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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.

Schizosaccharomyces pombe is an attractive model organism with which to study core principles of conserved molecular cell biology processes. The ability to monitor protein behavior following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) underpins much of this activity. Here we describe a robust protocol for the preparation of protein samples for analysis by SDS-PAGE.

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.

Schizosaccharomyces pombe cells are rod shaped, and they grow by tip elongation. Growth ceases during mitosis and cell division; therefore, the length of a septated cell is a direct measure of the timing of mitotic commitment, and the length of a wild-type cell is an indicator of its position in the cell cycle. A large number of documented stage-specific changes can be used as landmarks to characterize cell cycle progression under specific experimental conditions. Conditional mutations can permanently or transiently block the cell cycle at almost any stage. Large, synchronously dividing cell populations, essential for the biochemical analysis of cell cycle events, can be generated by induction synchrony (arrest-release of a cell cycle mutant) or selection synchrony (centrifugal elutriation or lactose-gradient centrifugation). Schizosaccharomyces pombe cell cycle studies routinely combine particular markers, mutants, and synchronization procedures to manipulate the cycle. We describe these techniques and list key landmarks in the fission yeast mitotic cell division cycle.

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.

Biochemical monitoring and interrogation of protein function is a critical component of most fission yeast studies. In particular, its small proteome size, high conservation of core molecular cell biology, and genetic malleability make Schizosaccharomyces pombe an excellent model organism in which to use mass spectrometry to conduct proteome-wide approaches. Here we discuss issues encountered during the analysis of fission yeast protein preparations.

We describe procedures for the immunoprecipitation (IP) of a molecule of interest from cell extracts under native or denaturing conditions. The methods are equally effective with antibodies that directly recognize the molecule of interest and those that recognize a generic peptide "epitope tag" that has been fused to sequences encoding the gene of interest. The diverse chemistry of intermolecular interactions and enzymatic activities means that a range of different buffer conditions must be assessed empirically to identify optimal conditions for the study of a specific target/complex in a particular assay. We describe three buffers that can serve as starting points for this empirical testing and discuss modifications that are commonly used in the optimization of assays based on immunoprecipitation.

We describe procedures for the immunoprecipitation (IP) of a molecule of interest from cell extracts under native or denaturing conditions. The methods are equally effective with antibodies that directly recognize the molecule of interest and those that recognize a generic peptide “epitope tag” that has been fused to sequences encoding the gene of interest. The diverse chemistry of intermolecular interactions and enzymatic activities means that a range of different buffer conditions must be assessed empirically to identify optimal conditions for the study of a specific target/complex in a particular assay. We describe three buffers that can serve as starting points for this empirical testing and discuss modifications that are commonly used in the optimization of assays based on immunoprecipitation.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system., Swiss National Science Foundation (www.snf.ch). École polytechnique fédérale de Lausanne (www.epfl.ch). Cancer Research UK [CRUK] C147/A6058.

Transient inactivation of the cdc25+ gene product by manipulation of the culture temperature for cdc25-22 cells is the most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes the inhibitory phosphate placed on Cdk1 by Wee1, inactivation of Cdc25 arrests cells at the G2/M boundary. Incubation at the restrictive temperature of 36°C for just over one generation time forces all cells in the culture to accumulate at the G2/M boundary. Restoration of Cdc25 function via a return to the permissive temperature or chemical inhibition of Wee1 activity at 36°C can then promote a highly synchronous wave of cell division throughout the culture. These approaches can be performed on any scale and thus support simultaneous assessment of numerous events within a single culture. After describing this simple and widely applicable procedure, we discuss frequently overlooked issues that can have a considerable impact on the interpretation of data from cdc25-22 induction-synchronized cultures.

Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive β-tubulin mutation was optimized by Mitsuhiro Yanagida’s laboratory to synchronize mitotic progression in an entire cell population. The inability to form microtubules following the loss of β-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of β-tubulin function by rewarming to 30°C (or higher) releases the arrest, generating a highly synchronous progression through mitosis. The viability of nda3-KM311 strains at 30°C makes it feasible to generate double mutants between nda3-KM311 and any temperature-sensitive mutant that can also grow at 30°C. These double mutants can be used in reciprocal shift analyses, in which cold-induced early mitotic arrest is relieved by a shift to 36°C, which then inactivates the product of the second mutant gene. The addition of microtubule depolymerizing drugs before the return to 36°C will maintain checkpoint signaling at 36°C transiently, permitting analysis of the impact of temperature-sensitive mutations on checkpoint function. Silencing the checkpoint of nda3-KM311-arrested cells at 20°C through chemical inhibition of aurora kinase is a powerful way to study checkpoint recovery pathways and mitotic exit without anaphase.

Division of Schizosaccharomyces pombe by medial fission produces identically sized daughter cells that grow by tip extension until their own division is prompted by reaching the same critical size for division as the parental cell. The fidelity of this size control in the absence of perturbation means that cells of the same size are at the same point in the cell cycle. Size selection of small cells from an asynchronous culture by centrifugal elutriation permits generation of synchronous cultures large enough for biochemical analysis. The changes observed in the synchronized cell cycle progression of such cultures are representative of those that accompany cell cycle progression of individual cells. Here, we describe how size selection with the Beckman Coulter JE-5.0 rotor can be used to generate synchronized cultures. Because of the continuous passage of medium through the rotor throughout the procedure, elutriation is considered to have less impact on the integrity of the cell cycle than other approaches. Two protocols are presented here: The first generates a 2-L culture ideal for detailed biochemical analysis, whereas the second allows rapid generation and simultaneous analysis of three smaller (200-mL) cultures.

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.