Fission yeast MO25 protein is localized at SPB and septum and is essential for cell morphogenesis

Cell morphogenesis is of fundamental significance in all eukaryotes for development, differentiation, and cell proliferation. In fission yeast, Drosophila Furry-like Mor2 plays an essential role in cell morphogenesis in concert with the NDR/Tricornered kinase Orb6. Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25-like Pmo25, GC kinase Nak1, Mor2, and Orb6, constitute a morphogenesis network that is important for polarity control and cell separation. Intriguingly, Pmo25 was localized at the mitotic spindle pole bodies (SPBs) and then underwent translocation to the dividing medial region upon cytokinesis. Pmo25 formed a complex with Nak1 and was required for both the localization and kinase activity of Nak1. Pmo25 and Nak1 in turn were essential for Orb6 kinase activity. Further, the Pmo25 localization at the SPBs and the Nak1-Orb6 kinase activities during interphase were under the control of the Cdc7 and Sid1 kinases in the septation initiation network (SIN), suggesting a functional linkage between SIN and the network for cell morphogenesis/separation following cytokinesis.     

Fission Yeast Germinal Center (GC) Kinase Ppk11 Interacts with Pmo25 and Plays an Auxiliary Role in Concert with the Morphogenesis Orb6 Network (MOR) in Cell Morphogenesis

How cell morphology and the cell cycle are coordinately regulated is a fundamental subject in cell biology. In fission yeast, 2 germinal center kinases (GCKs), Sid1 and Nak1, play an essential role in septation/cytokinesis and cell separation/cell polarity control, respectively, as components of the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR). Here we show that a third GCK, Ppk11, is also required for efficient cell separation particularly, at a high temperature. Although Ppk11 is not essential for cell division, this kinase plays an auxiliary role in concert with MOR in cell morphogenesis. Ppk11 physically interacts with the MOR component Pmo25 and is localized to the septum, by which Ppk11 is crucial for Pmo25 targeting/accumulation to the septum. The conserved C-terminal WDF motif of Ppk11 is essential for both septum accumulation of Pmo25 and efficient cell separation. In contrast its kinase activity is required only for cell separation. Thus, both interaction of Ppk11 with Pmo25 and Ppk11 kinase activity are critical for efficient cell separation.     

A method for Pmo25-associated kinase assay in fission yeast: the activity is dependent on two gC kinases Nak1 and Sid1

In fission yeast, the conserved proteins, MO25/Pmo25, GC kinase/Nak1, Furry/Mor2, NDR kinase/Orb6, and Mob2, constitute the morphogenesis Orb6 network (MOR). Previously we showed that Pmo25 functions as an upstream component of MOR and that it plays a connecting role between the septation initiation network (SIN) and MOR. Here we establish a Pmo25-associated kinase assay and show that the activity is dependent on Nak1/MOR and Sid1/SIN.     

Determination of Lipase Activity in AOT-Isooctane Reversed Micelles

In fission yeast, the conserved proteins, MO25/Pmo25, GC kinase/Nak1, Furry/Mor2, NDR kinase/Orb6, and Mob2, constitute the morphogenesis Orb6 network (MOR). Previously we showed that Pmo25 functions as an upstream component of MOR and that it plays a connecting role between the septation initiation network (SIN) and MOR. Here we establish a Pmo25-associated kinase assay and show that the activity is dependent on Nak1/MOR and Sid1/SIN.     

Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype

BACKGROUND: Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP)proteins, first identified in baculoviruses. Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division. To determine the function of Survivin in mammals, we analyzed the pattern of localization of Survivin protein during the cell cycle, and deleted its gene by homologous recombination in mice. RESULTS: In human cells, Survivin appeared first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocated to the spindle midzone during anaphase/telophase, and disappeared at the end of telophase. In the mouse, Survivin was required for mitosis during development. Null embryos showed disrupted microtubule formation, became polyploid, and failed to survive beyond 4.5days post coitum. This phenotype, and the cell-cycle localization of Survivin, resembled closely those of INCENP. Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division. CONCLUSIONS: In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.     

Fission yeast Aip3p (spAip3p) is required for an alternative actin-directed polarity program

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Delta strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae.     

Cpc2, a fission yeast homologue of mammalian RACK1 protein, interacts with Ran1 (Pat1) kinase To regulate cell cycle progression and meiotic development

The Schizosaccharomyces pombe ran1/pat1 gene regulates the transition between mitosis and meiosis. Inactivation of Ran1 (Pat1) kinase is necessary and sufficient for cells to exit the cell cycle and undergo meiosis. The yeast two-hybrid interaction trap was used to identify protein partners for Ran1/Pat1. Here we report the identification of one of these, Cpc2. Cpc2 encodes a homologue of RACK1, a WD protein with homology to the beta subunit of heterotrimeric G proteins. RACK1 is a highly conserved protein, although its function remains undefined. In mammalian cells, RACK1 physically associates with some signal transduction proteins, including Src and protein kinase C. Fission yeast cells containing a cpc2 null allele are viable but cell cycle delayed. cpc2Delta cells fail to accumulate in G(1) when starved of nitrogen. This leads to defects in conjugation and meiosis. Copurification studies show that although Cpc2 and Ran1 (Pat1) physically associate, Cpc2 does not alter Ran1 (Pat1) kinase activity in vitro. Using a Ran1 (Pat1) fusion to green fluorescent protein, we show that localization of the kinase is impaired in cpc2Delta cells. Thus, in parallel with the proposed role of RACK1 in mammalian cells, fission yeast cpc2 may function as an anchoring protein for Ran1 (Pat1) kinase. All defects associated with loss of cpc2 are reversed in cells expressing mammalian RACK1, demonstrating that the fission yeast and mammalian gene products are indeed functional homologues.     

Control of cell polarity in fission yeast by association of Orb6p kinase with the highly conserved protein methyltransferase Skb1p

In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.     

Genetic and molecular characterization of Skb15, a highly conserved inhibitor of the fission yeast PAK, Shk1

The p21-activated kinase, Shk1, is essential for viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Here we describe the characterization of a highly conserved, WD repeat protein, Skb15, which negatively regulates Shk1 in fission yeast. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution.     

A Link between Aurora Kinase and Clp1/Cdc14 Regulation Uncovered by the Identification of a Fission Yeast Borealin-Like Protein

The chromosomal passenger complex (CPC) regulates various events in cell division. This complex is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin. Together, these four subunits interdependently regulate CPC function, and they are highly conserved among eukaryotes. However, a Borealin homologue has never been characterized in the fission yeast, Schizosaccharomyces pombe. Here, we isolate a previously uncharacterized S. pombe protein through association with the Cdc14 phosphatase homologue, Clp1/Flp1, and identify it as a Borealin-like member of the CPC. Nbl1 (novel Borealin-like 1) physically associates with known CPC components, affects the kinase activity and stability of the S. pombe Aurora B homologue, Ark1, colocalizes with known CPC subunits during mitosis, and shows sequence similarity to human Borealin. Further analysis of the Clp1–Nbl1 interaction indicates that Clp1 requires CPC activity for proper accumulation at the contractile ring (CR). Consistent with this, we describe negative genetic interactions between mutant alleles of CPC and CR components. Thus, this study characterizes a fission yeast Borealin homologue and reveals a previously unrecognized connection between the CPC and the process of cytokinesis in S. pombe.     

Characterization of CaGSP1, the Candida albicans RAN/GSP1 homologue

Gsp1p is a small nuclear-located GTP binding protein from the yeast Saccharomyces cerevisiae. It is highly conserved among eucaryotic cells and is involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules. To learn more about the GSP1 structure/function, we have characterized its Candida albicans homologue. CaGsp1p is 214 amino acids long and displays 91% identity to the ScGsp1p. There is functional complementation in S. cerevisiae, and its mRNA is constitutively expressed in the diploid C. albicans grown under various physiological conditions. Disruption of both alleles was not possible, suggesting that it could be an essential gene, but heterozygous mutants exhibited genomic instability.     

禾谷镰孢菌蛋白激酶FgCdc15功能的研究

细胞分裂是真核细胞生长发育的重要环节。本研究利用生物信息学的方法,通过使用酵母菌Cdc15蛋白激酶序列比对,发现禾谷镰孢菌中只存在一个Cdc15蛋白激酶。基因缺失的功能研究表明FgCDC15（FGSG_10381）基因敲除后,突变体菌落生长速度减慢,对小麦和玉米无致病力。FgCDC15基因敲除突变体产生的分生孢子外观形态正常,但隔膜数量变少,同时发现该基因在分生孢子阶段表达量最高。在有性生殖阶段,FgCDC15基因敲除突变体可产生极少量的子囊壳,但不产生子囊和子囊孢子。本文研究表明,禾谷镰孢菌蛋白激酶FgCdc15可能参与了有性和无性阶段的细胞分裂和生长,同时影响禾谷镰刀菌的致病毒力。     

Cell Cycle Regulated Interaction of a Yeast Hippo Kinase and Its Activator MO25/Hym1

Hippo pathways are ancient signaling systems that contribute to cell growth and proliferation in a wide diversity of eukaryotes, and have emerged as a conserved regulator of organ size control in metazoans. In budding yeast, a Hippo signaling pathway called the Regulation of Ace2 and Morphogenesis (RAM) network promotes polarized cell growth and the final event in the separation of mother and daughter cells. A crucial regulatory input for RAM network control of cell separation is phosphorylation of a conserved hydrophobic motif (HM) site on the NDR/LATS family kinase Cbk1. Here we provide the first direct evidence that the Hippo-like kinase Kic1 in fact phosphorylates the HM site of Cbk1, and show that Kic1 is allosterically activated by Hym1, a highly conserved protein related to mammalian MO25. Using the structure of mammalian MO25 in complex with the Kic1-related pseudokinase STRAD, we identified conserved residues on Kic1 that are required for interaction with Hym1. We find that Kic1 and Hym1 protein levels remain constant throughout the cell cycle but the proteins’ association is regulated, with maximal interaction coinciding with peak Cbk1 HM site phosphorylation. We show that this association is necessary but not sufficient for this phosphorylation, suggesting another level of regulation is required to promote the complex to act upon its substrates. This work presents a previously undiscovered cell cycle regulated interaction between a Hippo kinase and a broadly conserved allosteric activator. Because of the conserved nature of this pathway in higher eukaryotes, this work may also provide insight into the modularity of Hippo signaling pathways.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.     

An anillin homologue,Mid2p,acts during fission yeast cytokinesis to organize the septin ring and promote cell separation

Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.     

Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

The role of polar localization in the function of an essential Caulobacter crescentus tyrosine kinase

DivL is an essential tyrosine kinase in Caulobacter crescentus that controls an early step in the cell division cycle. We show here that DivL dynamically localizes to the stalk-distal cell pole and less frequently to the stalked cell pole during the S-phase. The kinase is subsequently released from the cell poles late in division and remains dispersed in the newly divided progeny stalk and swarmer cells. Mutational analysis of DivL in a DivL-GFP fusion protein demonstrated that the extreme C-terminus and residues in the conserved four-helix bundle, which is the phosphorylation-dimerization domain, are important for localization. We speculate that the four-helix bundle of the core catalytic domain may serve as a recognition site for the "localization machinery". Unexpectedly, a DivL protein with mutations in the C-terminal localization sequence, and an intact catalytic domain, efficiently complemented a divL null mutation. Thus, subcellular localization of DivL is not essential to its function in cell division regulation. Regulation of cell division by DivL does, however, depend on its localization in the cell membrane.     

Dual function of the germinal centre kinase Don3 during mitosis and cytokinesis in Ustilago maydis

Septum formation is a crucial step of cytokinesis in fungi. In the basidiomycete Ustilago maydis, the germinal centre kinase Don3 triggers initiation of a secondary septum necessary for cell separation after cytokinesis. Here we show that oligomerization of Don3 via a putative coiled-coil domain is critical for secondary septum formation. Within the Don3 sequence we detected a characteristic C-terminal sequence motif (T-motif), which determines the subcellular localization of Don3 but is not required for regulation of cell separation. This motif defines a novel family of fungal protein kinases including Sid1p, an essential component of the septation initiation network (SIN) in Schizosaccharomyces pombe. Using the yeast two-hybrid system we isolated the Don3-interacting protein Dip1, which is similar to S. pombe Cdc14p, another member of the SIN. Remarkably, deletion of dip1 did not interfere with cytokinesis in U. maydis, but both dip1 and don3 mutants were affected in nuclear envelope breakdown (NEBD) during mitosis. This phenotype has already been described for mutants, which lack the small GTPase Ras3, the U. maydis homologue of the SIN component Spg1p. We propose that the Don3 kinase exerts a dual function in the regulation of cell separation and NEBD.