SIN activation in interphase arrests nuclear division and inhibits the polarity machinery
To examine how the SIN affects interphase polarity and cell cycle progression we ectopically activated the SIN in interphase. The SIN can be turned on by inactivation of Cdc16, which is the GAP for the Spg1 GTPase (Fankhauser et al., 1993
; Schmidt et al., 1997
). If the SIN is activated in asynchronous cells, the cells stop polarized growth because they cease to elongate, and arrest as mononucleate or binucleate cells with multiple septa (Minet et al., 1979
; Fankhauser et al., 1993
). The disruption of polarized growth in these cells could be an indirect effect caused by ectopic septa formation, which could interfere with growth at cell ends or cause cell death by cutting the nuclei in half. To remove potential complications due to formation of multiple septa, the SIN was activated using the cdc16-116
temperature-sensitive mutant in either the cdc3-124
temperature-sensitive mutant background. The cdc3-124
mutations block cytokinesis and septum formation by disrupting actomyosin ring assembly, but do not interfere with SIN signaling (Balasubramanian et al., 1994
; Fankhauser et al., 1995
; Sparks et al., 1999
). The cdc16-116 cdc3-124
and cdc16-116 cdc15-140
strains were shifted to restrictive conditions for 4 h (almost two cell cycles) and were scored for cell length and number of nuclei. Because the cdc3-124
mutations block cytokinesis, the cdc16-116 cdc3-124
and cdc16-116 cdc15-140
cells should be tetranucleate and twice the length of normal wild-type cells if nuclear division and cell growth proceeded normally. Interestingly, the cdc16-116 cdc3-124
and cdc16-116 cdc15-140
double-mutant cells arrested as both mono- and binucleate cells and failed to elongate past the size of asynchronous wild-type cells (, ). These cells also displayed an increase in cell diameter consistent with a disruption in polarized growth (). FACS analysis showed that the cells were arrested with 2C and 4C peaks of similar size to the mono- and binucleate peaks, consistent with each nuclei arresting with a G2 DNA content (Fig. S1 A
). Thus, SIN activation blocked cell elongation and mitotic entry in interphase cells.
Figure 1. SIN activation arrests nuclear division and inhibits the interphase polarity machinery. (A) Cells of the indicated genotypes were grown at 25°C and then shifted to 36°C for 4 h, methanol fixed, stained with DAPI, and scored for number (more ...)
Activation of the SIN inhibits polarized cell growth
Cell elongation in interphase requires polarization of actin patches to the cell tips. To test if the block in cell elongation upon SIN activation is due to inhibition of the interphase polarity machinery, we looked at the actin distribution in the cdc3-124 cdc16-116 and cdc15-140 cdc16-116 cells. Upon shift to the restrictive temperature, the double-mutant cells had a dispersed actin cytoskeleton ( and Fig. S1, B and C), suggesting that SIN signaling disrupted interphase polarity. To rule out the possibility that Cdc16 inactivation could trigger an alternative pathway besides the SIN or that the cdc3-124 ring mutation disrupts interphase polarity, we inactivated the most downstream SIN component Sid2 using the sid2-250 mutation. The cdc3-124 cdc16-116 sid2-250 cells showed no block in cell elongation and nuclear division at the restrictive temperature and became long and multi-nucleate with actin polarized at the cell tips (; Fig. S1, B and C; ), confirming that the effects of Cdc16 inactivation are caused by ectopic activation of SIN signaling.
The G2 arrest observed after SIN activation is presumably mediated by inhibitory phosphorylation on Cdk1 because the nuclear division arrest was lost when the Cdk1 inhibitory kinase Wee1 was inactivated (). Interestingly, examination of cdc3-124 cdc16-116 wee1-50 cells at the restrictive temperature showed that although the wee1-50 mutation overcame the nuclear division block caused by SIN signaling, these cells failed to elongate and had depolarized actin (; ; Fig. S1, B and C), showing that the wee1-50 mutation did not overcome the SIN-mediated inhibition of polarized growth. Thus, SIN inhibition of nuclear division can be uncoupled from its inhibition of interphase actin organization.
The SIN inhibits the activity of the MOR pathway kinase Orb6
The phenotype caused by ectopic activation of the SIN is very similar to that observed in MOR pathway loss of function mutants, which arrest with depolarized actin, fail to elongate, and undergo a Wee1-dependent G2 arrest (Hirata et al., 2002
; Kanai et al., 2005
). In fact, inactivation of the most downstream MOR pathway component Orb6 caused a block in cell elongation and nuclear division along with an increase in cell diameter in cdc3-124 cdc16-116 sid2-250
cells similar to that in the cdc3-124 cdc16-116
double mutant ( and ). Consistent with the block in cell elongation, the actin cytoskeleton was depolarized in the cdc3-124 cdc16-116 sid2-250 orb6-25
cells (). These results suggested that the SIN might disrupt interphase polarity by inhibiting the MOR pathway.
The most downstream component of the MOR pathway is the NDR family kinase Orb6, allowing the kinase activity of Orb6 to serve as a read-out for the functional status of the MOR pathway. To directly test if SIN activation blocks MOR signaling, we examined the kinase activity of Orb6 after ectopic activation of the SIN using the cdc16-116 mutation. As before, the cdc15-140 mutation was included to overcome complications due to constitutive septation in the cdc16-116 cells. In contrast to asynchronous wild-type and G2-arrested cdc25-22 cells, the cdc16-116 cells, which also arrest in G2, showed reduced Orb6 activity, with or without the cdc15-140 mutation (). We next examined if SIN activation had a similar effect in interphase-arrested cells. Interestingly, although cdc15-140 cdc25-22 cells maintained high Orb6 activity consistent with their bipolar growth in interphase, SIN activation in this mutant background using the cdc16-116 mutation inhibited Orb6 activity (). Hence, SIN activation in interphase results in loss of Orb6 kinase activity and blocks MOR signaling.
Figure 2. The SIN has a dual role in regulation of the Orb6 kinase activity. (A) Ectopic activation of the SIN in interphase inhibits Orb6 kinase activity. All strains expressed mob2-13Myc from the chromosomal promoter. Cells were grown at 25°C, then shifted (more ...)
We next examined the relationship between the SIN and Orb6 activity under more physiological circumstances. In wild-type cells, Orb6 kinase activity peaks in G2 phase and then decreases as cells go through mitosis and septation (Kanai et al., 2005
). Interestingly, the interphase peak in Orb6 activity after cell division was shown to require the activity of some of the SIN components such as Cdc7 in the previous mitosis (Kanai et al., 2005
), apparently contradicting our results showing that ectopic SIN activation inhibits Orb6. However, careful examination of Orb6 kinase activity in synchronous cdc7-24
mutant cells resolved the apparent discrepancy. Consistent with the earlier results, Cdc7 was required for the large peak in the Orb6 activity after completion of mitosis (, compare 200–240-min time points in wild-type and cdc7-24
cells). However, cdc7-24
cells maintained moderate levels of Orb6 activity, consistent with the continued polarized growth observed in cdc7-24
mutants. Thus, the SIN is not essential for Orb6 activity, per se, but is required for the peak in activity associated with the onset of bipolar growth in the following cell cycle. We next examined Orb6 activity in synchronous cdc15-140
mutants. The cdc15-140
mutant cells have defects in actomyosin ring assembly, which triggers the cytokinesis checkpoint, causing them to arrest as binucleate cells with activated SIN (Liu et al., 2000
; Mishra et al., 2004
). Interestingly, these cells maintained very low Orb6 kinase activity consistent with the SIN inhibiting Orb6 (; 160–240 min in cdc15-140
). If Cdc7 is inactivated in these cells, they regained moderate levels of Orb6 kinase activity (, compare 160–240-min time points in cdc15-140 cdc7-24
cells) but lacked the peak in the following interphase (, compare 200–240-min time points in cdc15-140 cdc7-24
and wild-type cells). Thus, SIN has a dual role in regulation of the MOR pathway. The SIN suppresses Orb6 activity during cytokinesis but is required for its later increase as cells begin new end growth in the next cell cycle after cytokinesis.
The SIN inhibits the MOR pathway by blocking Nak1-mediated activation of Orb6
We next wanted to understand the mechanism by which the SIN inhibits Orb6 kinase activity. Similar to other members of the NDR kinase family, Orb6 kinase is activated by the GC kinase Nak1 (Kanai et al., 2005
; Hergovich et al., 2006
). Thus, the SIN could either inhibit the activity of Nak1, or it could inhibit the ability of Nak1 to activate Orb6. A previous study suggested that the SIN does not inhibit Nak1 because Nak1 activity remained high during cytokinesis when the SIN is active (Kanai et al., 2005
). To test this possibility in another way, we examined Nak1 kinase activity in cdc3-124 cdc16-116
cells, which arrest with active SIN. Consistent with earlier results, upon SIN activation there was no significant reduction in Nak1 kinase activity (). This result suggested that the SIN did not alter Nak1 kinase activity, per se, but possibly disrupted the ability of Nak1 to activate Orb6.
Figure 3. Fusion of Nak1 to Orb6 bypasses SIN inhibition of cell elongation. (A) The SIN does not inhibit Nak1 activity. Wild-type and cdc3-124 cdc16-116 cells expressing Nak1-3GFP from the chromosomal promoter were grown at 25°C and then shifted to 36°C (more ...)
The exact mechanism by which Nak1 activates Orb6 remains unclear. Although interaction between the two proteins was not observed by coimmunoprecipitation, a two-hybrid interaction was observed (Kanai et al., 2005
), suggesting a physical association between the two proteins. This raised the possibility that the SIN might interfere with Nak1-mediated activation of Orb6 by preventing association of the two proteins. To test this hypothesis, we constructed a Nak1–Orb6 fusion (). The Nak1–Orb6 fusion was able to rescue the growth defects of both the Nak1 and Orb6 single mutants (), suggesting that the individual proteins in the fusion retained functionality. The fusion did not rescue a mutant in the upstream regulator Pmo25, consistent with the known dependence of Nak1 activity on Pmo25 (Kanai et al., 2005
). Interestingly, the fusion, but not the Nak1 and Orb6 proteins individually or in combination, completely rescued a mutation in the scaffold protein Mor2, suggesting that the key function of Mor2 is to bring Nak1 and Orb6 together ().
Rescue of MOR pathway mutants by the Nak1–Orb6 fusion
To test whether the Nak1–Orb6 fusion could bypass the block in cell elongation when the SIN is activated, we expressed the Nak1–Orb6 fusion in cdc3-124 cdc16-116 background. Unlike cdc3-124 cdc16-116 cells carrying the vector control plasmid, which did not elongate, the cdc3-124 cdc16-116 cells containing the Nak1–Orb6 fusion plasmid were able to undergo significant elongation (). The cdc3-124 cdc16-116 cells expressing the Nak1–Orb6 fusion were able to polarize actin to the cell tips, but also showed medial actin distribution consistent with the cells trying to carry out both the SIN and MOR actin polarization programs (). Expression of Nak1 or Orb6 alone or coexpression of both Nak1 and Orb6 in the cdc3-124 cdc16-116 cells did not bypass the cell elongation and actin polarization defects observed in these cells (), showing that fusion of Nak1 and Orb6 was required to bypass the SIN-mediated inhibition of polarized growth. Furthermore, the kinase activity of Nak1 was required in the fusion because a kinase inactivating mutation in Nak1 blocked the ability of the fusion to drive polarized growth when the SIN is active (). Interestingly, the Nak1–Orb6 fusion also partially overrode the block in nuclear division caused by SIN activation, as seen by the reduction in mononucleate and increase in bi- and tetranucleate cells compared with controls at the 7-h time point (). Because either MOR inactivation or SIN activation blocks both nuclear division and cell elongation, the ability of the Nak1–Orb6 fusion to partially bypass both of these blocks when the SIN is active suggests that the SIN might block both nuclear division and cell growth by inhibiting the MOR. The failure of the fusion to completely bypass the SIN-mediated block in growth and nuclear division suggests that the SIN can still partially inhibit the MOR in the presence of the fusion, or the SIN can affect nuclear division and cell growth through an additional mechanism besides inhibition of the MOR.
Failure to inhibit MOR pathway signaling interferes with cytokinesis
The previous results showed that the SIN blocks polarized growth during cytokinesis by inhibiting the MOR pathway. We next wanted to address why the SIN inhibits the MOR. One possibility is that the SIN might inhibit the MOR to keep it from interfering with cytokinesis by titrating shared cytoskeletal elements such as actin away from the cell division site and toward the cell tips. To test whether loss of SIN inhibition of the MOR caused defects in cytokinesis, we examined the effects of expressing the Nak1–Orb6 fusion in different S. pombe
strains. The fusion protein caused a very slight increase in cell length (Fig. S5 C
), and surprisingly, expression of the Nak1–Orb6 fusion had only mild effects on cytokinesis in wild-type cells (unpublished data), perhaps because expression of the fusion does not totally bypass SIN inhibition. To see if expression of the Nak1–Orb6 fusion might interfere with cytokinesis in a more sensitized background, we expressed the fusion in cells with compromised SIN signaling. Interestingly, expression of the fusion was lethal when expressed in the temperature-sensitive SIN mutant sid2-250
at the semi-permissive temperature of 29°C (). This result suggested that MOR inhibition becomes essential when cytokinesis is partially compromised. To further test this hypothesis we used an alternative way to interfere with the cell division machinery. Low doses of the actin-depolymerizing drug, latrunculin B (Lat B) causes a cell division delay in wild-type cells (Trautmann and McCollum, 2005
). During the delay the SIN remains active, causing an arrest in polarized growth and nuclear division until cytokinesis is complete (Mishra et al., 2004
). We found that expression of the Nak1–Orb6 fusion in wild-type cells treated with low doses of Lat B is lethal (). Examination of similarly treated wild-type cells in liquid culture showed that wild-type cells with the vector control initially accumulate binucleate cells because of the delay in cytokinesis, but are eventually able to divide as judged by their ability to maintain a population of mononucleate cells and failure to accumulate multinucleate cells (). In contrast, wild-type cells expressing the fusion protein are unable to complete cytokinesis as seen by the loss of mononucleate cells and the accumulation of multinucleate cells (). This phenotype could be caused by loss of SIN signaling, which also causes cells to fail cytokinesis and become multinucleate when cytokinesis is delayed (Mishra et al., 2004
), or the Nak1–Orb6 fusion could be interfering with the cytokinetic apparatus, keeping cells from completing cytokinesis. We do not think that expression of the fusion protein is interfering with SIN signaling because the cells expressing the fusion maintained similar levels of SIN activity as those with the control vector, as judged by the presence of the SIN kinase Cdc7 at the SPB.
Figure 4. MOR inhibition becomes essential when cytokinesis is perturbed. (A) sid2-250 cells expressing the vector alone or the Nak1–Orb6 fusion transgene were grown in medium lacking thiamine for 14 h at 25°C to induce expression of the fusion (more ...)
We also used another approach to test whether the Nak1–Orb6 fusion was affecting SIN signaling. We completely blocked cytokinesis using the cdc3-124
profilin mutant, which cannot form actomyosin rings. After shift to restrictive temperature, these cells undergo a prolonged SIN-dependent arrest as binucleates but eventually leak past the nuclear division arrest and become multinucleate (Trautmann et al., 2001
). Expression of the Nak1–Orb6 fusion in these cells did not significantly interfere with the ability of these cells to arrest as binucleates, suggesting that it was not affecting SIN signaling (), which is required to maintain the nuclear division arrest. Furthermore, examination of the kinase activity of the most downstream component of the SIN pathway, Sid2, did not show a significant change when the Nak1–Orb6 fusion was expressed (). Along similar lines, we did not observe any evidence for persistent SIN signaling when the MOR was inhibited in orb6-25
mutants (Fig. S4
). Together, these results suggest that the MOR pathway may interfere with actomyosin ring assembly and constriction downstream of the SIN, and that MOR inhibition is essential when the cell division machinery is compromised and cytokinesis is delayed.
Reduction in MOR activity allows weak SIN signaling to promote cytokinesis
If the MOR pathway interferes with the ability of the SIN to promote cytokinesis, then reduction in MOR pathway activity might be predicted to enhance cytokinesis in SIN mutants. Therefore, we tested the phenotype of double mutants between the MOR mutant orb6-25 and various SIN mutants. Interestingly, the orb6-25 temperature-sensitive mutant was able to rescue the growth defect of some temperature-sensitive SIN mutants at 29°C and 33°C, which are semi-permissive temperatures for orb6-25 (). Although the orb6-25 mutant dies at 36°C and was thus not able to rescue SIN mutants at this temperature, examination of double mutants between orb6-25 and various SIN mutants at 36°C showed that the orb6-25 mutation was able to partially rescue the septum formation defect in all SIN mutants tested (). We also examined this phenotype using sid2-250, orb6-25, and sid2-250 orb6-25 cells that had been synchronized in G2 and then shifted to restrictive temperature (Fig. S5 A). Consistent with results from asynchronous cells, the sid2-250 orb6-25 cells showed a substantial increase in septation compared with the sid2-250 single-mutant cells, which totally failed in septation. Although MOR inactivation allowed septation in the sid2-250 orb6-25 cells, absence of a completely functional SIN pathway could explain the reduced septation index in these cells relative to orb6-25 alone. Inactivation of orb6 was unable to promote septum formation in SIN double-mutant cells with mutations in two different SIN components (sid2-250 sid1-239 and sid2-250 cdc11-123), which presumably completely ablates SIN signaling (), suggesting that inactivation of orb6 does not bypass the requirement of the SIN in septum formation, but instead allows residual weak SIN signaling to promote cytokinesis.
Figure 5. The MOR pathway mutant orb6-25 rescues growth and cell division defects of SIN mutants. (A) Cells of the indicated genotypes were grown at 25°C and their growth was tested at the indicated temperatures by spotting 10-fold serial dilutions on YE (more ...)
To examine how loss of MOR activity rescued SIN mutants in more detail, we observed the dynamics of actomyosin ring assembly and septum formation in orb6-25 sid2-250
double-mutant cells. Previous studies have shown that SIN mutants form actomyosin rings that fall apart in anaphase and the cells do not form septa (Krapp and Simanis, 2008
). Therefore, we examined actomyosin ring constriction and septum formation in the sid2-250 orb6-25
double-mutant cells expressing the GFP-tagged actomyosin ring component Rlc1 (Le Goff et al., 2000
; Naqvi et al., 2000
) at the restrictive temperature of 36°C using time-lapse microscopy. As expected, in wild-type cells, actomyosin rings formed then constricted (15/15 cells; ). In contrast, actomyosin rings formed in sid2-250
single-mutant cells, but failed to constrict and then disassembled in 16 out of 21 cells observed (). Unlike sid2-250
mutant cells but similar to wild-type cells, actomyosin rings formed and constricted in sid2-250 orb6-25
double mutants (21/21 cells; ). Thus, loss of Orb6 activity allows SIN mutants to maintain actomyosin ring stability and complete cytokinesis. Thus, loss of MOR activity allows weak SIN signaling to promote actomyosin ring constriction and septum formation.
Interestingly, MOR inactivation also enhanced the ability of weak SIN signaling to promote ectopic septation. The Spg1-GFP allele has a weakly activated SIN phenotype, which causes occasional formation of interphase septa, or additional rounds of septum formation after normal cytokinesis (García-Cortés and McCollum, 2009
). When spg1-GFP
was combined with orb6-25
, or any other MOR mutant, the resulting double-mutant cells showed an increased rate of ectopic septum formation (). Together, these results show that reduction in MOR pathway activity enhances the ability of weak SIN signaling to promote cytokinesis.