RESUMO
Cytokinesis, as the last stage of the cell division cycle, is a tightly controlled process amongst all eukaryotes, with defective division leading to severe cellular consequences and implicated in serious human diseases and conditions such as cancer. Both mammalian cells and the fission yeast Schizosaccharomyces pombe use binary fission to divide into two equally sized daughter cells. Similar to mammalian cells, in S. pombe, cytokinetic division is driven by the assembly of an actomyosin contractile ring (ACR) at the cell equator between the two cell tips. The ACR is composed of a complex network of membrane scaffold proteins, actin filaments, myosin motors and other cytokinesis regulators. The contraction of the ACR leads to the formation of a cleavage furrow which is severed by the endosomal sorting complex required for transport (ESCRT) proteins, leading to the final cell separation during the last stage of cytokinesis, the abscission. This review describes recent findings defining the two phases of cytokinesis in S. pombe: ACR assembly and constriction, and their coordination with septation. In summary, we provide an overview of the current understanding of the mechanisms regulating ACR-mediated cytokinesis in S. pombe and emphasize a potential role of ESCRT proteins in this process.
RESUMO
Cytokinesis is the final stage of cell division cycle when cellular constituents are separated to produce two daughter cells. This process is driven by the formation and constriction of a contractile ring. Progression of these events is controlled by mechanisms and proteins that are evolutionary conserved in eukaryotes from fungi to humans. Genetic and molecular studies in different model organisms identified essential cytokinesis genes, with several conserved proteins, including the anillin/Mid1p proteins, constituting the core cytokinetic machinery. The fission yeast Schizosaccharomyces pombe represents a well-established model organism to study eukaryotic cell cycle regulation. Cytokinesis in fission yeast and mammalian cells depends on the placement, assembly, maturation, and constriction of a medially located actin-myosin contractile ring (ACR). Here, we review aspects of the ACR assembly and cytokinesis process in fission yeast and consider the regulation of such events in mammalian cells. First, we briefly describe the role of anillin during mammalian ACR assembly and cytokinesis. Second, we describe different aspects of the anillin-like protein Mid1p regulation during the S. pombe cell cycle, including its structure, function, and phospho-regulation. Third, we briefly discuss Mid1pindependent ACR assembly in S. pombe. Fourth, we highlight emerging studies demonstrating the roles of anillin in human tumourigenesis introducing anillin as a potential drug target for cancer treatment. Collectively, we provide an overview of the current understanding of medial division and cytokinesis in S. pombe and suggest the implications of these observations in other eukaryotic organisms, including humans.
Assuntos
Neoplasias , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Humanos , Citocinese , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas Contráteis/metabolismo , Actinas/metabolismoRESUMO
Cytokinesis is the final stage of the cell cycle which separates cellular constituents to produce two daughter cells. Using the fission yeast Schizosaccharomyces pombe we have investigated the role of various classes of proteins involved in this process. Central to these is anillin/Mid1p which forms a ring-like structure at the cell equator that predicts the site of cell separation through septation in fission yeast. Here we demonstrate a direct physical interaction between Mid1p and the endosomal sorting complex required for transport (ESCRT)-associated protein Vps4p, a genetic interaction of the mid1 and vps4 genes essential for cell viability, and a requirement of Vps4p for the correct cellular localization of Mid1p. Furthermore, we show that Mid1p is phosphorylated by aurora kinase, a genetic interaction of the mid1 and the aurora kinase ark1 genes is essential for cell viability, and that Ark1p is also required for the correct cellular localization of Mid1p. We mapped the sites of phosphorylation of Mid1p by human aurora A and the polo kinase Plk1 and assessed their importance in fission yeast by mutational analysis. Such analysis revealed serine residues S332, S523 and S531 to be required for Mid1p function and its interaction with Vps4p, Ark1p and Plo1p. Combined these data suggest a physical interaction between Mid1p and Vps4p important for cytokinesis, and identify phosphorylation of Mid1p by aurora and polo kinases as being significant for this process.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Contráteis/metabolismo , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais/genética , Aurora Quinases/genética , Aurora Quinases/metabolismo , Sobrevivência Celular/genética , Análise Mutacional de DNA/métodos , Genes Fúngicos , Microrganismos Geneticamente Modificados/metabolismo , Mitose/genética , Mutação , Fosforilação/genética , Transporte Proteico/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The fission yeast Schizosaccharomyces pombe, an ascomycete fungus, is an established model organism for studying eukaryotic molecular and cellular events such as the cell cycle due to its powerful genetics, a sequenced genome, and the ease of molecular manipulation (Wood et al., Nature 415:871-880, 2002; Hoffman et al., Genetics 201:403-423, 2015). This chapter describes genetic and cytological methods to study endosomal sorting complex required for transport (ESCRT) function during the cell cycle in fission yeast. These include tetrad analysis to allow the creation of double mutants to test for genetic interactions by synthetic phenotype characterization, such as cellular growth and the analysis of division septa by calcofluor-white staining.
Assuntos
Ciclo Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Microscopia Intravital/métodos , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Benzenossulfonatos/química , Técnicas de Cultura de Células/métodos , Corantes Fluorescentes/química , Técnicas de Genotipagem/métodos , Microscopia de Fluorescência/métodos , Mutação , Coloração e Rotulagem/métodosRESUMO
Recent studies have revealed exciting new functions for forkhead transcription factors in cell proliferation and development. Cell proliferation is a fundamental process controlled by multiple overlapping mechanisms, and the control of gene expression plays a major role in the orderly and timely division of cells. This occurs through transcription factors regulating the expression of groups of genes at particular phases of the cell division cycle. In this way, the encoded gene products are present when they are required. This review outlines recent advances in our understanding of this process in yeast model systems and describes how this knowledge has informed analysis in more developmentally complex eukaryotes, particularly where it is relevant to human disease.
RESUMO
The division cycle of unicellular yeasts is completed with the activation of a cell separation program that results in the dissolution of the septum assembled during cytokinesis between the 2 daughter cells, allowing them to become independent entities. Expression of the eng1(+) and agn1(+) genes, encoding the hydrolytic enzymes responsible for septum degradation, is activated at the end of each cell cycle by the transcription factor Ace2. Periodic ace2(+) expression is regulated by the transcriptional complex PBF (PCB Binding Factor), composed of the forkhead-like proteins Sep1 and Fkh2 and the MADS box-like protein Mbx1. In this report, we show that Ace2-dependent genes contain several combinations of motifs for Ace2 and PBF binding in their promoters. Thus, Ace2, Fkh2 and Sep1 were found to bind in vivo to the eng1(+) promoter. Ace2 binding was coincident with maximum level of eng1(+) expression, whereas Fkh2 binding was maximal when mRNA levels were low, supporting the notion that they play opposing roles. In addition, we found that the expression of eng1(+) and agn1(+) was differentially affected by mutations in PBF components. Interestingly, agn1(+) was a major target of Mbx1, since its ectopic expression resulted in the suppression of Mbx1 deletion phenotypes. Our results reveal a complex regulation system through which the transcription factors Ace2, Fkh2, Sep1 and Mbx1 in combination control the expression of the genes involved in separation at the end of the cell division cycle.
Assuntos
Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/metabolismoRESUMO
Mammalian cytokinesis proceeds by constriction of an actomyosin ring and furrow ingression, resulting in the formation of the midbody bridge connecting two daughter cells. At the centre of the midbody resides the Flemming body, a dense proteinaceous ring surrounding the interlocking ends of anti-parallel microtubule arrays. Abscission, the terminal step of cytokinesis, occurs near the Flemming body. A series of broad processes govern abscission: the initiation and stabilisation of the abscission zone, followed by microtubule severing and membrane scission-The latter mediated by the endosomal sorting complex required for transport (ESCRT) proteins. A key goal of cell and developmental biologists is to develop a clear understanding of the mechanisms that underpin abscission, and how the spatiotemporal coordination of these events with previous stages in cell division is accomplished. This article will focus on the function and dynamics of the ESCRT proteins in abscission and will review recent work, which has begun to explore how these complex protein assemblies are regulated by the cell cycle machinery.
Assuntos
Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Mitose , Proteínas Quinases/metabolismo , Animais , Humanos , Modelos Biológicos , Schizosaccharomyces/enzimologiaRESUMO
Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.
Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Epistasia Genética , Mitose , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Schizosaccharomyces/citologia , Proteínas de Ciclo Celular , Endossomos/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Mutação , Fenótipo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases , Transporte Proteico , Proteínas Proto-Oncogênicas , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismo , Quinase 1 Polo-LikeRESUMO
Here we report the result of a genetic screen for mutants resistant to the microtubule poison methyl benzimidazol-2-yl carbamate (MBC) that were also temperature sensitive for growth. In total the isolated mutants were distributed in ten complementation groups. Cloning experiments revealed that most of the mutants were in essential genes encoding various 26S proteasome subunits. We found that the proteasome mutants are multi-drug resistant due to stabilization of the stress-activated transcription factor Pap1. We show that the ubiquitylation and ultimately the degradation of Pap1 depend on the Rhp6/Ubc2 E2 ubiquitin conjugating enzyme and the Ubr1 E3 ubiquitin-protein ligase. Accordingly, mutants lacking Rhp6 or Ubr1 display drug-resistant phenotypes.
Assuntos
Complexo de Endopeptidases do Proteassoma/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Brefeldina A/farmacologia , Cafeína/farmacologia , Resistência a Múltiplos Medicamentos , Proteínas Associadas a Pancreatite , Estaurosporina/farmacologia , UbiquitinaçãoRESUMO
Temporal changes in transcription programs are coupled to control of cell growth and division. We here report that Mediator, a conserved coregulator of eukaryotic transcription, is part of a regulatory pathway that controls mitotic entry in fission yeast. The Mediator subunit cyclin-dependent kinase 8 (Cdk8) phosphorylates the forkhead 2 (Fkh2) protein in a periodic manner that coincides with gene activation during mitosis. Phosphorylation prevents degradation of the Fkh2 transcription factor by the proteasome, thus ensuring cell cycle-dependent variations in Fkh2 levels. Interestingly, Cdk8-dependent phosphorylation of Fkh2 controls mitotic entry, and mitotic entry is delayed by inactivation of the Cdk8 kinase activity or mutations replacing the phosphorylated serine residues of Fkh2. In addition, mutations in Fkh2, which mimic protein phosphorylation, lead to premature mitotic entry. Therefore, Fkh2 regulates not only the onset of mitotic transcription but also the correct timing of mitotic entry via effects on the Wee1 kinase. Our findings thus establish a new pathway linking the Mediator complex to control of mitotic transcription and regulation of mitotic entry in fission yeast.
Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Mitose , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Ativação Enzimática , Fatores de Transcrição Forkhead/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Schizosaccharomyces/citologiaAssuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Citocinese/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas Tirosina Fosfatases/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The regulation of gene expression through the mitotic cell cycle, so that genes are transcribed at particular cell cycle times, is widespread among eukaryotes. In some cases, it appears to be important for control mechanisms, as deregulated expression results in uncontrolled cell divisions, which can cause cell death, disease, and malignancy. In this review, I describe the current understanding of such regulated gene expression in two established simple eukaryotic model organisms, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In these two yeasts, the global pattern of cell cycle gene expression has been well described, and most of the transcription factors that control the various waves of gene expression, and how they are in turn themselves regulated, have been characterized. As related mechanisms occur in all other eukaryotes, including humans, yeasts offer an excellent paradigm to understand this important molecular process.
Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Evolução Biológica , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , Genes Fúngicos , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Checkpoints monitor the successful completion of cell cycle processes, such as DNA replication, and also regulate the expression of cell cycle-dependent genes that are required for responses. In the model yeast Schizosaccharomyces pombe G 1/S phase-specific gene expression is regulated by the MBF (also known as DSC1) transcription factor complex and is also activated by the mammalian ATM/ATR-related Rad3 DNA replication checkpoint. Here, we show that the Yox1 homeodomain transcription factor acts to co-ordinate the expression of MBF-regulated genes during the cell division cycle. Moreover, our data suggests that Yox1 is inactivated by the Rad3 DNA replication checkpoint via phosphorylation by the conserved Cds1 checkpoint kinase. Collectively, our data has implications for understanding the mechanisms underlying the coordination of cell cycle processes in eukaryotes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Replicação do DNA , Regulação Fúngica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/genética , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The control of gene expression at certain times during the mitotic cell division cycle is a common feature in eukaryotes. In fission yeast, at least five waves of gene expression have been described, with one transcribed at the M-G1 interval under the control of the PBF transcription factor complex. PBF consists of at least three transcription factors, two forkhead-like proteins Sep1p and Fkh2p, and a MADS box-like protein Mbx1p, and binds to PCB motifs found in the gene promoters. Mbx1p is under the direct control of the polo-like kinase Plo1p and the Cdc14p-like phosphatase Clp1p (Flp1p). Here, we show that M-G1 gene expression in fission yeast is also regulated by the anillin-like protein, Mid1p (Dmf1p). Mid1p binds in vivo to both Fkh2p and Sep1p, and to the promoter regions of M-G1 transcribed genes. Mid1p promoter binding is dependent on Fkh2p, Plo1p and Clp1p. The absence of mid1(+) in cells results in partial loss of M-G1 specific gene expression, suggesting that it has a negative role in controlling gene expression. This phenotype is exacerbated by also removing clp1(+), suggesting that Mid1p and Clp1p have overlapping functions in controlling transcription. As mid1(+) is itself expressed at M-G1, these observations offer a new mechanism whereby Mid1p contributes to controlling cell cycle-specific gene expression as part of a feedback loop.
Assuntos
Fase G1/genética , Regulação Fúngica da Expressão Gênica , Mitose/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Transcrição Gênica , Genes Fúngicos/genética , Modelos Genéticos , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Regulated gene expression makes an important contribution to cell cycle control mechanisms. In fission yeast, a group of genes is coordinately expressed during a late stage of the cell cycle (M phase and cytokinesis) that is controlled by common cis-acting promoter motifs named pombe cell cycle boxes (PCBs), which are bound by a trans-acting transcription factor complex, PCB binding factor (PBF). PBF contains at least three transcription factors, a MADS box protein Mbx1p and two forkhead transcription factors, Sep1p and Fkh2p. Here we show that the fission yeast Cdc14p-like phosphatase Clp1p (Flp1p) controls M-G1 specific gene expression through PBF. Clp1p binds in vivo both to Mbx1p, a MADS box-like transcription factor, and to the promoters of genes transcribed at this cell cycle time. Because Clp1p dephosphorylates Mbx1p in vitro, and is required for Mbx1p cell cycle-specific dephosphorylation in vivo, our observations suggest that Clp1p controls cell cycle-specific gene expression through binding to and dephosphorylating Mbx1p.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Fase G1/genética , Genes Fúngicos/genética , Mitose/genética , Modelos Genéticos , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
In fission yeast the expression of several genes during M-G1 phase is controlled by binding of the PCB binding factor (PBF) transcription factor complex to Pombe cell cycle box (PCB) promoter motifs. Three components of PBF have been identified, including two forkhead-like proteins Sep1p and Fkh2p, and a MADS-box-like protein, Mbx1p. Here, we examine how PBF is controlled and reveal a role for the Polo kinase Plo1p. plo1(+) shows genetic interactions with sep1(+), fkh2(+) and mbx1(+), and overexpression of a kinase-domain mutant of plo1 abolishes M-G1-phase transcription. Plo1p binds to and directly phosphorylates Mbx1p, the first time a Polo kinase has been shown to phosphorylate a MADS box protein in any organism. Fkh2p and Sep1p interact in vivo and in vitro, and Fkh2p, Sep1p and Plo1p contact PCB promoters in vivo. However, strikingly, both Fkh2p and Plo1p bind to PCB promoters only when PCB-controlled genes are not expressed during S- and G2-phase, whereas by contrast Sep1p contacts PCBs coincident with M-G1-phase transcription. Thus, Plo1p, Fkh2p and Sep1p control M-G1-phase gene transcription through a combination of phosphorylation and cell-cycle-specific DNA binding to PCBs.
Assuntos
Divisão Celular , Fatores de Transcrição Forkhead/genética , Fase G1 , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cdc10p is a major component of the cell cycle transcription factor complex MBF that controls G1-S phase specific gene expression in the fission yeast Schizosaccharomyces pombe. Here, we describe the identification of a new binding partner to Cdc10p and Pol5p. Pol5p was discovered through a 2-hybrid screen, with the direct interaction confirmed by in vitro "pull-down" experiments with bacterially expressed proteins. Pol5p appears to have no role in cell cycle gene expression, but is instead required for rRNA production. Pol5p is an essential gene, expressed constitutively throughout both the mitotic and meiotic life cycles, and localises to the nucleus. Over-expressing Pol5p has no phenotype, but reducing levels of Pol5p inhibits rRNA production. Pol5p is shown to bind to rDNA promoter fragments. Potentially, we have identified a mechanism by which Cdc10p controls rDNA gene expression, therefore linking the cell cycle with cellular growth.
Assuntos
Proteínas de Ciclo Celular/metabolismo , RNA Fúngico/biossíntese , RNA Ribossômico/biossíntese , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ciclo Celular , Proteínas de Ciclo Celular/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Modelos Biológicos , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genéticaRESUMO
V-ATPases play multiple roles in eukaryotes: in Drosophila, null mutations are recessive lethal. Here, mutations underlying five extant lethal alleles of vha55, encoding the B subunit, were identified, including a premature termination codon and two mutations very close to residues thought to participate in the catalytic site of the enzyme. Lethality of these alleles could be reverted by transformation of flies with a wild type vha55::GFP fusion, confirming that the lethal phenotype described for these alleles was due to defects in V-ATPase function. The chimeric protein was correctly localised to the apical domain of the Malpighian (renal) tubule, and restored fluid transport function to wild-type levels. No dominant-negative phenotype was apparent in heterozygotes. When the vha55::GFP fusion was driven ubiquitously, fluorescent protein was only detectable in tissues known to contain high levels of V-ATPase, suggesting that vha55 requires stoichometric co-expression of other subunits to be stable. Yeast (Saccharomyces cerevisiae) deleted for the corresponding gene (Deltavma2) demonstrated a pH-sensitive growth phenotype that was rescued by the vha55::GFP construct. Deltavma2 yeast could not be rescued with fly cDNAs encoding any of the mutant vha55 alleles, confirming the functional significance of the mutated residues. In yeast, bafilomycin-sensitive ATPase activity and growth rate correlated with the ability of different constructs to rescue the pH-sensitive conditional-lethal phenotype. These classical Drosophila mutants thus identify residues that are essential for function in organisms with wide phylogenetic separation.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/fisiologia , Alelos , Animais , Sequência Conservada/fisiologia , Drosophila melanogaster/genética , Regulação Enzimológica da Expressão Gênica , Genes Letais , Proteínas de Fluorescência Verde/genética , Concentração de Íons de Hidrogênio , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
Much scientific research has focused on characterising regulatory pathways and mechanisms responsible for cell integrity, growth and division. This area of study is of direct relevance to human medicine as uncontrolled growth and division underlies many diseases, most strikingly cancer. In cancer cells, normal regulatory mechanisms for growth and division are often altered, or even fail to exist. This review summarises the mechanisms that control the genes and gene products regulating cytokinesis and cell separation in the fission yeast Schizosaccharomyces pombe, as well as highlighting conserved aspects in the budding yeast Saccharomyces cerevisiae and higher eukaryotes. Particular emphasis is put on the role of gene expression, the Polo-like kinases (Plks), and the signal transduction pathways that control these processes.