A novel role of Dma1 in regulating forespore membrane assembly and sporulation in fission yeast

In fission yeast Schizosaccharomyces pombe, a diploid mother cell differentiates into an ascus containing four haploid ascospores following meiotic nuclear divisions, through a process called sporulation. Several meiosis-specific proteins of fission yeast have been identified to play essential roles in meiotic progression and sporulation. We report here an unexpected function of mitotic spindle checkpoint protein Dma1 in proper spore formation. Consistent with its function in sporulation, expression of dma1(+) is up-regulated during meiosis I and II. We showed that Dma1 localizes to the SPB during meiosis and the maintenance of this localization at meiosis II depends on septation initiation network (SIN) scaffold proteins Sid4 and Cdc11. Cells lacking Dma1 display defects associated with sporulation but not nuclear division, leading frequently to formation of asci with fewer spores. Our genetic analyses support the notion that Dma1 functions in parallel with the meiosis-specific Sid2-related protein kinase Slk1/Mug27 and the SIN signaling during sporulation, possibly through regulating proper forespore membrane assembly. Our studies therefore revealed a novel function of Dma1 in regulating sporulation in fission yeast.     

Ced-9 inhibits Al-induced programmed cell death and promotes Al tolerance in tobacco

Our previous data showed that apoptotic suppressors inhibit aluminum (Al)-induced programmed cell death (PCD) and promote Al tolerance in yeast cells, however, very little is known about the underlying mechanisms, especially in plants. Here, we show that the Caenorhabditis elegans apoptotic suppressor Ced-9, a Bcl-2 homologue, inhibited both the Al-induced PCD and Al-induced activity of caspase-like vacuolar processing enzyme (VPE), a crucial executioner of PCD, in tobacco. Furthermore, we show that Ced-9 significantly alleviated Al inhibition of root elongation, decreased Al accumulation in the root tip and greatly inhibited Al-induced gene expression in early response to Al, leading to enhancing the tolerance of tobacco plants to Al toxicity. Our data suggest that Ced-9 promotes Al tolerance in plants via inhibition of Al-induced PCD, indicating that conserved negative regulators of PCD are involved in integrated regulation of cell survival and Al-induced PCD by an unidentified mechanism.     

Vacuole Partitioning during Meiotic Division in Yeast

We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y, were also detected outside spores after meiotic division. Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores. These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores. When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores. Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division.     

Differential requirement for phospholipase D/Spo14 and its novel interactor Sma1 for regulation of exocytotic vesicle fusion in yeast meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.     

植物发育过程中的细胞程序性死亡

细胞程序性死亡(PCD)是植物发育过程中必不可少的一部分,近年来对植物发育过程中的细胞程序性死亡机制的研究已经广泛开展。植物发育过程中的PCD对植物自身形态建成和组织分化有重要意义。一般认为动、植物的PCD有很大的相似性,但植物发育过程也有着独特的PCD机制,例如依靠有裂解功能的液泡来参与PCD。通过比较植物和其他生物发育过程中的PCD,可对植物发育过程中PCD的特征有着更深入的了解。说明植物发育过程中PCD的研究将在理论和生产上有重大意义。     

Prospore Membrane Formation Defines a Developmentally Regulated Branch of the Secretory Pathway in Yeast

Spore formation in yeast is an unusual form of cell division in which the daughter cells are formed within the mother cell cytoplasm. This division requires the de novo synthesis of a membrane compartment, termed the prospore membrane, which engulfs the daughter nuclei. The effect of mutations in late-acting genes on sporulation was investigated. Mutation of SEC1, SEC4, or SEC8 blocked spore formation, and electron microscopic analysis of the sec4-8 mutant indicated that this inability to produce spores was caused by a failure to form the prospore membrane. The soluble NSF attachment protein 25 (SNAP-25) homologue SEC9, by contrast, was not required for sporulation. The absence of a requirement for SEC9 was shown to be due to the sporulation-specific induction of a second, previously undescribed, SNAP-25 homologue, termed SPO20. These results define a developmentally regulated branch of the secretory pathway and suggest that spore morphogenesis in yeast proceeds by the targeting and fusion of secretory vesicles to form new plasma membranes in the interior of the mother cell. Consistent with this model, the extracellular proteins Gas1p and Cts1p were localized to an internal compartment in sporulating cells. Spore formation in yeast may be a useful model for understanding secretion-driven cell division events in a variety of plant and animal systems.     

Vacuolar cell death in plants

Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by a dramatic growth of lytic vacuoles, which gradually digest cytoplasmic content leading to self-clearance of dying cells. Our recent data demonstrate that vacuolar PCD critically requires autophagy and its upstream regulator, a caspase-fold protease metacaspase. Furthermore, both components lie downstream of the point of no return in the cell-death pathway. Here we consider the possibilities that i) autophagy could have both cytotoxic and cytoprotective roles in the vacuolar PCD, and ii) metacaspase could augment autophagic flux through targeting an as yet unknown autophagy repressor.     

The essential role of yeast topoisomerase III in meiosis depends on recombination

Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5' topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Delta mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Delta mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Delta mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination.     

Mating Type and Sporulation in Yeast. II. Meiosis, Recombination, and Radiation Sensitivity in an α_entitystart_#945_entit Diploid with Altered Sporulation Control

In wild-type S. cerevisiae, diploid cells must be heterozygous at the mating-type locus in order to sporulate. In the preceding paper, we described a number of mutants (CSP mutants), isolated from nonsporulating aa and αα parent strains, in which sporulation appeared to be uncoupled from control by mating type. The characterization of one of these mutants (CSP1) is now extended to other processes controlled by mating type. This mutant is indistinguishable from αα cells and unlike aα cells for mating factor production and response, zygote formation, intragenic mitotic recombination, and for X-ray sensitivity. The mutant apparently undergoes a full round of DNA synthesis in sporulation medium, but with delayed kinetics. Only 20% of the cells complete sporulation. Among spores in completed asci, the frequency of both intra- and intergenic recombination is the same as it is for spores produced by aα cells. However, experiments in which cells were shifted from sporulation medium back to minimal growth medium gave a frequency of meiotic recombination between ade2 or leu2 heteroalleles only 25% to 29% as high for CSP1 αα diploid or CSP1 aa disomic cells as for aα diploid or disomic cells. Because the latter result, indicating recombination defectiveness, measured recombinant production in the entire cell population, whereas the result indicating normal recombination sampled only completed spores, we infer that all meiotic recombination events occurring in the population of CSP1 αα cells are concentrated in those few cells which complete sporulation. This high degree of correlation between meiotic recombination and the completion of meiosis and sporulation suggests that recombination may be required for proper meiotic chromosome segregation in yeast just as it appears to be in maize and in Drosophila     

Sporulation in the budding yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.     

<Emphasis Type="Italic">SSP2</Emphasis>, a sporulation-specific gene necessary for outer spore wall assembly in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Sporulation in yeast consists of two highly coordinated processes. First, a diploid cell that is heterozygous at the mating-type locus undergoes meiosis, in which one round of DNA replication is followed by two rounds of nuclear division. Second, the meiotic products are packaged into spore cells that remain within the mother cell. A large number of genes are induced specifically during sporulation, and their products carry out different sporulation-specific events. Expression of these sporulation-specific genes is controlled by several regulators which function at different stages of the sporulation program, resulting in a cascade of gene expression following induction of meiosis. Here we describe one sporulation-specific gene, SSP2, which is induced midway through meiosis. Ssp2 shows significant homology to the predicted product of a hypothetical ORF in Candida albicans. Homozygous mutant ssp2 diploid cells fail to sporulate. In the mutant background, meiotic recombination and nuclear divisions remain normal; however, viability declines rapidly. Following meiosis, ssp2 cells form the prospore membrane, but fail to form the outer layer of the spore wall. The Ssp2 protein localizes to the spore wall after meiosis II. In addition, the ssp2 defect is also associated with delayed and reduced expression of late sporulation-specific genes. Our results suggest that SSP2 function is required after meiosis II and during spore wall formation.     

Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia

Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active and rapid nuclear degradation is triggered by vacuole rupture.     

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae

A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.     

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.Key words: actin microfilament, cell cycle, cryptogein, microtubules, nuclei, programmed cell death, tobacco BY-2 cells, vacuoles     

Approach to the early sporangial development in angiosperms considering meiosis control and cellular differentiation

 Applying current data on cell differentia- tion and meiosis control to the early sporangial development in angiosperms, a strict relationship between cell lineage and its differentiation fate is rejected. An evaluation of cytological features indicative of a meiotic (sporogenous) fate discards the sterilization phenomenon and introduces the premeiotic cellular differentiation (PCD) concept. The early sporangial development comprises 5 basic steps and 4 cellular stages, where PCD and meiosis extension and gradient are related to mechanisms of spore mother cell selection. Concepts here discussed explain the exceptions to the normal early sporangial development and allow a precise definition of archesporium and archespore. PCD and meiotic extension and gradient recover more information of the early sporangial development, distinguishing different developmental patterns leading to the same final result and retaining slight developmental differences. However, there are no early developmental characteristics distinctive of andro- or gynosporangia. Therefore, the heterosporic condition is not related to early developmental changes. Received January 5, 2001 Accepted August 29, 2001     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed "naked" nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei from meiosis. In 2--4 spored asci usually four products of meiosis in form of enclosed and free nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.     

Isolation and properties of merodiploid strains with F-factor including the pts-region of the Escherichia coli K-12 chromosome]

A technique of hybridization of haploid methanol-utilizing yeast Pichia pinus MH4 is worked out using UV- and N-nitrosoguanidine-induced auxotrophic mutants. Vegetative diploid cultures are isolated. Tetrad analysis and random spore analysis have revealed a meiotic nature of spores, recombination of genetic material in the process of sporulation and the chromosomal nature of some mutations. A possibility to construct a genetic map of the yeast Pichia pinus MH4 is demonstrated on the basis of tetrad analysis. Three linkage groups are revealed. The life cycle in a homothalic haploid yeast, Pichia pinus, was demonstrated. They are capable to form zygotes and meiotic spores under conditions preventing vegetative growth.     

Identification of yeast meiosis-specific genes by differential display

Meiosis, a specialized cell division process, occurs in all sexually reproducing organisms. During this process a diploid cell undergoes a single round of DNA replication followed by two rounds of nuclear division to produce four haploid gametes. In yeast, the meiotic products are packaged into four spores that are enclosed in a sac known as an ascus. To enhance our understanding of the meiotic developmental pathway and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae. Such comparative analyses have identified five different classes of genes that are expressed at different stages of the sporulation program. We identified several meiosis-specific genes including some already known to be induced during meiosis. Here we describe one of these previously uncharacterized genes, SSP1, which plays an essential role in meiosis and spore formation. SSP1 is induced midway through meiosis, and the homozygous mutant-diploid cells fail to sporulate. In ssp1 cells, meiosis is delayed, nuclei fragment after meiosis II, and viability declines rapidly. The ssp1 defect is not related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of meiotic divisions. Our results suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation. Functional analysis of other uncharacterized genes is underway.