The kinetochore proteins Pcs1 and Mde4 and heterochromatin are required to prevent merotelic orientation

BACKGROUND: Accurate chromosome segregation depends on the establishment of correct-amphitelic-kinetochore orientation. Merotelic kinetochore orientation is an error that occurs when a single kinetochore attaches to microtubules emanating from opposite spindle poles, a condition that hinders segregation of the kinetochore to a spindle pole in anaphase. To avoid chromosome missegregation resulting from merotelic kinetochore orientation, cells have developed mechanisms to prevent or correct merotelic attachment. A protein called Pcs1 has been implicated in preventing merotelic attachment in mitosis and meiosis II in the fission yeast S. pombe. RESULTS: We report that Pcs1 forms a complex with a protein called Mde4. Both Pcs1 and Mde4 localize to the central core of centromeres. Deletion of mde4(+), like that of pcs1(+), causes the appearance of lagging chromosomes during the anaphases of mitotic and meiosis II cells. We provide evidence that the kinetochores of lagging chromosomes in both pcs1 and mde4 mutant cells are merotelically attached. In addition, we find that lagging chromosomes in cells with defective centromeric heterochromatin also display features consistent with merotelic attachment. CONCLUSIONS: We suggest that the Pcs1/Mde4 complex is the fission yeast counterpart of the budding yeast monopolin subcomplex Csm1/Lrs4, which promotes the segregation of sister kinetochores to the same pole during meiosis I. We propose that the Pcs1/Mde4 complex acts in the central kinetochore domain to clamp microtubule binding sites together, the centromeric heterochromatin coating the flanking domains provides rigidity, and both systems contribute to the prevention of merotelic attachment.     

Spo5/Mug12, a putative meiosis-specific RNA-binding protein, is essential for meiotic progression and forms Mei2 dot-like nuclear foci

We report here a functional analysis of spo5(+)(mug12(+)) of Schizosaccharomyces pombe, which encodes a putative RNA-binding protein. The disruption of spo5(+) caused abnormal sporulation, generating inviable spores due to failed forespore membrane formation and the absence of a spore wall, as determined by electron microscopy. Spo5 regulates the progression of meiosis I because spo5 mutant cells display normal premeiotic DNA synthesis and the timely initiation of meiosis I but they show a delay in the peaking of cells with two nuclei, abnormal tyrosine 15 dephosphorylation of Cdc2, incomplete degradation of Cdc13, retarded formation and repair of double strand breaks, and a reduced frequency of intragenic recombination. Immunostaining showed that Spo5-green fluorescent protein (GFP) appeared in the cytoplasm at the horsetail phase, peaked around the metaphase I to anaphase I transition, and suddenly disappeared after anaphase II. Images of Spo5-GFP in living cells revealed that Spo5 forms a dot in the nucleus at prophase I that colocalized with the Mei2 dot. Unlike the Mei2 dot, however, the Spo5 dot was observed even in sme2Delta cells. Taken together, we conclude that Spo5 is a novel regulator of meiosis I and that it may function in the vicinity of the Mei2 dot.     

A Gip1p-Glc7p phosphatase complex regulates septin organization and spore wall formation.

Sporulation of Saccharomyces cerevisiae is a developmental process in which a single cell is converted into four haploid spores. GIP1, encoding a developmentally regulated protein phosphatase 1 interacting protein, is required for spore formation. Here we show that GIP1 and the protein phosphatase 1 encoded by GLC7 play essential roles in spore development. The gip1Delta mutant undergoes meiosis and prospore membrane formation normally, but is specifically defective in spore wall synthesis. We demonstrate that in wild-type cells, distinct layers of the spore wall are deposited in a specific temporal order, and that gip1Delta cells display a discrete arrest at the onset of spore wall deposition. Localization studies revealed that Gip1p and Glc7p colocalize with the septins in structures underlying the growing prospore membranes. Interestingly, in the gip1Delta mutant, not only is Glc7p localization altered, but septins are also delocalized. Similar phenotypes were observed in a glc7-136 mutant, which expresses a Glc7p defective in interacting with Gip1p. These results indicate that a Gip1p-Glc7p phosphatase complex is required for proper septin organization and initiation of spore wall formation during sporulation.     

End3p-mediated endocytosis is required for spore wall formation in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, vesicles transported to the vicinity of spindle pole bodies are fused to each other to generate bilayered prospore membranes (PSMs). PSMs encapsulate the haploid nuclei that arise from the meiotic divisions and serve as platforms for spore wall deposition. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3Delta, sporulated poorly and the spores produced lost resistance to ether vapor, suggesting that END3-mediated endocytosis is important for sporulation. End3p-GFP localized to cell and spore peripheries in vegetative and sporulating cells and colocalized with actin structures. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3Delta. Analysis of meiosis in end3Delta mutants revealed that the meiotic divisions occurred with wild-type kinetics. Furthermore, PSMs were assembled normally. However, the levels of proteins required for spore wall synthesis and components of the spore wall layers at spores were reduced, indicating that end3Delta mutants are defective in spore wall synthesis. Thus, END3-mediated endocytosis is important for spore wall formation. Additionally, cytological analyses suggest that trafficking between the plasma membrane and PSMs is important earlier during sporulation.     

Ady3p links spindle pole body function to spore wall synthesis in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Delta/ady3Delta asci that do form contain fewer than four spores. The sporulation defect in ady3Delta/ady3Delta cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Delta/ady3Delta cells. In mpc70Delta/mpc70Delta cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.     

The Fission Yeast Synaptobrevin Ortholog Syb1 Plays an Important Role in Forespore Membrane Formation and Spore Maturation

Synaptobrevin, also called vesicle-associated membrane protein (VAMP), is a component of the plasma membrane N-methylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which plays a key role in intracellular membrane fusion. Previous studies have revealed that, similar to synaptobrevin in other organisms, the fission yeast synaptobrevin ortholog Syb1 associates with post-Golgi secretory vesicles and is essential for cytokinesis and cell elongation. Here, we report that Syb1 has a role in sporulation. After nitrogen starvation, green fluorescent protein (GFP)-Syb1 is found in intracellular dots. As meiosis proceeds, GFP-Syb1 accumulates around the nucleus and then localizes at the forespore membrane (FSM). We isolated a syb-S1 mutant, which exhibits a defect in sporulation. In syb1-S1 mutants, the FSM begins to form but fails to develop a normal morphology. Electron microscopy shows that an abnormal spore wall is often formed in syb1-S1 mutant spores. Although most syb1-S1 mutant spores are germinated, they are less tolerant to ethanol than wild-type spores. The syb1-S1 allele carries a missense mutation, resulting in replacement of a conserved cysteine residue adjacent to the transmembrane domain, which reduces the stability and abundance of the Syb1 protein. Taken together, these results indicate that Syb1 plays an important role in both FSM assembly and spore wall formation.     

Synthesis of alpha-glucans in fission yeast spores is carried out by three alpha-glucan synthase paralogues, Mok12p, Mok13p and Mok14p

Fission yeast possesses a family of (1,3)-alpha-glucan synthase-related genes; one of them, mok1+/ags1+, plays an essential function in morphogenesis during vegetative growth. Here we show that three mok1+ paralogues -mok12+, mok13+ and mok14+- are required for sporulation to succeed, acting at different stages of the spore wall maturation process. Mutation of mok12+ affected the efficiency of spore formation and spore viability. Deletion of mok13+ does not affect spore viability but the spores showed reduced resistance to stress conditions. mok14Delta mutant spores failed to accumulate the amylose-like spore wall-specific polymer. mok12+, mok13+ and mok14+ expression was restricted to sporulating cells and the proteins localized to the spore envelope but with different timing. mok11+ was also induced during the sporulation process although its deletion did not show apparently a sporulation defect. In vegetative cells, beta-glucans are more abundant than alpha-glucans (55% versus 28%). In spores, the situation was the opposite, alpha-glucans accounted for 46% while beta-glucans were approximately 38% of the total polysaccharides. We found at least two types of alpha-glucan polymers, Mok12p and Mok13p, were involved in the synthesis of the greater part of alpha-glucan in the spores envelope, a polymer that is mainly digested with alpha-1,3 glucanase, while Mok14p, homologous to starch synthases, was required for the synthesis of the iodine-reactive polymer that is made of alpha-1,4 glucose residues.     

Role of GerD in germination of Bacillus subtilis spores

Spores of a Bacillus subtilis strain with a gerD deletion mutation (Delta gerD) responded much slower than wild-type spores to nutrient germinants, although they did ultimately germinate, outgrow, and form colonies. Spores lacking GerD and nutrient germinant receptors also germinated slowly with nutrients, as did Delta gerD spores in which nutrient receptors were overexpressed. The germination defect of Delta gerD spores was not suppressed by many changes in the sporulation or germination conditions. Germination of Delta gerD spores was also slower than that of wild-type spores with a pressure of 150 MPa, which triggers spore germination through nutrient receptors. Ectopic expression of gerD suppressed the slow germination of Delta gerD spores with nutrients, but overexpression of GerD did not increase rates of spore germination. Loss of GerD had no effect on spore germination induced by agents that do not act through nutrient receptors, including a 1:1 chelate of Ca2+ and dipicolinic acid, dodecylamine, lysozyme in hypertonic medium, a pressure of 500 MPa, and spontaneous germination of spores that lack all nutrient receptors. Deletion of GerD's putative signal peptide or change of its likely diacylglycerylated cysteine residue to alanine reduced GerD function. The latter findings suggest that GerD is located in a spore membrane, most likely the inner membrane, where the nutrient receptors are located. All these data suggest that, while GerD is not essential for nutrient germination, this protein has an important role in spores' rapid response to nutrient germinants, by either direct interaction with nutrient receptors or some signal transduction essential for germination.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.     

一种展示SARS冠状病毒受体结合区的重组枯草杆菌芽孢的制备及免疫原性分析

目的：利用枯草杆菌芽孢呈递技术制备表达SARS冠状病毒S蛋白受体结合区（RBD）的重组芽孢。方法：将枯草杆菌 CotB 基因构建到基因组整合质粒pDG1664中,再将 RBD 基因连接到 CotB 基因的下游,构建成重组质粒pDG1664-CotB-RBD,通过同源重组整合到PY-79枯草杆菌基因组中;利用红霉素抗性筛选重组菌并进行PCR和DNA测序鉴定,Western印迹鉴定重组菌芽孢表面RBD蛋白的表达情况;用表达RBD的重组芽孢以口服方式免疫小鼠,通过ELISA和流式细胞术检测重组芽孢的免疫原性。结果：制备出枯草杆菌基因组整合了RBD抗原基因的重组菌株RS1931,形成的重组芽孢表达相对分子质量约62×103的CotB-RBD融合蛋白;重组芽孢免疫的小鼠血清RBD抗原特异性IgG抗体滴度在末次免疫后2周可达1∶10880,重组芽孢初免后18周的小鼠脾细胞中IFN-γ＋CD4^＋、IL-4＋CD4^＋和IFN-γ＋CD8^＋T细胞比例上调,表明重组芽孢经口服免疫产生良好的体液免疫和细胞免疫应答。结论：针对SARS冠状病毒S蛋白RBD建立了枯草杆菌芽孢呈递技术方法,制备出在枯草杆菌芽孢表面稳定表达外源RBD蛋白的重组株,获得的重组芽孢具有良好的免疫原性,为开发芽孢呈递型SARS疫苗奠定了基础。     

Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.     

Nud1p, the yeast homolog of Centriolin, regulates spindle pole body inheritance in meiosis

Nud1p, a protein homologous to the mammalian centrosome and midbody component Centriolin, is a component of the budding yeast spindle pole body (SPB), with roles in anchorage of microtubules and regulation of the mitotic exit network during vegetative growth. Here we analyze the function of Nud1p during yeast meiosis. We find that a nud1-2 temperature-sensitive mutant has two meiosis-related defects that reflect genetically distinct functions of Nud1p. First, the mutation affects spore formation due to its late function during spore maturation. Second, and most important, the mutant loses its ability to distinguish between the ages of the four spindle pole bodies, which normally determine which SPB would be preferentially included in the mature spores. This affects the regulation of genome inheritance in starved meiotic cells and leads to the formation of random dyads instead of non-sister dyads under these conditions. Both functions of Nud1p are connected to the ability of Spc72p to bind to the outer plaque and half-bridge (via Kar1p) of the SPB.     

A zinc finger protein controls the onset of premeiotic DNA synthesis of fission yeast in a Mei2-independent cascade.

In the fission yeast Schizosaccharomyces pombe, meiosis is initiated by the action of Mei2 in a complex cascade activated following conjugation. We have isolated a new gene named rep1+ that is required for the initiation of premeiotic DNA synthesis. rep1+ encodes a 53 kDa protein with one zinc finger motif that is essential for function, and effectively rescues a null mutant of the res1+ gene but only partially a temperature-sensitive mutant of the cdc10+ gene, both of which are required for the onset of mitotic, as well as premeiotic, S phase. Deletion of rep1+ has no apparent effects on the mitotic cell cycle or conjugation, but blocks the initiation of premeiotic DNA synthesis. However, this defect is partially suppressed when rapidly growing cells are induced to conjugate, indicating that the rep1+ function is at least partly substituted by those of the genes controlling the 'start' of the mitotic cell cycle. The rep1 null mutant fails to induce the res2+ gene, a newly identified res1+ homolog cooperating with Cdc10 and acting for the onset of mitotic and premeiotic DNA synthesis, as well as for meiotic division. The rep1+ gene itself is induced moderately during nitrogen starvation but highly during conjugation, and this induction is dependent on both ste11+ and mating pheromones but independent of mei2+. Thus, rep1+ controls the initiation of premeiotic DNA synthesis via induction and/or activation of Res2 and some other essential factors in a cascade independent of Mei2.