Radicicol Binding to Swo1/Hsp90 and Inhibition of Growth of Specific Temperature-sensitive Cell Cycle Mutants of Fission Yeast

A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expresion partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.     

The S. pombe cdc16 gene is required both for maintenance of p34cdc2 kinase activity and regulation of septum formation: a link between mitosis and cytokinesis?

In the fission yeast Schizosaccharomyces pombe, septum formation and cytokinesis are dependent upon the initiation, though not the completion of mitosis. A number of cell cycle mutants which show phenotypes consistent with a defect in the regulation of septum formation have been isolated. A mutation in the S. pombe cdc16 gene leads to the formation of multiple septa without cytokinesis, suggesting that the normal mechanisms that limit the cell to the formation of a single septum in each cycle do not operate. Mutations in the S. pombe early septation mutants cdc7, cdc11, cdc14 and cdc15 lead to the formation of elongated, multinucleate cells, as a result of S phase and mitosis continuing in the absence of cytokinesis. This suggests that in these cells, the normal mechanisms which initiate cytokinesis are defective and that they are unable to respond to this by preventing further nuclear cycles. Genetic analysis has implied that the products of some of these genes may interact with that of the cdc16 gene. To understand how the processes of septation and cytokinesis are regulated and coordinated with mitosis we are studying the early septation mutants and cdc16. In this paper, we present the cloning and analysis of the cdc16 gene. Deletion of the gene shows that it is essential for cell proliferation: spores lacking a functional cdc16 gene germinate, complete mitosis and form multiple septa without undergoing cell cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sce3, a suppressor of the Schizosaccharomyces pombe septation mutant cdc11, encodes a putative RNA-binding protein.

In the fission yeast Schizosaccharomyces pombe, the cdc11 gene is required for the initiation of septum formation at the end of mitosis. The sce3 gene was cloned as a multi-copy suppressor of the heat-sensitive mutant cdc11-136. When over-expressed, it rescues all mutants of cdc11 and also a heat-sensitive allele of cdc14, but not the cdc14 null mutant. Deletion shows that sce3 is not essential for cell proliferation. It encodes a putative RNA-binding protein which shows homology to human eIF4B. Immunolocalisation indicates that Sce3p is located predominantly in the cytoplasm. Elevated expression of sce3 increases the steady-state level of cdc14 mRNA. Possible mechanisms of its action are discussed.     

Periodic accumulation of cdc15 mRNA is not necessary for septation in Schizosaccharomyces pombe

Analysis of Schizosaccharomyces pombe mutants that are defective in septum formation and cytokinesis has identified the product of the cdc15 gene as a key element in formation of a division septum. S. pombe cells lacking cdc15p function cannot assemble a functional medial ring, and do not make a division septum. cdc15 mRNA accumulates periodically during the cell cycle, peaking after entry into mitosis, and increased expression of the gene in G2-arrested cells can promote F-actin ring formation. Here, we have investigated the effects of mutations that block cell division upon the expression of cdc15 in synchronised cell populations, and analysed the expression of cdc15 when septum formation is induced by ectopic activation of the septation signalling network. We concluded the following: (i) the septation signalling network genes are not required for periodic accumulation of cdc15 mRNA; (ii) induction of septum formation in G2-arrested cells by activation of the septation signalling network does not result in accumulation of cdc15 mRNA, which is therefore not a prerequisite for septum formation; (iii) failure to turn off septum formation at the end of mitosis results in continued expression of cdc15; and (iv) periodic accumulation of cdc15 mRNA is mediated by a 97 bp region 5' to the mRNA start site.     

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.     

The Schizosaccharomyces pombe cdc14 gene is required for septum formation and can also inhibit nuclear division.

A conditional heat-sensitive mutation in the cdc14 gene of the fission yeast Schizosaccharomyces pombe results in failure to form a septum. Cells become highly elongated and multinucleate as growth and nuclear division continue in the absence of cell division. This article describes the cloning of the cdc14 gene and the identification of its product, a protein of 240 amino acids, p28cdc14. A null allele of the cdc14 gene shows that the gene is essential for septum formation and completion of the cell-division cycle. Overexpression of the gene product, p28cdc14, causes cell-cycle arrest in late G2 before mitosis. Cells leaking past the block activate p34cdc2 kinase and show condensed chromosomes, but the normal rearrangements of the microtubules and microfilaments that are associated with the transition from interphase to mitosis do not occur. Overexpression of p28cdc14 in mutants, in which the timing of mitosis is altered, suggests that these effects may be mediated upstream of the mitotic inhibitor wee1. These data are consistent with the idea that p28cdc14 may play a role in both the initiation of mitosis and septum formation and, by doing so, be part of the mechanism that coordinates these two cell-cycle events.     

Isolation and characterization of new fission yeast cytokinesis mutants.

Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring. To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel. Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring. Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized. Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118. The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively. The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p. In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation.     

Ultrastructure and cell wall composition in cell division cycle mutants ofSchizosaccharomyces pombe deficient in septum formation

A number of temperature-sensitive cdc^- mutants ofSchizosaccharomyces pombe that are affected in septum formation were analyzed with respect to their ultrastructure and the composition of their cell wall polymers. One mutant strain, cdc 16–116, has a cell wall composition similar to the wild type (strain 972 h^-). However two other mutants, cdc 4 and cdc 7, show a higher galactomannan content and a lower -glucan content. In all the mutants tested, total glucose incorporation, protein, RNA and DNA synthesis increased similarly to wild type over 3 1/2 h. After 2–3 h of incubation at the non permissive temperature-35°C-, cell numbers remained constant although, increases in optical densities at 600 nm were observed. According to scanning electron microscopy, the mutants had aberrant shapes after 5h of incubation at 35°C. Transmission electron microscopy showed that cdc 3 is unable to complete septum formation. cdc 4 showed the most varied morphological shapes and aberrant depositions of cell wall material. cdc 8 exhibited a deranged plasma membrane and cell wall regions near of cell poles; an abnormal septum and several nuclei. cdc 7 showed elongated cells with several nuclei and with an apparently normal cell wall completely lacking in septum and septal material. cdc 16 showed more than one septum per cell.     

S. pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body.

BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic. RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis. CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.     

The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F- actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.     

Regulation of mating in the cell cycle of Saccharomyces cerevisiae

The capacity of haploid a yeast cells to mate (fuse with a haploid strain of alpha mating type followed by nuclear fusion to produce a diploid cell) was assessed for a variety of temperature-sensitive cell division cycle (cdc) mutants at the permissive and restrictive temperatures. Asynchronous populations of some mutants do not mate at the restrictive temperature, and these mutants define genes (cdc 1, 4, 24, and 33) that are essential both for the cell cycle and for mating. For most cdc mutants, asynchronous populations mate well at the restrictive temperature while populations synchronized at the cdc block do not. Populations of a mutant carrying the cdc 28 mutation mate well at the restrictive temperature after synchronization at the cdc 28 step. These results suggest that mating can occur from the cdc 28 step, the same step at which mating factors arrest cell cycle progress. The cell cycle interval in which mating can occur may or may not extend to the immediately succeeding and diverging steps (cdc 4 and cdc 24). High frequency mating does not occur in the interval of the cell cycle extending from the step before the initiation of DNA synthesis (cdc 7) through DNA synthesis (cdc 2, 8, and 21), medial nuclear division (cdc 13), and late nuclear division (cdc 14 and 15).     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

The Scw1 RNA-binding domain protein regulates septation and cell-wall structure in fission yeast

Loss of the nonessential RNA-binding domain protein, Scw1, increases resistance to cell-wall-degrading enzymes in fission yeast. Surprisingly, scw1 null mutations also suppress the lethality of mutations (cdc11-136, cdc7-24, cdc14-118, sid1-239, sid2-250, sid3-106, sid4-A1, and mob1-1) at all levels of the sid pathway. This pathway forms part of the septation initiation network (SIN), which regulates the onset of septum formation and ensures the proper coupling of mitosis to cytokinesis. In contrast, scw1(-) mutations do not suppress ts alleles of the rng genes, cdc12 or cdc15. These mutations also prevent the formation of a septum and in addition block assembly and/or function of the contractile acto-myosin ring. sid mutants exhibit a hyper-sensitivity to cell-wall-degrading enzymes that is suppressed by loss of Scw1. Furthermore, scw1(-)-mediated rescue of sid mutants is abolished in the presence of calcofluor white, a compound that interferes with cell-wall synthesis. These data suggest that Scw1 acts in opposition to the SIN as a negative regulator of cell-wall/septum deposition. Unlike components of the SIN, Scw1 is predominantly a cytoplasmic protein and is not localized to the spindle pole body.     

Formation of septum-like structures at locations remote from the budding sites in cytokinesis-defective mutants of Saccharomyces cerevisiae.

Cell wall structures that partition membrane-bound portions of cytoplasm were formed at sites along the peripheral wall when a cytokinesis-defective cell division cycle mutant (cdc3) of Saccharomyces cerevisiae was grown at a restrictive temperature. The appearance of these structures, as observed in electron micrographs, was similar to that of normal septa. Aberrant septa were also detected in cytokinesis mutants harboring mutations cdc10, cdc11, and cdc12, after growth at 37 degrees C. Formation of the abnormal septa was abolished by the introduction, in a cdc3-containing strain, of additional cell cycle mutations that precluded events leading to cytokinesis and cell division. These results showed that septum formation can occur in the absence of cytokinesis. Formation of the abnormal structures was controlled by the same sequences of cell cycle events as formation of normal septa but was not subject to the spatial controls that ensure association of the septum with the budding site.     

CDC37 is required for p60v-src activity in yeast.

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.     

Sequence, expression, and characterization of the first archaeal ATP-dependent 6-phosphofructokinase, a non-allosteric enzyme related to the phosphofructokinase-B sugar kinase family, from the hyperthermophilic crenarchaeote Aeropyrum pernix

The onset of septum formation in the fission yeast Schizosaccharomyces pombe is signaled via the spglp GTPase-switch, which is part of the septation initiation network. This is negatively regulated by the two-component GTPase-activating protein (GAP) comprised of the products of the cdc16 and byr4 genes. Loss-of-function mutations in either of these genes result in multiple rounds of septum formation without cell cleavage. In this work, we demonstrate that attenuation of the protein kinase cdc7p can rescue the lethality of a null allele of cdc16. This observation provides the basis for selection of chromosomal mutations and multicopy suppressors that attenuate the signaling of septation. Using this screen, mutations in all the previously described septation initiation network genes were obtained, with the exception of byr4, sid4 and plo1. We also demonstrate that increased expression of the dma1 gene can rescue the lethality of a null allele of cdc16. The implications for the regulation of septum formation in fission yeast are discussed.     

Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.     

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.     

The protein phosphatase 2A B'-regulatory subunit par1p is implicated in regulation of the S. pombe septation initiation network.

In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway. One of these encodes the protein phosphatase 2A (PP2A) B'-regulatory subunit par1p. Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants. Our data suggest that PP2A-par1p acts as a negative regulator of SIN signalling.