RESUMO
The septation initiation network (SIN) is a conserved signal transduction network, which is important for cytokinesis in Schizosaccharomyces pombe. The SIN component Etd1p is required for association of some SIN proteins with the spindle pole body (SPB) during anaphase and for contractile ring formation. We show that tethering of Cdc7p or Sid1p to the SIN scaffold Cdc11p at the SPB, rescues etd1-Δ. Analysis of a suppressor of the mutant etd1-M9 revealed that SIN signalling is influenced by the carbon source of the cell. Growth on a non-fermentable carbon source glycerol reduces the requirement for SIN signalling but does not bypass it. The decreased need for SIN signalling is mediated largely by reduction of protein kinase A activity, and it is phenocopied by deletion of pka1 on glucose medium. We conclude that protein kinase A is an important regulator of the SIN, and that SIN signalling is regulated by the carbon source of the cell.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Schizosaccharomyces , Proteínas Quinases Dependentes de AMP Cíclico/genética , Schizosaccharomyces/genética , Citoesqueleto de Actina , Carbono , Transdução de SinaisRESUMO
The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis, and asymmetric association of SIN proteins with the mitotic spindle pole bodies (SPBs) is important for its regulation. Here, we have used semi-automated image analysis to study SIN proteins in large numbers of wild-type and mutant cells. Our principal conclusions are: first, that the association of Cdc7p with the SPBs in early mitosis is frequently asymmetric, with a bias in favour of the new SPB; second, that the early association of Cdc7p-GFP to the SPB depends on Plo1p but not Spg1p, and is unaffected by mutations that influence its asymmetry in anaphase; third, that Cdc7p asymmetry in anaphase B is delayed by Pom1p and by activation of the spindle assembly checkpoint, and is promoted by Rad24p; and fourth, that the length of the spindle, expressed as a fraction of the length of the cell, at which Cdc7p becomes asymmetric is similar in cells dividing at different sizes. These data reveal that multiple regulatory mechanisms control the SIN in mitosis and lead us to propose a two-state model to describe the SIN.
Assuntos
GTP Fosfo-Hidrolases/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Schizosaccharomyces pombe/genética , Fuso Acromático/genética , Corpos Polares do Fuso/genética , Proteínas de Ciclo Celular/genética , Citocinese/genética , Proteínas de Fluorescência Verde/genética , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose/genética , Proteínas Quinases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Fuso Acromático/fisiologiaRESUMO
The Schizosaccharomyces pombe septation initiation network (SIN) is required for cytokinesis during vegetative growth and for spore formation during meiosis. Regulation of the SIN during mitosis has been studied extensively, but less is known about its meiotic regulation. Here, we show that several aspects of SIN regulation differ between mitosis and meiosis. First, the presence of GTP-bound Spg1p is not the main determinant of the timing of Cdc7p and Sid1p association with the spindle pole body (SPB) during meiosis. Second, the localisation dependencies of SIN proteins differ from those in mitotic cells, suggesting a modified functional organisation of the SIN during meiosis. Third, there is stage-specific degradation of SIN components in meiosis; Byr4p is degraded after meiosis I, whereas the degradation of Cdc7p, Cdc11p and Sid4p occurs after the second meiotic division and depends upon the ubiquitin ligase Dma1p. Finally, Dma1p-dependent degradation is not restricted to the SIN, as we show that Dma1p is needed for the degradation of Mcp6p (also known as Hrs1p) during meiosis I. Taken together, these data suggest that stage-specific targeted proteolysis plays an important role in regulating meiotic progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Meiose/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Proteínas de Ciclo Celular/genética , Proteínas Repressoras/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transdução de SinaisRESUMO
Meiosis is a specialised form of the cell cycle that gives rise to haploid gametes. In Schizosaccharomyces pombe, the products of meiosis are four spores, which are formed by encapsulation of the four meiosis II nuclei within the cytoplasm of the zygote produced by fusion of the mating cells. The S. pombe spindle pole body is remodelled during meiosis II and membrane vesicles are then recruited there to form the forespore membrane, which encapsulates the haploid nucleus to form a prespore. Spore wall material is then deposited, giving rise to the mature spore. The septation initiation network is required to coordinate cytokinesis and mitosis in the vegetative cycle and for spore formation in the meiotic cycle. We have investigated the role of the SIN regulator dma1p in meiosis; we find that although both meiotic divisions occur in the absence of dma1p, asci frequently contain fewer than four spores, which are larger than in wild-type meiosis. Our data indicate that dma1p acts in parallel to the leading-edge proteins and septins to assure proper formation for the forespore membrane. Dma1p also contributes to the temporal regulation of the abundance of the meiosis-specific SIN component mug27p.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Mutantes/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/genética , Contagem de Colônia Microbiana , Meiose/genética , Fusão de Membrana/genética , Proteínas Mutantes/genética , Transporte Proteico/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais , Esporos Fúngicos/genética , Esporos Fúngicos/isolamento & purificação , Transgenes/genéticaRESUMO
Meiotic progression in S. pombe is regulated by stage-specific gene expression and translation, changes in RNA stability, expression of anti-sense transcripts, and targeted proteolysis of regulatory proteins. We have used SILAC labeling to examine the relative levels of proteins in diploid S. pombe cells during meiosis. Among the 3,268 proteins quantified at all time points, the levels of 880 proteins changed at least 2-fold; the majority of proteins showed stepwise increases or decreases during the meiotic divisions, while some changed transiently. Overall, we observed reductions in proteins involved in anabolism and increases in proteins involved in catabolism. We also observed increases in the levels of proteins of the ESCRT-III complex and revealed a role for ESCRT-III components in chromosome segregation and spore formation. Correlation with studies of meiotic gene expression and ribosome occupancy reveals that many of the changes in steady-state protein levels are post-transcriptional.
Assuntos
Segregação de Cromossomos/fisiologia , Cromossomos Fúngicos/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Meiose/fisiologia , Proteoma/biossíntese , Proteínas de Schizosaccharomyces pombe/biossíntese , Schizosaccharomyces/metabolismo , Cromossomos Fúngicos/genética , Proteoma/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
A novel mutant screen in fission yeast has identified the 'ethanol dependent' protein etd1p as a potential link between the septation initiation network (SIN), which initiates cytokinesis, and the actomyosin contractile ring that drives separation of the two daughter cells at the end of mitosis.
Assuntos
Actomiosina/metabolismo , Divisão Celular/fisiologia , Citocinese/fisiologia , Modelos Biológicos , Proteínas de Ciclo Celular/metabolismo , Etanol/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The septation initiation network (SIN) triggers the onset of cytokinesis in the fission yeast Schizosaccharomyces pombe by promoting contraction of the medially placed F-actin ring. SIN signaling is regulated by the polo-like kinase plo1p and by cdc2p, the initiator of mitosis, and its activation is co-ordinated with other events in mitosis to ensure that cytokinesis does not begin until chromosomes have been separated. Though the SIN controls the contractile ring, the signal originates from the poles of the mitotic spindle. Recent studies suggest that the spindle pole body may act as a dynamic assembly site for active SIN signaling complexes. In the budding yeast Saccharomyces cerevisiae the counterpart of the SIN, called the MEN, mediates both mitotic exit and cytokinesis, in part through regulating activation of the phosphoprotein phosphatase Cdc14p. Flp1p, the S. pombe ortholog of Cdc14p, is not essential for mitotic exit, but may contribute to an orderly mitosis-G1 transition by regulating the destruction of the mitotic inducer cdc25p.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Transdução de Sinais/fisiologia , Leveduras/fisiologia , Actinas/metabolismo , Citocinese/fisiologia , Fuso Acromático/fisiologiaRESUMO
The fission yeast septation initiation network (SIN) triggers the onset of septum formation and cytokinesis. SIN proteins signal from the spindle pole body (SPB), to which they bind in a cell cycle-dependent manner, via the scaffold proteins sid4p and cdc11p. cdc11p becomes hyperphosphorylated during anaphase, when the SIN is active. We have investigated the phosphorylation state of cdc11p during mitosis in various mutant backgrounds. We show that association of cdc11p with the spindle pole body is required for its phosphorylation and that ectopic activation of the SIN results in hyperphosphorylation of cdc11p. We demonstrate that mitotic hyperphosphorylation of cdc11p requires the activity of cdc7p and that its dephosphorylation at the end of mitosis requires PP2A-par1p. Furthermore, spindle checkpoint arrest prevents cdc11p hyperphosphorylation. Finally, we show that the septation inhibitor byr4p interacts preferentially with hypophosphorylated cdc11p. We conclude that cdc11p hyperphosphorylation correlates with activation of the SIN and that this may be mediated primarily by cdc7p in vivo.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Genes Fúngicos , Mitose , Mutação , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fuso Acromático/metabolismoRESUMO
Cytokinesis in fission yeast is controlled by the Septation Initiation Network (SIN), a protein kinase signaling network using the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this paper, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.
Assuntos
Citocinese/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes/genética , Modelos Genéticos , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Mutação , Reprodutibilidade dos Testes , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais/genéticaRESUMO
The initiation of cytokinesis in the fission yeast Schizosaccharomyces pombe is signalled by the septation initiation network (SIN). Signalling originates from the spindle pole body (SPB), where SIN proteins are anchored by a scaffold composed of cdc11p and sid4p. Cdc11p links the other SIN proteins to sid4p and the SPB. Homologues of cdc11p have been identified in Saccharomyes cerevisiae (Nud1p) and human cells (Centriolin). We have defined functional domains of cdc11p by analysis of deletion mutants. We demonstrate that the C-terminal end of cdc11p is necessary for SPB localisation. We also show that the N-terminal domain is necessary and sufficient for signal transduction, since tethering of this domain to the SPB will substitute for cdc11p in SIN function.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/metabolismo , Transdução de Sinais , Proteínas de Ciclo Celular/química , Divisão Celular , Proteínas do Citoesqueleto/química , Desoxirribonucleases/metabolismo , Proteínas de Drosophila/metabolismo , Deleção de Genes , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático , Temperatura , tRNA MetiltransferasesRESUMO
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.
Assuntos
Engenharia Genética/métodos , Schizosaccharomyces/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Vetores Genéticos/genética , Genoma Bacteriano/genética , Schizosaccharomyces/efeitos dos fármacos , Homologia de Sequência do Ácido Nucleico , Estreptotricinas/farmacologia , Transformação GenéticaRESUMO
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.
Assuntos
Fator Promotor de Maturação/metabolismo , Mitose , Morfogênese , Schizosaccharomyces/fisiologia , Centrossomo , Ativação Enzimática , Estabilidade Enzimática , Retroalimentação Fisiológica , Fase G2 , Proteínas de Fluorescência Verde/metabolismo , Meia-Vida , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático/metabolismo , Imagem com Lapso de TempoRESUMO
A new study of fission yeast cell division has revealed a coupling between cytoplasmic partitioning and the turning-off of cytokinesis signalling that may be mediated by asymmetric protein distribution.
Assuntos
Citocinese , Schizosaccharomyces/citologia , Epistasia Genética , Schizosaccharomyces/genética , Transdução de SinaisRESUMO
The fission yeast septation initiation network, or SIN, is a signal transduction network that is required for septum formation in Schizosaccharomyces pombe. Its activity is tightly regulated through the cell cycle, to ensure proper co-ordination of mitosis and cytokinesis. SIN signalling requires three protein kinases for its function and is mediated by a ras-superfamily GTPase. We discuss the elements of the SIN and how they are regulated.
Assuntos
Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Divisão Celular , Meiose , Transporte Proteico , Schizosaccharomyces/crescimento & desenvolvimentoRESUMO
The protein kinase Cdc2p is the master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. It is required both for entry into mitosis and for onset of DNA replication. Cdc2p must be inactivated to permit exit from mitosis, licensing of replication origins and cytokinesis. To study the role of Cdc2p in greater detail, we generated a cdc2 allele that is sensitive to an inhibitory ATP analogue. We show that the inhibitor-induced cell cycle arrest is reversible and examine the effect of inhibiting Cdc2p on the regulation of the septation initiation network (SIN), which controls the initiation of cytokinesis in S. pombe. We found that specific inactivation of Cdc2p in a mitotically arrested cell promotes the asymmetrical recruitment of SIN proteins to the spindle poles and the recruitment of the most downstream SIN components and beta-(1,3) glucan synthase to the contractile ring. Thus, we conclude that inactivation of Cdc2p is sufficient to activate the SIN and promote cytokinesis.
Assuntos
Proteína Quinase CDC2/fisiologia , Citocinese , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/citologia , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/genética , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/fisiologia , Meiose , Mitose , Mutação , Proteínas Quinases/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Cytokinesis in Schizosaccharomyces pombe begins at mitotic entry, when the site of division is defined by formation of the contractile acto-myosin ring (CAR) at the cell cortex. Contraction of the CAR and formation of the division septum are triggered at the end of mitosis by septation initiation network (SIN) proteins associated with the spindle pole body (SPB). SIN signalling requires activation of the GTPase Spg1p, which is regulated by the bipartite GTPase-activating protein (GAP) Byr4p-Cdc16p. We show that, for Spg1p to associate with the SPB, it must be bound to its GAP or to its mitotic effector, the protein kinase Cdc7p. Analysis of the GAP proteins reveals that the steady-state level of Byr4p reflects that of Spg1p. Furthermore, if the interaction of Byr4p with Spg1p is compromised, the level of Byr4p decreases dramatically. The adaptation of the level of Byr4p to that of Spg1p requires the presence of Cdc16p and is mediated by proteasome-dependent destruction. It requires neither association with the SPB nor an active SIN. We propose a mechanism that limits the amount of the Byr4p-Cdc16p GAP to the amount required to inhibit Spg1p signalling.
Assuntos
Citocinese/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Homeostase/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , GTP Fosfo-Hidrolases/genética , Proteínas Ativadoras de GTPase/genética , Mitose/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/fisiologia , Fuso Acromático/genética , Fuso Acromático/metabolismo , Regulação para Cima/fisiologiaRESUMO
When nutrients are abundant, S. pombe cells grow as rods, dividing by fission after formation of a medially placed cell wall or division septum. Septum formation is triggered by a group of proteins, called the septation initiation network or SIN, that trigger contraction of the acto-myosin contractile ring at the end of mitosis. Ectopic activation of the SIN can uncouple septum formation from other cell-cycle events, whereas loss of SIN signalling gives rise to multinucleated cells due to the failure of cytokinesis. When starved, S. pombe cells of opposite mating types fuse to form a diploid zygote that undergoes meiosis and produces four spores. No septa or contractile rings are formed during meiosis. In this study, we have investigated the role of the SIN in meiosis. Our data show that, whereas the meiotic divisions appear normal, SIN mutants cannot form spores. Forespore membrane formation is initiated, but the nuclei are not encapsulated properly. The SIN proteins localise to the spindle pole body in meiosis. The protein kinases Sid1p and Cdc7p do not associate with the spindle pole body until meiosis II, when forespore membrane deposition begins. These data indicate a role for the SIN in regulating spore formation during meiosis.
Assuntos
Meiose , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Esporos Fúngicos/citologia , Esporos Fúngicos/metabolismo , Membrana Celular/metabolismo , Diploide , Mutação/genética , Transporte Proteico , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Mitotic exit in Saccharomyces cerevisiae and septation in Schizosaccharomyces pombe are regulated by a conserved signaling network called the mitotic exit and septum initiation networks (SIN), respectively. The network is active on one of the two anaphase B spindle-pole bodies (SPBs). Whereas the inherent asymmetry of growth by budding accounts for elements of the asymmetry in S. cerevisiae, it has been unclear how, or why, the pathway is asymmetric in S. pombe. We show that elements of SPB duplication in S. pombe are conservative, and that the SIN is active on the new SPB. SIN association with the new SPB persists after transient depolymerization of microtubules. The localization of the NIMA-related kinase, Fin1, reveals further complexity in SPB inheritance. Fin1 associates with the SPB bearing the older components in all cells and with the "new" SPB in half of the population. Fin1 only binds the new SPB when this new SPB has arisen from the duplication of an SPB that is two or more cycles old. Thus, each of the four SPBs generated over two consecutive cell cycles are different, because they have distinct fates in the next cell cycle. Fin1 binds the SPB once the SIN is active and the association requires the SIN inhibitors Byr4 and Cdc16. Fin1 physically associates with Byr4. Compromising Fin1 function leads to SIN activation on both anaphase B SPBs and promotes septation, indicating that Fin1 restrains SIN activity on the old SPB.
Assuntos
Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Ciclo Celular , Centríolos/enzimologia , Genes Fúngicos , Modelos Biológicos , Mutação , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Fuso Acromático/enzimologiaRESUMO
In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is signalled via the septation initiation network (SIN) involving several protein kinases and a GTPase. Arabidopsis thaliana and Brassica napus proteins homologous to fission yeast spg1p (AtSGP1, AtSGP2), cdc7p (AtMAP3K epsilon 1, AtMAP3K epsilon 2, BnMAP3K epsilon 1) and sid1p (AtMAP4K alpha 1, AtMAP4K alpha 2, BnMAP4K alpha 2) exhibit a significant similarity. The plant proteins AtSGP1/2 and BnMAP4K alpha 2 are able to complement the S. pombe mutant proteins spg1-B8 and sid1-239, respectively and to induce mutisepta when overexpressed in wild-type yeast. Yeast two-hybrid assays demonstrated interactions both between plant proteins and between plant and yeast proteins of the SIN pathway. However, the primary structure of the proteins as well as the partial complementation of yeast mutants indicates that plant homologous proteins and their yeast counterparts have diverged during evolution. Real-time RT-PCR studies demonstrated plant SIN-related gene expression in all organs tested and a co-expression pattern during the cell cycle, with a higher accumulation at G(2)-M. During interphase, the plant SIN-related proteins were found to co-localise predominantly in the nucleolus of the plant cells, as shown by fusions to green fluorescent protein. These data suggest the existence of a plant SIN-related pathway.