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Events at the fission yeast equivalent of the centrosome, the spindle pole body, determine the timing of mitotic commitment and mitotic exit. Previous work has established that events on Cut12 drive commitment, whereas events on a distinct scaffold, Sid4 drive exit. Chan et al. now show how signaling on Sid4 influences commitment to explain the rationale for using the centrosome as a signaling center; centrosomal signaling supports integration of outputs from distinct inputs., The fission yeast scaffold molecule Sid4 anchors the septum initiation network to the spindle pole body (SPB, centrosome equivalent) to control mitotic exit events. A second SPB-associated scaffold, Cut12, promotes SPB-associated Cdk1–cyclin B to drive mitotic commitment. Signals emanating from each scaffold have been assumed to operate independently to promote two distinct outcomes. We now find that signals from Sid4 contribute to the Cut12 mitotic commitment switch. Specifically, phosphorylation of Sid4 by NIMAFin1 reduces Sid4 affinity for its SPB anchor, Ppc89, while also enhancing Sid4’s affinity for casein kinase 1δ (CK1δ). The resulting phosphorylation of Sid4 by the newly docked CK1δ recruits Chk2Cds1 to Sid4. Chk2Cds1 then expels the Cdk1–cyclin B antagonistic phosphatase Flp1/Clp1 from the SPB. Flp1/Clp1 departure can then support mitotic commitment when Cdk1–cyclin B activation at the SPB is compromised by reduction of Cut12 function. Such integration of signals emanating from neighboring scaffolds shows how centrosomes/SPBs can integrate inputs from multiple pathways to control cell fate.

Summary Background Activation of the Cdk1/cyclin B complex, also known as mitosis-promoting factor (MPF), drives commitment to mitosis. Interphase MPF is inhibited through phosphorylation of Cdk1 by Wee1-related kinases. Because Cdc25 phosphatases remove this phosphate, Cdc25 activity is an essential part of the switch that drives cells into mitosis. The generation of a critical "trigger" of active MPF promotes a positive feedback loop that employs Polo kinase to boost Cdc25 activity and inhibit Wee1, thereby ensuring that mitotic commitment is a bistable switch. Mutations in the spindle pole body (SPB) component Cut12 suppress otherwise lethal deficiencies in Cdc25. Results Cut12 harbors a bipartite protein phosphatase 1 (PP1) docking domain. Mutation of either element alone suppressed the temperature-dependent lethality of cdc25.22 , whereas simultaneous ablation of both allowed cells to divide in the complete absence of Cdc25. Late G2 phase phosphorylation between the two elements by MPF and the NIMA kinase Fin1 blocked PP1 Dis2 recruitment, thereby promoting recruitment of Polo to Cut12 and the SPB and elevating global Polo kinase activity throughout the cell. Conclusions PP1 recruitment to Cut12 sets a threshold for Polo's feedback-loop activity that locks the cell in interphase until Cdc25 pushes MPF activity through this barrier to initiate mitosis. We propose that events on the SPB (and, by inference, the centrosome) integrate inputs from diverse signaling networks to generate a coherent decision to divide that is appropriate for the particular environmental context of each cell. PP1 recruitment sets one or more critical thresholds for single or multiple local events within this switch.

PMCID: PMC3549529.-- et al., Activation of mitosis-promoting factor (MPF) drives mitotic commitment. In human cells active MPF appears first on centrosomes. We show that local activation of MPF on the equivalent organelle of fission yeast, the spindle pole body (SPB), promotes Polo kinase activity at the SPBs long before global MPF activation drives mitotic commitment. Artificially promoting MPF or Polo activity at various locations revealed that this local control of Plo1 activity on G2 phase SPBs dictates the timing of mitotic commitment. Cytokinesis of the rod-shaped fission yeast cell generates a naive, new, cell end. Growth is restricted to the experienced old end until a point in G2 phase called new end take off (NETO) when bipolar growth is triggered. NETO coincided with MPF activation of Plo1 on G2 phase SPBs (ref. 4). Both MPF and Polo activities were required for NETO and both induced NETO when ectopically activated at interphase SPBs. NETO promotion by MPF required polo. Thus, local MPF activation on G2 SPBs directs polo kinase to control at least two distinct and temporally separated, cell-cycle transitions at remote locations., This work was supported by Cancer Research UK (CRUK) grant number C147/A6058. V.S. and A.K. were supported by the Swiss National Science Foundation and EPFL.

Mitotic exit integrates the reversal of the phosphorylation events initiated by mitotic kinases with a controlled cytokinesis event that cleaves the cell in two. The mitotic exit network (MEN) of budding yeast regulates both processes, whereas the fission yeast equivalent, the septum initiation network (SIN), controls only the execution of cytokinesis. The components and architecture of the SIN and MEN are highly conserved. At present, it is assumed that the functions of the core SIN-MEN components are restricted to their characterized roles at the end of mitosis. We now show that the NDR (nuclear Dbf2-related) kinase component of the fission yeast SIN, Sid2-Mob1, acts independently of the other known SIN components in G2 phase of the cell cycle to control the timing of mitotic commitment. Sid2-Mob1 promotes mitotic commitment by directly activating the NIMA (Never In Mitosis)-related kinase Fin1. Fin1's activation promotes its own destruction, thereby making Fin1 activation a transient feature of G2 phase. This spike of Fin1 activation modulates the activity of the Pom1/Cdr1/Cdr2 geometry network towards Wee1.

Genome annotation is a synthesis of computational prediction and experimental evidence. Small genes are notoriously difficult to detect because the patterns used to identify them are often indistinguishable from chance occurrences, leading to an arbitrary cutoff threshold for the length of a protein-coding gene identified solely by in silico analysis. We report a systematic reappraisal of the Schizosaccharomyces pombe genome that ignores thresholds. A complete six-frame translation was compared to a proteome data set, the Pfam domain database, and the genomes of six other fungi. Thirty-nine novel loci were identified. RT-PCR and RNA-Seq confirmed transcription at 38 loci; 33 novel gene structures were delineated by 5′ and 3′ RACE. Expression levels of 14 transcripts fluctuated during meiosis. Translational evidence for 10 genes, evolutionary conservation data supporting 35 predictions, and distinct phenotypes upon ORF deletion (one essential, four slow-growth, two delayed-division phenotypes) suggest that all 39 predictions encode functional proteins. The popularity of S. pombe as a model organism suggests that this augmented annotation will be of interest in diverse areas of molecular and cellular biology, while the generality of the approach suggests widespread applicability to other genomes.

PP1 holoenzymes are composed of a small number of catalytic subunits and an array of regulatory, targeting, subunits. The Schizosaccharomyces pombe genome encodes two highly related catalytic subunits, Dis2 and Sds21. The gene for either protein can be individually deleted, however, simultaneous deletion of both is lethal. We fused enhanced green fluorescent protein (EGFP) coding sequences to the 5′ end of the endogenous sds21+ and dis2+ genes. Dis2.NEGFP accumulated in nuclei, associated with centromeres, foci at cell tips and endocytic vesicles. This actin-dependent endocytosis occurred between nuclei and growing tips and was polarised towards growing tips. When dis2+ was present, Sds21.NEGFP was predominantly a nuclear protein, greatly enriched in the nucleolus. When dis2+ was deleted, Sds21.NEGFP levels increased and Sds21.NEGFP was then clearly detected at centromeres, endocytic vesicles and cell tips. Dis2.NEGFP was recruited to cell tips by the formin binding, stress pathway scaffold Wsh3 (also known as Tea4). Wsh3/Tea4 modulates polarised tip growth in unperturbed cell cycles and governs polarised growth following osmotic stress. Mutating the PP1 recruiting RVXF motif in Wsh3/Tea4 blocked PP1 binding, altered cell cycle regulated growth to induce branching, induced branching from existing tips in response to stress, and blocked the induction of actin filaments that would otherwise arise from Wsh3/Tea4 overproduction.

Microtubules grow by incorporating αβ-tubulin heterodimers into the dynamic plus ends while their minus ends are often anchored at microtubule organizing centers. Plus ends exhibit a stochastic “dynamic instability,” so that neighboring microtubules oscillate between phases of growth and shrinkage independently of one another. The frequency with which microtubules change from growth to shrinkage is influenced by a variety of stimuli so that the distribution of the microtubule cyto-skeleton changes in response to developmental or positional cues (Kirschner and Mitchison 1986). The class of microtubule-associated proteins (MAPs) known as +TIPs plays a critical role in regulating microtubule dynamics. +TIPs associate with the dynamic, plus ends to modulate microtubule dynamics to mediate interactions between microtubule ends and specific docking sites within the cell (Akhmanova and Hoogenraad 2005; Galjart 2005). All major metazoan +TIPs studied so far including the dynactin component p150glued, Lis1, CLASP (CLIP170-associated protein), and EB1 are physically or functionally linked with the founder +TIP CLIP170 (Coquelle et al. 2002; Goodson et al. 2003; Lansbergen et al. 2004; Mimori-Kiyosue et al. 2005). Although there are functional distinctions between different +TIPs, they do physically interact with one another and so presumably influence each other's function. Importantly, an individual +TIP can interact with two partner +TIPs by distinct mechanisms (Coquelle et al. 2002; Goodson et al. 2003; Mimori-Kiyosue et al. 2005; Wolyniak et al. 2006), highlighting the complexity of the integration of +TIP function at microtubule ends. CLASP molecules were first identified by genetic approaches (Pasqualone and Huffaker 1994; Inoue et al. 2000) but subsequently gained their name by virtue of their physical association with CLIP170 and CLIP115 (Akhmanova et al. 2001). CLASPs are required to maintain a stem cell component in Drosophila and for ordered cell migration in vertebrates (Mathe et al. 2003; Lee et al. 2004) and can be critically important for localized modulation of microtubule dynamics (Inoue et al. 2004). Mammalian CLASPs stabilize the association of microtubules and the cell cortex (Mimori-Kiyosue et al. 2005). The simple fission yeast cytoskeleton offers unique opportunities for the analysis of conserved aspects of microtubule function. A differentiated interphase cytoskeleton, in which the plus ends extend away from a centrally placed nucleus, distributes mitochondria, maintains the central position of the nucleus, and delivers cell polarity determinants such as the Kelch domain protein Tea1 to the cell tips (Mata and Nurse 1997; Hagan 1998). Tea1 is transported to cell tips as a result of its association with the +TIPs Tea2 (kinesin-related protein), Tip1 (CLIP170), and Mal3 (EB1) (Browning et al. 2000, 2003; Brunner and Nurse 2000). Exactly how they mediate Tea1 transport remains to be established; however, all four molecules move along the microtubule lattice as well as associating with the growing plus ends. Although Tip1 can associate with microtubules in cells from which the tea2 + gene has been deleted (Busch and Brunner 2004), a large part of the movement of the Tea1/ Tip1/Mal3 complex along the microtubule lattice is mediated by Tea2 activity, and the physical association between Mal3 and Tea2 stimulates the ATPase activity of the kinesin (Browning et al. 1998, 2000; Browning and Hackney 2005). We now show that the Schizosaccharomyces pombe CLASP Peg1 destabilized microtubules in interphase and yet promoted microtubule stability in mitosis. Focusing on interphase, we did not find a strong link between the function of Peg1 and that of Mal3 or Tip1; rather, we revealed a strong functional link between Peg1 and dynein.

Summary Background Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion of arrays of cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into a single radial MT (rMT) array associated with the spindle pole body (SPB), the major MTOC during meiotic prophase. The rMT is then dissolved upon the onset of meiosis I when a bipolar spindle emerges to conduct chromosome segregation. Structural features and molecular mechanisms that govern these dynamic MT rearrangements are poorly understood. Results Electron tomography of the SPBs showed that the rMT emanates from a newly recognized amorphous structure, which we term the rMTOC. The rMTOC, which resides at the cytoplasmic side of the SPB, is highly enriched in γ-tubulin reminiscent of the pericentriolar material of higher eukaryotic centrosomes. Formation of the rMTOC depends on Hrs1/Mcp6, a meiosis-specific SPB component that is located at the rMTOC. At the onset of meiosis I, Hrs1/Mcp6 is subject to strict downregulation by both proteasome-dependent degradation and phosphorylation leading to complete inactivation of the rMTOC. This ensures rMT dissolution and bipolar spindle formation. Conclusions Our study reveals the molecular basis for the transient generation of a novel MTOC, which triggers a program of MT rearrangement that is required for meiotic differentiation.

Insertion into and release of the cytoplasmic domain of the Schizosaccharomyces pombe spindle pole body from a nuclear envelope fenestra during mitosis requires Brr6., The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.

The Aspergillus nidulans protein kinase NIMA regulates mitotic commitment, while the human and Xenopus equivalents influence centrosome function. Two recessive, temperature-sensitive mutations in the Schizosaccharomyces pombe NIMA homologue, Fin1, blocked spindle formation at 37 degrees C. One of the two spindle pole bodies (SPBs) failed to nucleate microtubules. This phenotype was reduced by accelerating mitotic commitment through genetic inhibition of Wee1 or activation of either Cdc25 or Cdc2. Polo kinase (Plo1) normally associates with the SPB of mitotic, but not interphase cells. cut12.s11 is a dominant mutation in an SPB component that both suppresses cdc25 mutants and promotes Plo1 association with the interphase SPB. Both cut12.s11 phenotypes were abolished by removing Fin1 function. Elevating Fin1 levels promoted Plo1 recruitment to the interphase SPB of wild-type cells and reduced the severity of the cdc25.22 phenotype. These data are consistent with Fin1 regulating Plo1 function during mitotic commitment. The fin1 mitotic commitment and spindle phenotypes resemble distinct nimA phenotypes in different systems and suggest that the function of this family of kinases may be conserved across species.