-EMCCD camera (Andor Technology). OBIS 488 nm LS 20 mW and OBIS 561 nm LS 20 mW lasers (Coherent) for excitation. The desired temperature was maintained during acquisition using a home-built version of the Therminator (Davies et al., 2014). Images were acquired using Micromanager 1.4 (Edelstein et al., 2014).

Acquisition settings for Pcp1-mEGFP Rlc1-tdTomato: 12 Z-slices were acquired with 0.5 μm spacing; 22 μW 561 nm laser power and 50 ms exposures; 35 μW 488 laser power and 50 ms exposures (power measured at the objective). An emission filter suitable for both tdTomato and mEGFP fluorescence was used to reduce the number of filter changes. All bgs1+ cells were imaged at 4 XY positions and 1-min intervals. Cells carrying bgs1D277N were imaged at 20 XY positions and 5-min intervals.

Acquisition settings for Rlc1-tdTomato Sfi1-mCherry mEGFP-Atb2: For cytokinesis timelines to determine the timing of spindle breakage, 12 Z-slices were acquired with 0.5 μm spacing at 3 XY positions and 1-minute intervals using 150 ms exposures with 32 μW 561 nm laser power and 200 ms exposures with 89 μW 488 laser power at the objective. Separate emission filters were used for tdTomato/mCherry and mEGFP fluorescence to avoid bleed through of the fluorophores with different spectral characteristics. To measure astral microtubule numbers and longevities, only mEGFP-Atb2 was imaged using 89 μW 488 laser power and 200 ms exposures of 10 Z-slices with 0.5 μm spacing at one XY position at 10-s intervals.

Acquisition settings for GFP-Bgs1 Rlc1-tdTomato Sfi1-mCherry: 12 Z-slices with 0.5 μm spacing were acquired at 3 XY positions and 1-min intervals using 150 ms exposures with 32 μW 561 nm laser power and 100 ms exposures with 89 μW 488 laser power at the objective. Separate emission filters were used for tdTomato/mCherry and mEGFP fluorescence to avoid bleed through of the fluorophores with different spectral characteristics. Strains bearing GFP-tagged proteins for the calibration curve were imaged under identical conditions. For camera noise correction, 100 images were acquired with 100 ms exposures and the shutter closed; for uneven illumination correction, dilute fluorescein was mounted on a 2% agarose EMM5S pad on the Therminator and 100 images were acquired using 50 ms exposures with 34 μW 488 laser power.

Image Analysis

All image visualization and analyses were done using Fiji (Schindelin et al., 2012). Macros used for processing can be downloaded from https://github.com/SDundon/ImageJ-Processing-Macros. Prior to analysis, the contrast of each image was adjusted so that the black point was 5% below extracellular background signal and the white point was 20% above maximum signal intensity. Representative images were selected based on similarity to median values of each data set.

Constriction rates and cytokinesis timeline measurements: Maximum-intensity projections were produced for each time-lapse acquisition and a kymograph was first produced for each ring. If necessary, the StackReg plugin (Thévenaz et al., 1998) was used to register images across all time points. A line was drawn parallel to the ring, the “reslice” tool in Fiji used, and the resliced image was maximum-intensity projected to create a kymograph. Ring circumference for each time point was calculated using the 180912_AutoCircum.ijm macro. Circumference was plotted against time as a scatter plot in Microsoft Excel, and a linear trendline fit to the slope to calculate the constriction rate. For analysis of cells bearing the bgs1D277N mutation (including cps1-191), cells were excluded from analysis if SPB separation occurred > 2 h after the start of acquisition. We found that rings formed after this time always constricted at an even slower rate, most likely due to effects of phototoxicity.

Cytokinesis milestones were defined as follows: “SPB separation” is the first frame in which two SPBs labeled with Pcp1-mEGFP could be resolved; “Complete assembly” is the first frame in which no Rlc1-tdTomato-labeled nodes were visible adjacent to the ring, because all had coalesced at the equator; “Constriction onset” is the first frame in which the Rlc1-tdTomato-labeled ring was smaller than its initial diameter and continued to decrease in size; “Constriction complete” is the frame in which the Rlc1-tdTomato signal reached its smallest size at the end of constriction. All milestones were temporally aligned with SPB separation set as time 0 and plotted as cumulative distributions. Spindle breakage was defined as the first time point in which there was no longer a single microtubule structure marked with GFP-Atb2 connecting the two SPBs. PAA appearance was defined as the first frame in which microtubules appeared at the equator distinct from the mitotic spindle.

Astral microtubule measurements: Astral microtubules were considered to be the same structure across consecutive frames (10 s intervals) if they a) originated from the same SPB and b) were oriented in the same direction ± 45° relative to the SPB in consecutive frames (Fig. 3A). The number of astral microtubules that originated from a single SPB during anaphase B was determined to exclude the intra-nuclear “astral” microtubules that form during earlier phases of mitosis (Zimmerman et al., 2004).

Rho biosensor and GFP-Bgs1 measurements: A ROI was selected for bleach correction reference using the following criteria: 1) must stay within the boundaries of one cell for the duration of the acquisition. 2) must be located within a cell that does not divide for the duration of the movie (to prevent differences in cytosolic concentration due to recruitment to the division site); 3) Pcp1-mEGFP-marked SPB must never enter the ROI. The same ROI was used for bleach correction of all channels. Bleach correction was conducted using the Fiji plugin for Exponential Fitting Method for the Bleach Correction. The intensity from all 12 Z-slices (0.5 μm apart) was then summed.

Rho biosensor measurements: Prior to analysis, the contrast of each image was adjusted so that the black point was 5% below extracellular background signal and the white point was 20% above maximum signal intensity of Pcp1-mEGFP, which was more consistent than pkc1(HR1-C2)-mEGFP signal. As previously observed, expression from the 3nmt1 promoter was highly variable. Therefore, cells were analyzed that had an average pkc1(HR1-C2)-mEGFP pixel intensity of >175,000 A.U. at SPB separation. This was calculated by drawing a spline-fit polygon ROI around the cell at the last time point prior to SPB separation. The integrated density was measured for this ROI, as well as a circular ROI covering the SPB. The SPB integrated density was subtracted from the cellular integrated density, and this value divided by the cell area with SPB area subtracted. This value (175,000 arbitrary units) was selected because cells of all genotypes below this threshold were found to have very low Peak Rho/Interphase Rho ratio values with a very different standard deviation than cells above this threshold. We reasoned that cells below this threshold express insufficient pkc1(HR1-C2)-mEGFP to enable reliable measurement of active Rho enrichment at the cell equator. After elimination of cells below this threshold, the data were homoscedastic when the ratio was plotted against average pixel intensity.

To calculate the Peak Rho/Interphase Rho ratio, a 3.75 x 1.95 μm ROI was positioned with the cytokinetic ring in the center (long axis to match the cell width). The integrated density was measured for this ROI for each time point. These values were divided by the integrated density of this ROI for the time point immediately prior to SPB separation to normalize the intensity by Pkc1(HR1-C2)-mEGFP expression. Normalized intensity was plotted against time using Microsoft Excel, all time points in which the SPB was located within the ROI were excluded. To measure peak duration, the peak fluorescence start was defined as the last time point of consistent signal increase across >3 time points. Peak end was defined as the last time point prior to consistent signal decrease below the peak start value across >3 time points. For cytokinesis peak signal, the maximum ratio between these points was determined. For separation peak signal, the maximum normalized signal within two time points of cell separation was determined.

Measurements of Bgs1 molecule numbers: Bleach-corrected summed intensity images were corrected for camera noise and uneven illumination. To generate the calibration curve, a spline-fit polygon ROI was drawn around wild-type (unlabeled) cells and the average intensity in the ROI was measured (n = 74 cells). Subsequently, ROIs were similarly drawn around cells bearing seven different GFP-tagged proteins for the calibration curve (n > 65 cells). For each cell, the wild-type background fluorescence was subtracted and the integrated density was calculated. These integrated density measurements were plotted against the average number of molecules per cell for each protein reported in Wu and Pollard, 2005 and fit with a linear regression. A 1.0 μm by 3.75 μm (cell width) ROI was positioned to cover the equatorial GFP-Bgs1 signal and the integrated density measured from cell cycle time zero until 30 min past the completion of constriction. The same ROI was used to measure cytoplasmic GFP-Bgs1 signal adjacent to the ring at constriction onset. Cytoplasmic GFP-Bgs1 integrated density was subtracted from the equatorial signal until the onset of constriction. The average constriction rate and average ring size were used to calculate a correction factor for the progressively smaller cytoplasmic volume within the plane of the constricting ring, which was applied to the value of the cytoplasmic signal and subtracted from the equatorial signal until constriction completion.

Statistical analysis

All statistical tests were carried out in R (R Core Team, 2018). Plotting was done using tidyverse and ggbeeswarm packages (Wickham, 2017; Clarke and Sherrill-Mix, 2017), except for pie charts in Fig. 2, which were generated using Microsoft Excel.

Acknowledgments

Research reported in this publication was supported by National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM026132 and an NRSA fellowship (F32GM125193) to S. Dundon. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank M. Balasubramanian, M. Das, and D. McCollum, for useful discussions; F. Chang, M. C. King, J. C. Ribas, K. E. Sawin, and J.-Q. Wu for generously sharing S. pombe strains; J. Berro and R. Fernandez for assistance with CRISPR; R. M. Dundon and T. J. Straub for assistance with genome analysis. The authors declare no competing financial interests.

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Posted November 14, 2019.
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Astral Microtubules Regulate Formation of the Cytokinetic Cleavage Furrow in Fission Yeast
Samantha E. R. Dundon, Thomas D. Pollard
bioRxiv 843110; doi: https://doi.org/10.1101/843110
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