The SIN consists of three protein kinases (cdc7p, sid1p, sid2p) and their regulatory subunits (spg1p, cdc14p and mob1p, respectively; Fig. 1). SIN signalling is thought to originate from the poles of the mitotic spindle, where SIN proteins are located during mitosis. They assemble at the spindle pole bodies (SPBs) on a scaffold composed of sid4p and cdc11p. Signalling is modulated by the nucleotide status of the GTPase spg1p, which is controlled by a two-component GAP byr4p ‑ cdc16p. SIN signalling is also regulated by the master cell cycle regulator cdc2p and the mitotic regulator plo1p. The mob1p‑sid2p protein kinase is also observed at the contractile ring late in mitosis. This is thought to be the trigger for ring contraction. If the SIN does not function, cells become elongated and multinucleated, as growth, DNA synthesis and mitosis continue in the absence of cytokinesis. In contrast, ectopic activation of the SIN can uncouple contractile ring formation and septation from other cell cycle events (Fig. 2). Regulation of the SIN is therefore important for proper co-ordination of mitosis with cytokinesis. Selected aspects of these studies of SIN regulation are summarised below.

Figure 1: Proteins of the SIN.
The protein kinases assembled at the spindle pole body bind to a scaffold composed of cdc11p and sid4p. Negative regulators are shown in red when essential and orange when non‑essential genes. Proteins required for signal transduction are shown in green.

Figure 2: The phenotypes resulting from modulation of SIN activity.
WT indicates unpertubed, dividing cells, stained with DAPI for DNA and Calcofluor for the medially-placed division septum. Elongated, multinucleated cells result from loss of the proteins required for signalling, while ectopic activation of the SIN by increased expression (OP) of some regulators, or loss of GAP activity, results in formation of multiple septa without cell cleavage.

Analysis of the SIN scaffold protein cdc11p

Binding of SIN proteins to the spindle pole body, from where signalling is thought to occur, requires the scaffold proteins sid4p and cdc11p, which form a complex. Cdc11p associates with the spindle pole body throughout the cell cycle, and becomes hyperphosphorylated during anaphase. To obtain a better understanding of how cdc11p regulated signalling, we studied its phosphorylation state during mitosis in various mutant backgrounds. We demonstrated that association of cdc11p with the spindle pole body is required for its phosphorylation, and that ectopic activation of the SIN results in hyperphosphorylation of cdc11p. We also showed that mitotic hyperphosphorylation of cdc11p requires the activity of plo1p, cdc7p, and spg1p, but not sid1p and sid2p. Furthermore, we showed that the septation inhibitor byr4p interacts preferentially with hypophosphorylated cdc11p. In cells that have been arrested early in mitosis by activation of the spindle assembly checkpoint, we have found that byr4p and cdc7p are both present on the spindle pole body, but cdc11p is in the interphase phosphorylation state, indicating that hyperphosphorylation of cdc11p occurs after chromosomes are properly attached to the spindle, and are under tension. We conclude that cdc11p hyperphosphorylation correlates with activation of the SIN, and that this may be mediated primarily by cdc7p in vivo. We have also demonstrated that the N‑terminus of cdc11p is sufficient for signal transduction, if recruited to the SPB as a fusion protein with its partner, sid4p.

The S. pombe E2 ubiquitin ligase ubc8p is a potential regulator of the SIN

Inappropriate activation of the SIN can uncouple septum formation from other cell cycle events, so regulators of the SIN are of considerable interest. Several studies have identified the product of the dma1 gene as a potent inhibitor of SIN signalling. Increased expression of dma1 inhibits SIN signalling and prevents cell division. To identify potential targets and mediators of this inhibition, multicopy suppressors of dma1 toxicity were identified. One of these, ubc8, is the subject of this paper. Genetic and molecular analyses are consistent with the view that ubc8p acts as an inhibitor of the SIN. The ubc8 gene is not essential, but in its absence cells are unable to prevent septum formation if progression through mitosis is impaired, suggesting that it may be an effector of the spindle assembly checkpoint.

The role of the SIN in meiosis

When starved, S. pombe cells of opposite mating types fuse to form a diploid zygote that undergoes meiosis producing four spores. No septa or contractile rings are formed during meiosis. Nonetheless, our previous studies of mob1 mutants suggested that defects in the SIN affected spore formation and viability. Therefore, we investigated whether the SIN plays a role in meiosis. We have discovered that, while the meiotic divisions appear normal, SIN mutants cannot form spores. Forespore membrane formation is initiated, but the nuclei are not encapsulated properly. The SIN proteins localise to the spindle pole body in meiosis. The protein kinases sid1p and cdc7p do not associate with the spindle pole body until meiosis II, when forespore membrane deposition begins. These data indicate a role for the SIN in regulating spore formation during meiosis.

Keywords

Cell cycle, cytokinesis, mitosis