Reduced Mad2 expression keeps relaxed kinetochores from arresting budding yeast in mitosis

Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1-Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.     

Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules

Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.     

Computational comparison of microtubule-stabilising agents laulimalide and peloruside with taxol and colchicine

Microtubule-stabilising agents laulimalide and peloruside have been compared with tubulin-interacting drugs paclitaxel and colchicine by different computational approaches. Docking and QSAR-based programs point to a favourable interaction with the beta tubulin paclitaxel binding site, although an additional, preferred binding site has been found at the alpha subunit of tubulin. All together provides a plausible rationalisation of the singular binding features of these microtubule stabilisers and paves the way for future structural studies.     

Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast

The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1- arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule- disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.     

Shp1, a regulator of protein phosphatase 1 Glc7, has important roles in cell morphogenesis, cell cycle progression and DNA damage response in Candida albicans

In yeast, the type 1 protein phosphatase (PP1) catalytic subunit Glc7 is involved in the regulation of multiple cellular processes and thought to achieve specificity through association with different regulatory subunits. Here, we report that the Glc7 regulator Shp1 plays important roles in cell morphogenesis, cell cycle progression and DNA damage response in Candida albicans. SHP1 deletion caused the formation of rod-shaped yeast cells with slow growth. Flow cytometry analysis revealed that shp1Δ cells showed a prolonged G(2)/M phase, which was rescued by deleting the spindle-checkpoint gene MAD2. Furthermore, shp1Δ cells were hypersensitive to heat and genotoxic stresses. Interestingly, depletion of Glc7 caused defects similar to the shp1Δ mutant such as arrest at G(2)/M transition; and the GLC7/glc7Δ heterozygous mutant exhibited increased sensitivity to genotoxic stresses, consistent with the recent finding that Saccharomyces cerevisiae Glc7 has a role in DNA damage response. We also show that Shp1 is required for the nuclear accumulation of Glc7, suggesting that Shp1 executes its cellular function partly by regulating Glc7 localization.     

FMP30 is required for the maintenance of a normal cardiolipin level and mitochondrial morphology in the absence of mitochondrial phosphatidylethanolamine synthesis

Mitochondria of the yeast Saccharomyces cerevisiae contain enzymes Crd1p and Psd1p, which synthesize cardiolipin (CL) and phosphatidylethanolamine respectively. A previous study indicated that crd1Δ is synthetically lethal with psd1Δ. In this study, to identify novel genes involved in CL metabolism, we searched for genes that genetically interact with Psd1p, and found that deletion of FMP30 encoding a mitochondrial inner membrane protein results in a synthetic growth defect with psd1Δ. Although fmp30Δ cells grew normally and exhibited a slightly decreased CL level, fmp30Δpsd1Δ cells exhibited a severe growth defect and an about 20-fold reduction in the CL level, as compared with the wild-type control. We found also that deletion of FMP30 caused a defect in mitochondrial morphology. Furthermore, FMP30 genetically interacted with seven mitochondrial morphology genes. These results indicated that Fmp30p is involved in the maintenance of mitochondrial morphology and required for the accumulation of a normal level of CL in the absence of mitochondrial phosphatidylethanolamine synthesis.     

Inhibitors and promoters of tubulin polymerization: synthesis and biological evaluation of chalcones and related dienones as potential anticancer agents

A series of dihalogenated chalcones and structurally related dienones were synthesized and evaluated for their antiproliferative activity in 10 different cancer cell lines and for their effect on microtubule assembly. All compounds showed cytotoxic activity, with IC(50) values in the 5-280 μM range depending on the chalcone structure and the cell line. Five of the compounds were found to be tubulin polymerization inhibitors. In contrast, one of the compounds was found to stabilize tubulin to the same extent as the anticancer drug docetaxel. Molecular modeling suggested that the tubulin inhibitors bind to the colchicine binding site of β-tubulin while the novel tubulin stabilization agent seems to interact with the paclitaxel binding site.     

Peloruside A, an antimitotic agent, specifically decreases tumor necrosis factor-alpha production by lipopolysaccharide-stimulated murine macrophages

Peloruside A (peloruside) is a naturally occurring compound isolated from a New Zealand marine sponge that, like the anticancer drug paclitaxel, stabilizes microtubules and inhibits mitosis. Paclitaxel is known to induce a proinflammatory response in murine macrophages; whereas, peloruside has never been tested for its immunomodulatory effects in these cells. Although the antimitotic effects of the two drugs appear to be similar, we found that peloruside, unlike paclitaxel, does not induce murine macrophages to produce the proinflammatory mediators interleukin-12p40 (IL-12p40), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay confirmed that the absence of cytokine production was not caused by cytotoxicity in these nondividing cells. Additionally, there was no effect on unstimulated splenocytes; whereas, both compounds inhibited proliferation after concanalavin A (Con A) stimulation. Finally, there was a significant decrease in TNF-alpha and nitric oxide but not IL-12p40 when macrophages were cultured with lipopolysaccharide (LPS) and either paclitaxel or peloruside. These results suggest that peloruside may prove to be an effective anti-inflammatory treatment, since it does not induce the production of proinflammatory mediators yet can downregulate TNF-alpha and nitric oxide production by LPS-stimulated macrophages, as well as inhibit lymphocyte proliferation.     

Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells

Saccharomyces cerevisiae cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, display a shortened lifespan and an increased sensitivity to oxidative stress. A lipidomic analysis revealed specific changes in sphingolipids that accompanied the premature ageing of Isc1p-deficient cells under severe calorie restriction conditions, including a decrease of dihydrosphingosine levels and an increase of dihydro-C(26) -ceramide and phyto-C(26) -ceramide levels, the latter raising the possibility of activation of ceramide-dependent protein phosphatases. Consequently, deletion of the SIT4 gene, which encodes for the catalytic subunit of type 2A ceramide-activated protein phosphatase in yeast, abolished the premature ageing and hydrogen peroxide sensitivity of isc1Δ cells. SIT4 deletion also abolished the respiratory defects and catalase A deficiency exhibited by isc1Δ mutants. These results are consistent with catabolic derepression associated with the loss of Sit4p. The overall results show that Isc1p is an upstream regulator of Sit4p and implicate Sit4p activation in mitochondrial dysfunction leading to the shortened chronological lifespan and oxidative stress sensitivity of isc1Δ mutants.     

猪链球菌2型全基因组DNA芯片的研制及基因表达谱技术平台的建立

目的：研制猪链球菌2型（SS2）全基因组DNA芯片,建立SS2基因表达谱技术平台。方法：利用SS2全基因组序列,挑选出2194条基因,经PCR扩增出2156条基因并将产物纯化,点样制备芯片;将芯片用于表达谱研究,采用实时定量PCR验证表达谱结果,对芯片进行可靠性分析。结果：芯片杂交数据与实时定量PCR验证显示了较高的相关性,二者相关系数r=0.87。结论：研制了一批SS2全基因组DNA芯片,并建立了基于DNA芯片的表达谱技术平台。     

KCS1 deletion in Saccharomyces cerevisiae leads to a defect in translocation of autophagic proteins and reduces autophagosome formation

Inositol phosphates are implicated in the regulation of autophagy; however, the exact role of each inositol phosphate species is unclear. In this study, we systematically analyzed the highly conserved inositol polyphosphate synthesis pathway in S. cerevisiae for its role in regulating autophagy. Using yeast mutants that harbored a deletion in each of the genes within the inositol polyphosphate synthesis pathway, we found that deletion of KCS1, and to a lesser degree IPK2, led to a defect in autophagy. KCS1 encodes an inositol hexakisphosphate/heptakisposphate kinase that synthesizes 5-IP 7 and IP 8; and IPK2 encodes an inositol polyphosphate multikinase required for synthesis of IP 4 and IP 5. We characterized the kcs1Δ mutant strain in detail. The kcs1Δ yeast exhibited reduced autophagic flux, which might be caused by both the reduction in autophagosome number and autophagosome size as observed under nitrogen starvation. The autophagy defect in kcs1Δ strain was associated with mislocalization of the phagophore assembly site (PAS) and a defect in Atg18 release from the vacuole membrane under nitrogen deprivation conditions. Interestingly, formation of autophagosome-like vesicles was commonly observed to originate from the plasma membrane in the kcs1Δ strain. Our results indicate that lack of KCS1 interferes with proper localization of the PAS, leads to reduction of autophagosome formation, and causes the formation of autophagosome-like structure in abnormal subcellular locations.     

Spindle assembly requires complete disassembly of spindle remnants from the previous cell cycle

Incomplete mitotic spindle disassembly causes lethality in budding yeast. To determine why spindle disassembly is required for cell viability, we used live-cell microscopy to analyze a double mutant strain containing a conditional mutant and a deletion mutant compromised for the kinesin-8 and anaphase-promoting complex-driven spindle-disassembly pathways (td-kip3 and doc1Δ, respectively). Under nonpermissive conditions, spindles in td-kip3 doc1Δ cells could break apart but could not disassemble completely. These cells could exit mitosis and undergo cell division. However, the daughter cells could not assemble functional, bipolar spindles in the ensuing mitosis. During the formation of these dysfunctional spindles, centrosome duplication and separation, as well as recruitment of key midzone-stabilizing proteins all appeared normal, but microtubule polymerization was nevertheless impaired and these spindles often collapsed. Introduction of free tubulin through episomal expression of α- and β-tubulin or introduction of a brief pulse of the microtubule-depolymerizing drug nocodazole allowed spindle assembly in these td-kip3 doc1Δ mutants. Therefore we propose that spindle disassembly is essential for regeneration of the intracellular pool of assembly-competent tubulin required for efficient spindle assembly during subsequent mitoses of daughter cells.     

A molecular docking study of anticancer drug paclitaxel and its analogues

Present study was aimed at finding a better alternative to paclitaxel, an anticancer chemotherapeutic drug. Two targets, tubulin beta-1 chain and apoptosis regulator Bcl-2 protein (202F) were used in the study. Of these, structure of tubulin beta-1 chain is not known and that of Bcl-2 was taken from protein data bank with ID 202F. Tertiary structure model of tubulin beta-1 chain was predicted and validated. The validated 3D structure of tubulin beta-1 chain and Bcl-2 protein was taken to study their interaction with paclitaxel. Molecular docking of paclitaxel and its analogues was performed with these targets separately. Results showed that out of 84 analogues taken from PubChem, CID_44322802 had glide score of -9.62, as compared to -5.86 of paclitaxel with tubulin beta-1 chain. It was also observed that CID_9919057 had glide score of -9.0, as compared to -8.24 of paclitaxel with Bcl-2 protein. However, further experimental and clinical verification is needed to establish these analogues as drug.     

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.     

The Yeast Deletion Collection: A Decade of Functional Genomics

The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general.     

A unique mode of microtubule stabilization induced by peloruside A

Microtubules are significant therapeutic targets for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside A, a macrolide isolated from New Zealand marine sponge Mycale hentscheli, is a microtubule-stabilizing agent that synergizes with taxoid drugs through a unique site and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A and present a structural model of the peloruside-binding site. Using a strategy involving comparative hydrogen-deuterium exchange mass spectrometry of different microtubule-stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the α/β-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of β-tubulin at a previously unknown ligand site, rather than on α-tubulin as suggested in earlier studies.     

Requirement of a specific group of sphingolipid-metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.     

Loss of heterozygosity at an unlinked genomic locus is responsible for the phenotype of a Candida albicans sap4Δ sap5Δ sap6Δ mutant

The diploid genome of the pathogenic yeast Candida albicans exhibits a high degree of heterozygosity. Genomic alterations that result in a loss of heterozygosity at specific loci may affect phenotypes and confer a selective advantage under certain conditions. Such genomic rearrangements can also occur during the construction of C. albicans mutants and remain undetected. The SAP2 gene on chromosome R encodes a secreted aspartic protease that is induced and required for growth of C. albicans when proteins are the only available nitrogen source. In strain SC5314, the two SAP2 alleles are functionally divergent because of differences in their regulation. Basal expression of the SAP2-2 allele, but not the SAP2-1 allele, provides the proteolytic degradation products that serve as inducers for full SAP2 induction. A triple mutant lacking the SAP4, SAP5, and SAP6 genes, which are located on chromosome 6, has previously been reported to have a growth defect on proteins, suggesting that one of the encoded proteases is required for SAP2 expression. Here we show that this sap4Δ sap5Δ sap6Δ mutant has become homozygous for chromosome R and lost the SAP2-2 allele. Replacement of one of the SAP2-1 copies in this strain by SAP2-2 and its regulatory region restored the ability of the sap4Δ sap5Δ sap6Δ mutant to utilize proteins as the sole nitrogen source. This is an illustrative example of how loss of heterozygosity at a different genomic locus can cause the mutant phenotype attributed to targeted deletion of a specific gene in C. albicans.