Import of extracellular ATP in yeast and man modulates AMPK and TORC1 signalling.

AMP-activated kinase (AMPK) and target of rapamycin (TOR) signalling coordinate cell growth, proliferation, metabolism and cell survival with the nutrient environment of cells. The poor vasculature and nutritional stress experienced by cells in solid tumours raises the question: how do they assimilate sufficient nutrients to survive? Here, we show that human and fission yeast cells import ATP and AMP from their external environment to regulate AMPK and TOR signalling. Exposure of fission yeast (Schizosaccharomyces pombe) and human cells to external AMP impeded cell growth; however, in yeast this restraining impact required AMPK. In contrast, external ATP rescued the growth defect of yeast mutants with reduced TORC1 signalling; furthermore, exogenous ATP transiently enhanced TORC1 signalling in both yeast and human cell lines. Addition of the PANX1 channel inhibitor probenecid blocked ATP import into human cell lines suggesting that this channel may be responsible for both ATP release and uptake in mammals. In light of these findings, it is possible that the higher extracellular ATP concentration reported in solid tumours is both scavenged and recognized as an additional energy source beneficial for cell growth.

A PP1-PP2A phosphatase relay controls mitotic progression.

The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1-cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.

Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe.

Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, (13)C(6) (15)N(4)-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.

Mutation of a conserved residue enhances the sensitivity of analogue-sensitised kinases to generate a novel approach to the study of mitosis in fission yeast.

The chemical genetic strategy in which mutational enlargement of the ATP-binding site sensitises of a protein kinase to bulky ATP analogues has proved to be an elegant tool for the generation of conditional analogue-sensitive kinase alleles in a variety of model organisms. Here, we describe a novel substitution mutation in the kinase domain that can enhance the sensitivity of analogue-sensitive kinases. Substitution of a methionine residue to phenylalanine in the +2 position after HRDLKxxN motif of the subdomain VIb within the kinase domain markedly increased the sensitivities of the analogue-sensitive kinases to ATP analogues in three out of five S. pombe kinases (i.e. Plo1, Orb5 and Wee1) that harbor this conserved methionine residue. Kinome alignment established that a methionine residue is found at this site in 5-9% of kinases in key model organisms, suggesting that a broader application of this structural modification may enhance ATP analogue sensitivity of analogue-sensitive kinases in future studies. We also show that the enhanced sensitivity of the wee1.as8 allele in a cdc25.22 background can be exploited to generate highly synchronised mitotic and S phase progression at 36°C. Proof-of-principle experiments show how this novel synchronisation technique will prove of great use in the interrogation of the mitotic or S-phase functions through temperature sensitivity mutation of molecules of interest in fission yeast.

Spatial control of mitotic commitment in fission yeast.

The activation of the Cdk1 (cyclin-dependent kinase 1)-cyclin B complex to promote commitment to mitosis is controlled by the phosphorylation status of the Cdk1 catalytic subunit. Cdk1 phosphorylation by Wee1 kinases blocks activation until Cdc25 (cell division cycle 25) phosphatases remove this phosphate to drive division. Feedback inhibition of Wee1 and promotion of Cdc25 activities by the newly activated Cdk1-cyclin B complexes ensure that the transition from interphase to mitosis is a rapid and complete bi-stable switch. Although this level of molecular understanding of the mitotic commitment switch has been clear for over two decades, it is still unclear how the switch is engaged to promote division at the right time for a particular context. We discuss recent work in fission yeast that shows how the spatial organization of signalling networks, in particular events on the centrosome equivalent, the spindle pole body, plays a key role in ensuring that the timing of cell division is coupled to environmental cues.

Elevated basal AMP-activated protein kinase activity sensitizes colorectal cancer cells to growth inhibition by metformin.

Expression and activity of the AMP-activated protein kinase (AMPK) α1 catalytic subunit of the heterotrimeric kinase significantly correlates with poor outcome for colorectal cancer patients. Hence there is considerable interest in uncovering signalling vulnerabilities arising from this oncogenic elevation of AMPKα1 signalling. We have therefore attenuated mammalian target of rapamycin (mTOR) control of AMPKα1 to generate a mutant colorectal cancer in which AMPKα1 signalling is elevated because AMPKα1 serine 347 cannot be phosphorylated by mTORC1. The elevated AMPKα1 signalling in this HCT116 α1.S347A cell line confers hypersensitivity to growth inhibition by metformin. Complementary chemical approaches confirmed this relationship in both HCT116 and the genetically distinct HT29 colorectal cells, as AMPK activators imposed vulnerability to growth inhibition by metformin in both lines. Growth inhibition by metformin was abolished when AMPKα1 kinase was deleted. We conclude that elevated AMPKα1 activity modifies the signalling architecture in such a way that metformin treatment compromises cell proliferation. Not only does this mutant HCT116 AMPKα1-S347A line offer an invaluable resource for future studies, but our findings suggest that a robust biomarker for chronic AMPKα1 activation for patient stratification could herald a place for the well-tolerated drug metformin in colorectal cancer therapy.

Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe.

Strand-specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3' termination and bidirectional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent 'horsetail' stage of meiosis. Antisense production had no impact at any of these loci when the RNA interference (RNAi) machinery was removed. Thus, far from being simply 'genome chatter', this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe.

Polo kinase links the stress pathway to cell cycle control and tip growth in fission yeast.

Stress-activated mitogen-activated protein kinase cascades instigate a range of changes to enable eukaryotic cells to cope with particular insults. In Schizosaccharomyces pombe these responses include the transcription of specific gene sets and inhibition of entry into mitosis. The S. pombe stress response pathway (SRP) also promotes commitment to mitosis in unperturbed cell cycles to allow cells to match their rate of division with nutrient availability. The nature of this SRP function in cell cycle control is unknown. Entry into mitosis is controlled by mitosis-promoting factor (MPF; Cdc2/cyclin B) activity. Inhibitory phosphorylation of Cdc2 by Wee1 kinase inactivates MPF until Cdc25 removes this phosphate to promote mitosis. The balance between Wee1 and Cdc25 activities is influenced by the recruitment of polo kinase (Plo1) to the spindle pole body (SPB). The SPB component Cut12 mediates this recruitment. Hyper-activating mutations in either cut12 or plo1 enable Cdc25-defective cells to enter mitosis. The hyperactive cut12.s11 mutation suppresses cdc25.22, as it promotes recruitment of active Plo1 to interphase SPBs. Here we show that the SRP promotes phosphorylation of Plo1 on Ser 402. In unperturbed cell cycles, SRP-mediated phosphorylation of Ser 402 promotes Plo1 recruitment to SPBs and thus commitment to mitosis. Ser 402 phosphorylation also ensures efficient reinitiation of cell tip growth and cell division during recovery from particular stresses. Thus, phosphorylation of Plo1 Ser 402 not only enables SRP signalling to modulate the timing of mitotic commitment in response to nutrient status in unperturbed cycles, but also promotes the return to normal cell cycle control after stress.

A TOR (target of rapamycin) and nutritional phosphoproteome of fission yeast reveals novel targets in networks conserved in humans.

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe. Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPKα (ssp2::ura4+), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

Suppression of the Schizosaccharomyces pombe cut12.1 cell-cycle defect by mutations in cdc25 and genes involved in transcriptional and translational control.

Cdc25 phosphatase primes entry to mitosis by removing the inhibitory phosphate that is transferred to mitosis promoting factor (MPF) by Wee1 related kinases. A positive feedback loop then boosts Cdc25 and represses Wee1 activities to drive full-scale MPF activation and commitment to mitosis. Dominant mutations in the Schizosaccharomyces pombe spindle pole body (SPB) component Cut12 enable cdc25.22 mutants to overcome a G2 arrest at 36 degrees and enter mitosis. The recessive temperature-sensitive cut12.1 mutation results in the formation of monopolar spindles in which the spindle pole marker Sad1 is enriched on the nonfunctional SPB at 36 degrees . We identified mutations at five loci that suppressed the lethality of the recessive cut12.1 mutation at 36 degrees and conferred lethality at 20 degrees . Three of the five mutations led to the formation of monopolar spindles at restrictive temperatures, affected cell size at commitment to mitosis, and generated multiple Sad1 foci at nuclear periphery. The five loci, tfb2.rt1, tfb5.rt5, pla1.rt3, rpl4301.rt4, and rot2.1, and multicopy suppressors, including tfb1(+) and dbp10(+), are involved in transcription, translation, or RNA processing, prompting us to establish that elevating Cdc25 levels with the dominant cdc25.d1 allele, suppressed cut12.1. Thus, rot mutants provide a further link between protein production and cell-cycle progression.

S. pombe aurora kinase/survivin is required for chromosome condensation and the spindle checkpoint attachment response.

The spindle checkpoint inhibits anaphase until all chromosomes have established bipolar attachment. Two kinetochore states trigger this checkpoint. The absence of microtubules activates the attachment response, while the inability of attached microtubules to generate tension triggers the tension/orientation response. The single aurora kinase of budding yeast, Ipl1, is required for the tension/orientation, but not attachment, response. In contrast, we find that the single aurora kinase of fission yeast, Ark1, is required for the attachment response. Having established that the initiator codon assigned to ark1(+) was incorrect and that Ark1-associated kinase activity depended upon survivin function and phosphorylation, we found that the loss of Ark1 from kinetochores by either depletion or use of a survivin mutant overides the checkpoint response to microtubule depolymerization. Ark1/survivin function was not required for the association of Bub1 or Mad3 with the kinetochores. However, it was required for two aspects of Mad2 function that accompany checkpoint activation: full-scale association with kinetochores and formation of a complex with Mad3. Neither the phosphorylation of histone H3 that accompanies chromosome condensation nor condensin recruitment to mitotic chromatin were seen when Ark1 function was compromised. Cytokinesis was not affected by Ark1 depletion or expression of the "kinase dead" ark1.K118R mutant.

Elementary Protein Analysis in Schizosaccharomyces pombe.

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.

Small-Scale Immunoprecipitation from Fission Yeast Cell Extracts.

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.

Preparation of Protein Extracts from Schizosaccharomyces pombe Using Trichloroacetic Acid Precipitation.

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.

Large-Scale Immunoprecipitation from Fission Yeast Cell Extracts.

We outline immunoprecipitation (IP) procedures to isolate the large quantities of a molecule of interest that are required to identify posttranslational modifications (PTMs) in subsequent targeted mass spectrometry analysis. In situ denaturation by trichloroacetic acid precipitation inhibits the activities of modifying enzymes that could alter the PTM profile to preserve the PTMs on a target of interest throughout the precipitation step. In contrast, isolation of the same molecule with the nondenaturing variation on this IP procedure can maintain associations with partner molecules whose PTMs can also be mapped, albeit with the caveat that modifications could have occurred during the extended IP period.

Dialogue between centrosomal entrance and exit scaffold pathways regulates mitotic commitment.

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.

The centrosomal kinase Nek2 displays elevated levels of protein expression in human breast cancer.

Aneuploidy and chromosome instability are common abnormalities in human cancer. Loss of control over mitotic progression, multipolar spindle formation, and cytokinesis defects are all likely to contribute to these phenotypes. Nek2 is a cell cycle-regulated protein kinase with maximal activity at the onset of mitosis that localizes to the centrosome. Functional studies have implicated Nek2 in regulation of centrosome separation and spindle formation. Here, we present the first study of the protein expression levels of the Nek2 kinase in human cancer cell lines and primary tumors. Nek2 protein is elevated 2- to 5-fold in cell lines derived from a range of human tumors including those of cervical, ovarian, breast, prostate, and leukemic origin. Most importantly, by immunohistochemistry, we find that Nek2 protein is significantly up-regulated in preinvasive in situ ductal carcinomas of the breast as well as in invasive breast carcinomas. Finally, by ectopic expression of Nek2A in immortalized HBL100 breast epithelial cells, we show that increased Nek2 protein leads to accumulation of multinucleated cells with supernumerary centrosomes. These data highlight the Nek2 kinase as novel potential target for chemotherapeutic intervention in breast cancer.

Immunofluorescence Microscopy of Schizosaccharomyces pombe Using Chemical Fixation.

Establishing the subcellular distribution of molecules of interest and the dynamics of their spatial control underpins all areas of cell and developmental biology. Although the ability to monitor the distribution of fluorescent fusion proteins has revolutionized cell and developmental biology, indirect immunofluorescence microscopy of fixed samples remains an essential complement to this approach. Immunofluorescence is often a more appropriate approach for the study of subcellular architecture. It avoids potential artifacts caused by studying fusion proteins, which might show altered function under stressful imaging conditions. Furthermore, the quantitative analysis of multiple cells in an unperturbed population by immunofluorescence invariably provides a more accurate assessment of the spatial and temporal control of a particular process than does the analysis of individual cells that is the hallmark of live-cell imaging. Parallel studies of living and fixed cells often provide complementary data sets, both of which can be considered necessary for a comprehensive understanding of molecular function. This protocol provides a method for the visualization of the Schizosaccharomyces pombe microtubule cytoskeleton by indirect immunofluorescence microscopy following chemical fixation with formaldehyde and glutaraldehyde. It includes discussion of common modifications used to monitor the distribution of other fission yeast antigens and forms a basis from which to develop protocols to localize new molecules of interest.

Chromatin and Cell Wall Staining of Schizosaccharomyces pombe.

Fission yeasts grow by tip extension, maintaining a constant width until they reach a critical size threshold and divide. Division by medial fission-which gives these yeast their name-generates a new end that arises from the site of cytokinesis. The old end, which was produced during the previous cell cycle, initiates progression of the new cell cycle, and in G2, the new end is activated in a process termed new-end takeoff (NETO). In this protocol, the fluorescent stains calcofluor and 4',6-diamidino-2-phenylindole (DAPI) are used to give a rapid and informative assessment of morphogenesis and cell-cycle progression in the fission yeast Schizosaccharomyces pombe Calcofluor reveals the timing of NETO because it stains the birth scars that are generated at new ends by cytokinesis less efficiently than the rest of the cell wall. Intense calcofluor staining of the septum and measurement of cell length are also widely used to identify dividing cells and to gauge the timing of mitotic commitment. Staining nuclei with DAPI identifies mono- and binucleated cells and complements the calcofluor staining procedure to evaluate the stages of the cell cycle and identify mitotic errors. Equally simple DAPI staining procedures reveal chromatin structure in higher resolution, facilitating more accurate staging of mitotic progression and characterization of mitotic errors.