Co-ordination of anaphase onset with cytokinesis, inactivation of mitotic CDK (cyclin-dependent kinase) activity, and spindle disassembly is important to assure faithful transmission of the genome from mother to daughter cells and maintenance of ploidy. Our knowledge of how this is achieved is most complete in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. In both organisms, the onset of cytokinesis is signalled by conserved signal transduction networks known as the septation initiation network (SIN) in S. pombe and the mitotic exit network (MEN) in S. cerevisiae (Bardin and Amon, 2001; Dumitrescu and Saunders, 2002; Geymonat et al., 2002; Glover et al., 1998; Jensen and Johnston, 2002; McCollum and Gould, 2001). The MEN has an additional role as part of a checkpoint whose role is to prevent inactivation of mitotic B-type cyclin activity if the mitotic spindle is not correctly oriented. This is a critical event for S. cerevisiae, where the site of bud formation and the subsequent cytokinesis, are established at the onset of the cell cycle. Thus, during mitosis, the cell must ensure that one end of the spindle, and hence one set of chromosomes, has traversed the bud neck and entered the daughter cell. In contrast, in S. pombe, a rod-shaped cell that divides by fission after formation of a medially placed division septum, the division site is determined by the centrally positioned interphase nucleus, and so in the course of a normal mitosis each cell should then receive a nucleus. Nonetheless, S. pombe also has a spindle orientation checkpoint, which delays anaphase B if the spindle is not oriented along the long axis of the cell. Whether the SIN plays a role in this is unclear. An extended discussion of the SIN and MEN is provided in the accompanying Commentary (Simanis, 2003).FIG1
Both the SIN and MEN are centered around a GTPase, which triggers signaling through protein kinases, and both the MEN and SIN are located on the spindle pole body, placing them close to other mitotic regulators, and providing an excellent means to monitor spindle position and mitotic progression.
The upper panel shows the regulatory circuits that comprise the MEN and influence its action. In the course of a normal cell cycle, mitotic exit is accompanied by inactivation of mitotic CDK activity. The cyclin subunit of the CDK complex is targeted for proteolytic destruction through ubiquitylation by the anaphase-promoting complex, also known as the cyclosome (APC/C). Specificity is conferred by association with the targeting subunit Cdc20p. The APC/C also targets the securin Pds1p, thereby activating the separase Esp1p to cleave the cohesin Scc1p, and permitting sister chromatid separation. The APC/C is prevented from acting by the spindle assembly checkpoint until all the sister chromatids have attached to the mitotic spindle in a bipolar fashion (Zhou et al., 2002). The key element for mitotic exit is the phosphoprotein phosphatase Cdc14p, which reverses CDK-mediated phosphorylation events. During most of the cell cycle it is sequestered in the nucleolus, where it is tethered to, and inhibited by, the RENT complex. Once the cell commits itself to anaphase, the fourteen early anaphase release (FEAR) network, which includes separase, releases Cdc14p from the nucleolus. The mechanism is presently unclear. The MEN then acts to keep Cdc14p outside the nucleolus. The early release of Cdc14p is also thought to activate the protein kinase Cdc15p, which is inhibited by CDK phosphorylation. Cdc15p in turn activates the kinase Dbf2p, which is thought to be the effector that bring about cytokinesis and mitotic exit, although its targets remain unknown. Once released and activated, Cdc14p dephosphorylates a number of proteins. These include the APC/C targeting subunit Cdh1p, which completes destruction of mitotic cyclins, and the transcription factor Swi5p, which can then enter the nucleus and activate its target genes. Also among these is the CDK inhibitor Sic1p, whose dephosphorylation by Cdc14p protects it from phosphorylation by CDKs, which would target it for ubiquitylation and destruction. The combined effect of these events is to reduce CDK activity and promote mitotic exit. This self-perpetuating loop must be broken to permit B-type cyclins to reaccumulate for the next cell cycle. Bfa1p, a component of the GAP for Tem1p, is a target of Cdc14p, which is thought to reactivate it. Activation of Cdc14p also leads to production of Amn1p, which competes with Cdc15p for binding to Tem1p. Activated APC/CCdh1p promotes destruction of Spo12p and Cdc5p. Together, these events reduce MEN and FEAR activity, and so shut off the mitotic exit machinery. Mitotic exit is also regulated by cell polarity determinants. Lte1p, which contains a GEF domain, associates with the cell cortex by binding to Ras2p. One model suggests that it is the GAP for Tem1p and that entry of the spindle pole body bearing Tem1p into the bud leads to its activation by contact with Lte1p. However, Lte1p is essential for mitotic exit only at low temperatures, and it is unclear whether it is the GEF for Tem1p, whether its effects are mediated through cell polarity proteins, or both [see the accompanying Commentary (Simanis, 2003)].
Like the MEN, the SIN is associated with the spindle pole body, and it is also negatively regulated by mitotic Cdc2p activity. The SIN is activated by the protein kinase Plo1p, and signaling is mediated by three protein kinases and their associated subunits. It has been suggested that they act in the order Cdc7p, Sid1p, Sid2p, although biochemical evidence for this is lacking. The SIN is not essential for mitotic exit, but is required for signalling the onset of septum formation. The targets of the SIN are unknown, but appearance of the kinase Sid2p at the contractile ring is thought be a critical event in signaling the initiation of septum formation. The critical effector of the MEN, Cdc14p, has a counterpart in S. pombe called Flp1p, but this is not the essential mediator of SIN signaling, since flp1 mutant cells can divide. Like Cdc14p, Flp1p is sequestered in the nucleolus and released early in mitosis, when it appears on the contractile ring and the mitotic spindle; it can also influence mitotic commitment. Although its in vivo substrates remain to be identified, its localization suggests that it co-ordinates events during mitosis. Dma1p, Zfs1p, Scw1p and PP2A-Par1p have all been identified as inhibitors of SIN signaling, mainly through genetics or multicopy suppression. It has been suggested that Dma1p prevents localization of Plo1p (Guertin et al., 2002), but the mode of action of the other proteins is unclear. Both the SIN and the MEN are inhibited by high levels of mitotic CDK activity. Some components of the SIN and MEN have counterparts in higher eukaryotes. In most cases, their functions are unclear, although the orthologue of Cdc14p has been implicated in cytokinesis (Gruneberg et al., 2002; Kaiser et al., 2002), and centrosome function (Kaiser et al., 2002; Mailand et al., 2002). Centriolin, a centrosomal protein that shares a domain with Cdc11p/Nud1p has been implicated in cytokinesis and G1 progression (Gromley et al., 2003). CHFR, an orthologue of Dma1p, has been implicated in a prophase microtubule integrity checkpoint (Cortez and Elledge, 2000; Scolnick et al., 2000). It seems reasonable to assume that these will not be the only SIN/MEN components conserved through evolution.
Work in my lab is funded by the Swiss Cancer League, The Swiss National Science Foundation, The Fondation Forez and ISREC. I apologise for citing reviews at the expense of all the relevant original literature. I am grateful to Caren Norden and Yves Barral (Department of Biochemistry, ETHZ, Switzerland) for providing the images of yeast actin rings shown in the figure. I am also grateful to members of my lab for many discussions about SIN and in particular to Andrea Krapp, Iain Hagan, and Richard Sever for critical reading of the text.