Journal of Molecular Cell Biology Advance Access published online on September 24, 2009
Journal of Molecular Cell Biology, doi:10.1093/jmcb/mjp020
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A Pom1 Gradient Is Made to Measure
Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore
* Correspondence to: Meredith E. Calvert, E-mail: meredith{at}tll.org.sg
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In order for cell division to proceed, fission yeast must first attain critical cell size. The mechanism by which size is detected had not been identified until two recent studies showed that cells utilize an intracellular gradient of Pom1p kinase to measure cell length.
The existence of a cell size requirement that must be met prior to entry into mitosis was first established in the fission yeast, Schizosaccharomyces pombe. This organism is an ideal model system for studying mitosis and cytokinesis since specific features of these processes are analogous between S. pombe and higher eukaryotes, such as centromeric structure, mechanisms of microtubule attachment and the process of symmetrical cell division. The cell division cycle mutants characterized by Paul Nurse and colleagues defined many highly conserved proteins involved in cell size regulation, including the mitotic inhibitor, Wee1p kinase. Wee1p prevents activation of the cyclin-dependent kinase Cdc2p by phosphorylating Cdc2p; once the necessary cell size has been obtained, this phosphorylation is removed by the phosphatase Cdc25p. Dephosphorylation by Cdc25p activates Cdc2p and promotes the phosphorylation of multiple mitotic substrates, thereby facilitating the entry into mitosis (Nurse, 1990). Although these findings were made more than 20 years ago, the intracellular mechanism that determines cell size has remained elusive until now. In a pair of studies published in tandem in Nature by Moseley et al. and Martin and Berthelot-Grosjean, the authors demonstrate how fission yeast cells sense length by reading an intracellular gradient of another kinase, Pom1p (Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009). This gradient transmits information regarding cell size to downstream mitotic regulatory factors. Specifically, Pom1p acts as a dose-dependent inhibitor of entry into mitosis by negatively regulating the Wee1p inhibitory kinase Cdr2p, thus preventing inactivation of the Wee1p until the cell has reached a critical size.
Pom1p kinase was originally identified in a screen for mutants with morphogenesis defects. Cells harboring mutations in pom1 frequently exhibited defects in polarized growth and cytokinesis. Whereas in wild-type strains cell growth initially occurs only at the old end of the cell and then later becomes bipolar, a pom1 mutant cell grows from either end with equal frequency and will continue growing only from this end (Bähler and Pringle, 1998). Disruption of Pom1p function can also lead to misplaced division septa and the formation of multiple contractile rings and this latter phenotype is observed more frequently in a cdc25 mutant background. Pom1p is concentrated at the tips of the cell and also localizes to the center of the cell during division, and this localization requires the microtubule-associated proteins Tea1p and Tea4p (Bähler and Pringle, 1998; Padte et al., 2006). Together, Tea1p, Tea4p and Pom1p make up the tip complex, a signaling module which is required for the inhibition of septum assembly at the cell ends. The role of Pom1p in septal positioning may involve annilin-like protein, Mid1p, since Pom1p is required for the correct localization to Mid1 to the medial cortex and the combined loss of functional Pom1p and Mid1p leads to cytokinetic ring assembly at the cell ends and lethality (Bähler and Pringle, 1998; Huang et al., 2007). Therefore, in carrying out its role in regulating morphogenesis and contractile ring placement, Pom1p is apparently transmitting positional information to both the cell tips and the medial region of cells.
Although the two papers arrive at the same conclusions regarding the newly described role for Pom1p in sensing cell size, the preliminary findings that kindled these studies were rather different. Following the observation that pom1
cells are smaller in both wild-type and cdc25-22 mutant backgrounds, Martin and Berthelot-Grosjean further determined that Pom1p was required for preventing mitotic entry in the absence of functional Cdc25p. In a cdc25-22 background, loss of Pom1p could suppress the temperature sensitivity of the strain, whereas ectopic overexpression of Pom1p caused synthetic sickness. Additionally, the deletion of Pom1p caused no further decrease in cell size in wee1 mutants, suggesting that Pom1p may be upstream of the Wee1p-inhibitory pathway. Given this new cell cycle regulatory role for Pom1p, the authors performed further epistatic analyses and identified Wee1p-inhibitory kinase Cdr2p as a target for negative regulation by Pom1p. In confirmation of this, Cdr2p phosphorylation and localization were both shown to be Pom1p-dependent. Finally, they demonstrated that both Pom1p overexpression and ectopic localization of Pom1p to the middle of the cell by a Pom1p-Cdr2p chimera could inhibit entry into mitosis. Since the Pom1p-mediated G2 delay is both localization and dose-dependent, the authors proposed that an inhibitory gradient of Pom1p emanates from either cell end and overlaps in the middle with Cdr2p. As cell growth proceeds during G2, this gradient becomes shallower and the signal is thus diluted. Once the cell has reached a certain length, the level of Pom1p at the medial region of the cell decreases sufficiently to permit activation of Cdr2p (Figure 1A).
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To uncover the Pom1p gradient, Moseley et al. started in the middle of the cell by characterizing the proteins contained on medial cortical nodes during interphase. The sequential recruitment of cytokinetic factors such as Mid1p and myosin II to the cortical nodes prior to formation of the contractile ring had been previously demonstrated (Wu et al., 2003). Cdr2p also localizes to these nodes, and using proteomics the authors identified a new protein, Blt1p, that colocalized with Cdr2p and Mid1p during G2 (Morrell et al., 2004). The assembly of Blt1p, Mid1p, Cdr1p and Wee1p at the interphase nodes was determined to require Cdr2p, and the authors hypothesized that the cortical nodes harbor a mitotic signaling network, dependent upon Cdr2p. To determine how this network might be spatially regulated, they looked for alterations in Cdr2p localization in a number of strains exhibiting polarity defects, including tea1
, tea4 and pom1. In pom1 cells, Cdr2p nodes became dispersed throughout at least half of the cell, across the medial region to the non-growing cell tip. As was shown by the other group, Pom1p was demonstrated to both alter the phosphorylation status of Cdr2p and genetically interact with Cdc25p to regulate entry into mitosis in a dose-dependent manner. To test their resulting hypothesis that medial Pom1p inhibits Cdr2p and cell cycle progression, the authors targeted Pom1p throughout the cortex and found that Cdr2p also became delocalized and led to a delayed entry into mitosis. This result is particularly intriguing; both ectopic cortical localization of Pom1p and the complete loss of Pom1p from cells alter the distribution of Cdr2p and this implies that Pom1p regulates both the localization and the activity of Cdr2p in a spatially restricted manner. The model proposed by Moseley et al. is essentially identical to that of Martin and Berthelot-Grosjean, whereby a polar gradient of Pom1p inhibits medial Cdr2p activity until cellular growth sufficiently attenuates the inhibitory signal, allowing activation of Cdr2p and entry into mitosis (Figure 1A). Together, these studies offer complementary evidence for the existence of a novel size-sensing mechanism long predicted to be present in dividing cells. Both Pom1p and Cdr2p are members of kinase families that are conserved throughout higher eukaryotes (DYRK and SAD kinases, respectively) and this mechanism for coordinating cell size with cell cycle regulation is likely to be conserved. These findings should certainly stimulate the hunt for analogous size-sensing mechanisms in metazoan cells. Still, within fission yeast, many questions remain. Of fundamental import to understanding this mechanism will be determining whether Cdr2p is a bona fide target for phosphorylation by Pom1p, or if intermediate factors are involved. Also unclear at this stage is if the localization of Cdr2p is directly regulated by Pom1p and if its activity is localization-dependent.
Two earlier studies characterizing Cdr2p demonstrated that overexpression of Cdr2p is toxic and results in a terminal phenotype of elongated cells with multiple division septa. These findings are of note for a number of reasons: first, that this phenotype is not observed following overexpression of Cdr1p, the other closely related Wee1p inhibitory kinase and secondly, in the absence of Wee1p, cells are no longer elongated but still display septation defects. Taken together, these results suggest that Cdr2p may have a Wee1p-independent function in the regulation of cytokinesis (Breeding et al., 1998; Kanoh and Russell, 1998). If we consider that when Cdc25p function is compromised, the loss of Pom1p (and consequently its inhibitory influence on Cdr2p) causes a similar cytokinetic phenotype to that seen following the overexpression of Cdr2p, these results imply that a stoichiometric balance of these interacting proteins is critical to their regulatory functions, as would be predicted by the gradient model (Padte et al., 2006) (Figure 1B). That Pom1p interacts with both the mitotic signaling pathway and the regulators of cytokinesis places it at the molecular intersection of these processes. Given the localization of Cdr2p to the interphase nodes, the structural precursors to the cytokinetic ring, this kinase is uniquely positioned to provide cross-talk between the mechanisms regulating mitosis and cytokinesis. These new findings therefore point toward a more complex mechanism coupling the successful entry into mitosis to the early stages of cytokinesis. It will be intriguing to learn whether this newly described function of Pom1p is truly independent from its role in regulating cell morphogenesis.
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