The septation initiation network controls the assembly of nodes containing Cdr2p for cytokinesis in fission yeast

During cell division, fungal and animal cells use a contractile ring composed of actin filaments and myosin to cleave in two. The contractile ring in the fission yeast Schizosaccharomyces pombe forms from discrete, protein structures called nodes (Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009; Paoletti and Chang, 2000; Wu et al., 2006). Two types of nodes appear at different times and places during interphase and combine to form the cytokinetic nodes that assemble into the contractile ring (Akamatsu et al., 2014). Type 1 nodes, which are marked by the kinases Cdr1p and Cdr2p, appear in early G2 in a broad band around each daughter nucleus (Morrell et al., 2004; Moseley et al., 2009; Paoletti and Chang, 2000; Wu et al., 2006). Cdr1p and Cdr2p negatively regulate the kinase Wee1p, which inhibits the cyclin-dependent kinase Cdk1p as part of the mechanism controlling the entry into mitosis (Moseley et al., 2009; Wu and Russell, 1993). The anillin Mid1p moves from the nucleus and joins type 1 nodes during G2 (Morrell et al., 2004; Moseley et al., 2009; Paoletti and Chang, 2000; Wu et al., 2006). Type 2 nodes, composed of contractile ring proteins Blt1p, Klp8p, Nod1p and Gef2p (Jourdain et al., 2013; Moseley et al., 2009; Zhu et al., 2013), emerge from the contractile ring as it disassembles at the end of cytokinesis. During interphase, type 2 nodes diffuse in the cortex from the previous division site until they bind stationary type 1 nodes around the cell equator to form cytokinetic nodes (Akamatsu et al., 2014). Early in mitosis, these nodes accumulate additional contractile ring proteins (Moseley et al., 2009; Saha and Pollard, 2012). During anaphase, the type 2 node proteins, Mid1p and other proteins condense into the contractile ring (Vavylonis et al., 2008), while type 1 nodes move away from the equator as their proteins disperse into the cytoplasm for the duration of mitosis, a time called the eclipse period (Akamatsu et al., 2014).

A signaling cascade called the septation initiation network (SIN) originates from spindle pole bodies (SPBs) and regulates the onset of cytokinesis and septation (Johnson et al., 2012; Sparks et al., 1999). A GTPase-activating protein (GAP) composed of Byr4p and Cdc16p negatively regulates the GTPase Spg1p at the top of the SIN. As a cell enters mitosis, cell cycle kinases Cdk1p and Plo1p phosphorylate Byr4p, inhibiting the GAP activity and activating the SIN (Rachfall et al., 2014). Activating Spg1p on one SPB during mitosis turns on a cascade of three kinases – Cdc7p, Sid1p and Sid2p (Fankhauser and Simanis, 1994; Guertin et al., 2002; Johnson et al., 2012; Krapp and Simanis, 2008; Sohrmann et al., 1998). Low Cdk1p activity during anaphase allows Sid1p to accumulate on the SPB (Guertin et al., 2000) and to activate Sid2p, which moves with its binding partner Mob1p to the contractile ring to initiate constriction (Hou et al., 2004; Sparks et al., 1999).

We recently found that Cdr2p does not disperse from type 1 nodes during mitosis in sid2-250 mutant cells, suggesting that the SIN controls the assembly of type 1 nodes (Akamatsu et al., 2014). Here, we use a conditional mutation to turn the SIN on and off to establish that the SIN is both sufficient and necessary to disperse type 1 nodes.

We used the presence of the SIN kinase Cdc7p–GFP on spindle pole bodies (SPBs) to track SIN activity across the cell cycle (García-Cortés and McCollum, 2009; Sohrmann et al., 1998). We define the separation of the SPBs as cell cycle time zero (Wu et al., 2003). Cdc7p–GFP was dispersed in the cytoplasm during interphase, but concentrated at the single or both duplicated SPBs early in mitosis at cell cycle time +2±1.5 min (±s.d.; Fig. 1A,C), marking activation of the SIN. Active Cdc7p–GFP remained on both SPBs throughout anaphase but disappeared from the old SPB approximately at the end of anaphase B when the mitotic spindle reached its maximum length (Fig. 1A) (García-Cortés and McCollum, 2009). The SIN kinase Sid2p–mEGFP was visible at the SPB during interphase and persisted on both SPBs through mitosis. At the end of anaphase, Sid2p–mEGFP partially relocalized to the contractile ring at time +30±4 min where it persisted until the daughter cells separated (Fig. 1A,C) (Hou et al., 2004; Hou et al., 2000; McCormick et al., 2013; Salimova et al., 2000; Sparks et al., 1999).

During the cell cycle the presence of type 1 nodes marked by the kinase Cdr2p was inversely correlated with SIN activity (Akamatsu et al., 2014), so nodes were present around the equator when SIN was inactive from early G2 phase until early mitosis (Fig. 1C). During anaphase, type 1 nodes separated from the forming contractile ring and then dispersed into the cytoplasm ∼20 min after Cdc7p appeared on SPBs (Fig. 1A), leaving a small fraction (∼15%) of Cdr2p–mEGFP at the division site (Fig. 1A) (Akamatsu et al., 2014; Morrell et al., 2004; Saha and Pollard, 2012). Cdr2p nodes reappeared in the cortex around the nuclei of the daughter cells at time +67±4 min, coincidently with disappearance of Cdc7p–GFP from the SPBs at +68±5 min (Fig. 1B,C). Cdr2p nodes reappeared asymmetrically in 75% of cells, first around the inactive mother SPB lacking Cdc7p–GFP and then around the active daughter SPB 3±2 min later as Cdc7p–GFP disappeared from that SPB (Fig. 1D).

We tested for a cause and effect relationship between SIN activity and the dispersal of type 1 nodes by turning the SIN on and off with a temperature sensitive cdc16-116 mutation of the SIN inhibitor GAP Cdc16p (Fig. 2A,B; supplementary material Fig. S2A). Starting 14 min after shifting interphase cdc16-116 cells to the restrictive temperature of 35°C, SIN turned on asynchronously in the population of cells as indicated by accumulation of Cdc7p–GFP on the single interphase SPBs (Fig. 2C,E; supplementary material Movie 1). The fraction of interphase cells with type 1 nodes marked with Cdr2p–mEGFP declined in direct proportion to the fraction of cells with Cdc7p–GFP on their SPB (Fig. 2G). Activating SIN during interphase also dispersed the anillin Mid1p from type 1 nodes (supplementary material Fig. S2). After 100 min at the restrictive temperature, ∼80% of interphase cells accumulated Cdc7p–GFP at the SPB and dispersed their type 1 nodes (Fig. 2E,G). In contrast to wild-type mitotic cells, these cells retained no Cdr2p–mEGFP around the middle of the cell when their nodes dispersed (Fig. 2C). On a cell-by-cell basis, Cdc7p–GFP appeared at the SPB (Fig. 2C) 5±3 min (±s.d.) before Cdr2p–mEGFP dispersed from nodes (Fig. 2E,G). Similarly, in early G2 cells still connected by a septum the SPB in one daughter cell accumulated Cdc7p–GFP 7±5 min before the second SPB followed by the disappearance of Cdr2p–mEGFP from type 1 nodes (supplementary material Fig. S1A).

Returning interphase cdc16-116 cells from 35°C to the permissive temperature of 23°C turned off the SIN and allowed type 1 nodes to reform with Cdr2p and Mid1p (Fig. 2B,D; supplementary material Fig. S2B; Movie 2). In a population of cells, the time that Cdc7p–GFP disappeared from SPBs was highly correlated with the time Cdr2p–mEGFP reappeared in type 1 nodes, with a delay of only 4±5 min (Fig. 2F,G).

During interphase, wild-type cells had broad bands of type 1 nodes around their equators and no Cdc7p at their SPBs at 23°C (Fig. 2A,D). When shifted to 35°C, wild-type cells did not accumulate Cdc7p–GFP at the SPBs or disperse Cdr2p–mEGFP from nodes (Fig. 2A,C).

We used cdc16-116 cells expressing Cdr2p–mEGFP and Sad1p–mCherry to investigate how SIN activity influences Cdr2p during mitosis. These cells entered mitosis normally at all temperatures, allowing for observation of the effect of the SIN activity on mitotic entry. At the restrictive temperature, where the SIN is hyperactive during mitosis, nodes dispersed at time 0±2 min (±s.d.) simultaneously with SPB separation in cells entering mitosis, far earlier than in wild-type cells in which this occurred at time +16±4 min (Fig. 3A) (Vavylonis et al., 2008). We do not know when Cdc7p associated with SPBs in these experiments, because genetic interactions between tagged Cdc7p–GFP and cdc16-116 during mitosis precluded including this Cdc7p marker. The cdc16-116 cells expressing Cdc7p–GFP, Cdr2p–mEGFP and Sad1p–mCherry entered mitosis normally at the permissive temperature of 23°C, but at the restrictive temperature no SPBs separated during 100 min of imaging. This mitotic defect impaired growth of the triple-tagged strain.

We took advantage of an effect of cdc16-116 to show that the SIN must be inactivated for type 1 nodes to reform at the end of mitosis. If cdc16-116 cells entered mitosis prior to shifting the temperature from 23°C to 35°C, Cdc7p–GFP never disappeared from the old SPB (Sohrmann et al., 1998) and the type 1 nodes did not reform (Fig. 3B; supplementary material Movie 3). This result was duplicated in the cdc16-116 strain without Cdc7p–GFP, confirming that continued SIN activation inhibited node reformation and that this was not a result of the genetic interaction between cdc16-116 and Cdc7p–GFP (supplementary material Fig. S1B).

The reversibility of SIN activation and node dispersal during interphase provides evidence that the cell cycle and node assembly are regulated independently. SIN activity alone disperses nodes in both mitosis and interphase.

The terminal SIN kinase, Sid2p, is part of the pathway that regulates nodes, given that nodes do not disperse during mitosis in cells containing the conditional allele sid2-250 (Akamatsu et al., 2014). One possible output pathway is from Sid2p to the NIMA kinase Fin1p (Grallert et al., 2012) and then Pom1p kinase. Pom1p phosphorylates Cdr2p, inhibiting its kinase activity (Bhatia et al., 2014; Deng et al., 2014; Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009) and restricting it to the medial cortex during interphase (Rincon et al., 2014). This pathway might also regulate the assembly of Cdr2p in type 1 nodes, but fin1Δ cells dispersed Cdr2p from nodes ∼4 min earlier (not later) and nodes reappeared ∼8 min earlier than in wild-type cells (Fig. 3C). This behavior is consistent with Fin1p influencing the time of mitotic entry rather than the dispersal of type 1 nodes. Thus, a parallel pathway from Sid2p to Cdr2p is likely to regulate the assembly of Cdr2p into nodes.

The presence of Cdr2p–mEGFP in type 1 nodes was inversely correlated with SIN activity, but Cdc7p always appeared at SPBs before Cdr2p dispersed. The 20 min lag in wild-type cells entering mitosis (Fig. 1C,D) shows that the pathway through the SIN to Cdr2p must have one or more slow steps. The lag is shorter when the SIN is hyperactivated at 37°C in cdc16-116 cells (Fig. 2G), so these delays are overwhelmed.

Several steps along the SIN cascade from Cdc7p to Sid1p and Sid2p might slow the signal before it reaches the level required to disperse Cdr2p. First, the slow accumulation of ∼400 Cdc7p molecules on SPBs over 40 min (Wu and Pollard, 2005) is a good candidate to be the rate-limiting factor in the pathway, and the timing in wild-type cells suggests that a threshold of ∼200 molecules of Cdc7p–mYFP on the active SPB is required to sustain enough SIN activity to disperse type 1 nodes. Second, high Cdk1p activity early in mitosis inhibits Sid1p, so that it localizes to the active SPB only after Cdk1p activity drops in anaphase (Guertin et al., 2000). The low Cdk1p activity during interphase allows the SIN to respond quickly to ectopic activation (Guertin et al., 2000), explaining the rapid dispersal of type 1 nodes during interphase (Fig. 2C,E,G). Strong SIN activation before mitosis might overwhelm the modulation of Sid1p during mitosis (Fig. 3A). Finally, Sid2p activation might also be rate limiting; SPBs have only ∼50 molecules of Sid2p in late interphase and then accumulate ∼200–300 Sid2p molecules during mitosis (McCormick et al., 2013).

We conclude that SIN activity is both necessary and sufficient to disperse type 1 nodes based on our previous observation that SIN-deficient cells do not disperse Cdr2p from nodes in mitosis and our present finding that hyperactivation of the SIN forces rapid dispersal of type 1 nodes in interphase and mitotic cells.

We constructed strains of S. pombe with genetically encoded fluorescent fusions proteins and temperature sensitive alleles by genetic crosses (supplementary material Table S1). We deleted fin1⁺ with custom primers using a PCR-based gene-targeting method (Bähler et al., 1998) and the pFA6a-kanMX6 plasmid JW85. Fluorescently tagged strains were obtained from laboratory stocks or by crossing laboratory stock strains. We tested strains depending on fluorescent fusion proteins for functionality by colony growth at 25°, 30° and 37°C and measuring the timing of cell cycle events (SPB separation, contractile ring constriction, septation and cell separation). Cdc16-116 cells expressing Cdr2p–mEGFP, Sad1p–mCherry and Cdc7p–GFP did not enter mitosis (n>50), but cells with of the same genotype but without a tag on Cdc7p entered mitosis normally.

Cells were grown for 36 h in liquid YE5S at 25°C prior to imaging on gelatin pads at 23°C. For imaging at elevated temperatures, cells were incubated at 37°C for 1 h in suspension prior to imaging on agar pads with an objective heater to maintain the temperature at 35°C. We used an Olympus IX-71 microscope (Olympus America, Center Valley, PA) with either a 100× 1.4 NA objective or a 60× 1.4 NA objective with a 1.6× optical magnifier, argon-ion lasers (Melles Griot), a spinning disk confocal head (Yokogawa CSU- X1) and an electron multiplying CCD camera (Andor iXon 897) using Andor iQ2 acquisition software.

Time lapse images were a collected as a z-series of 20 confocal slices at 360-nm intervals encompassing the entire cell every 3 min at 23°C or every 2 min at 35°C. Images were visualized as maximum intensity or sum projections using ImageJ (National Institutes of Health, Bethesda, MD). We defined SPB separation as time zero of the cellular clock (Wu et al., 2003). We defined type 1 nodes as punctate Cdr2p–mEGFP or Mid1p–mEGFP structures near the medial cortex in two or more sequential images. We judged Cdc7p–GFP to be present in a SPB if it colocalized with Sad1p–mCherry in at least two sequential images.

Outcome plots in Fig. 1C,D, Fig. 2E,F and Fig. 3C were constructed by plotting the cumulative proportion of cells with the specified characteristics against time. Cell populations were drawn from at least two fields of view, but for each given plot are limited to a subset of cells, e.g. those in interphase (Fig. 2E,F) or those that undergo mitosis (Fig. 1C). The plot in Fig. 2G was constructed by plotting (white circles) the timing of node disappearance versus the timing of Cdc7p–GFP appearance on the SPB, or (black circles) the timing of node appearance versus the timing of Cdc7p–GFP disappearance on a cell-by-cell basis.

The authors thank the Raymond and Beverly Sackler Institute for support, Rajesh Arasada for helpful discussions, Julien Berro for sharing ImageJ plugins and Chad McCormick for sharing strains. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.