Ady4p and Spo74p are components of the meiotic spindle pole body that promote growth of the prospore membrane in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division. Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis. We report here the identification of two new components of the MOP, Ady4p and Spo74p. Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II. MOP assembly and prospore membrane formation are abolished in spo74Δ/spo74Δ cells and occur aberrantly in ady4Δ/ady4Δ cells. Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis. In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p. Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.     

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.     

Role of the spindle pole body of yeast in mediating assembly of the prospore membrane during meiosis

Spindle pole bodies (SPBs) are the centrosome equivalents in yeast, required for microtubule organization. In yeast, the SPB further serves as the attachment sites of the prospore membrane during meiosis. Here we report the identification of two new meiosis-specific components of the SPB, Mpc54p and Mpc70p, and the first protein specific for the prospore membrane, Don1p. Mpc54p and Mpc70p are not present in mitotic SPBs, and during meiosis II they are components of a meiosis-specific structural alteration of the outer plaque of the SPB. Both proteins are dispensable for the meiotic divisions but are essentially required for the formation of the prospore membrane. In the mpc54 and mpc70 mutants, the Don1p-containing precursors of the prospore membrane can still be found in the cytoplasm and associated with the SPB. Unexpectedly, however, the assembly of the precursors to a continuous membrane system is affected. Thus, the meiotic SPB is directly involved in the formation of a specialized membrane system, the membrane of the prospore.     

Meiotic spindle pole bodies acquire the ability to assemble the spore plasma membrane by sequential recruitment of sporulation-specific components in fission yeast

The spindle pole body (SPB) of Schizosaccharomyces pombe is required for assembly of the forespore membrane (FSM) during meiosis. Before de novo biogenesis of the FSM, the meiotic SPB forms outer plaques, an event referred to as SPB modification. A constitutive SPB component, Spo15, plays an indispensable role in SPB modification and sporulation. Here, we analyzed two sporulation-specific genes, spo13(+) and spo2(+), which are not required for progression of meiotic nuclear divisions, but are essential for sporulation. Spo13 is a 16-kDa coiled-coil protein, and Spo2 is a 15-kDa nonconserved protein. Both Spo13 and Spo2 specifically associated with the meiotic SPB. The respective deletion mutants are viable, but defective in SPB modification and in the onset of FSM formation. Spo13 and Spo2 localized on the cytoplasmic side of the SPB in close contact with the nascent FSM. Localization of Spo13 to the SPB was dependent on Spo15 and Spo2; that of Spo2 depended only on Spo15, suggesting that their recruitment to the SPB is strictly controlled. Spo2 physically associated with both Spo15 and Spo13, but Spo13 and Spo15 did not interact directly. Taken together, these observations indicate that Spo2 is recruited to the SPB during meiosis and then assists in the localization of Spo13 to the outer surface of the SPB.     

Conservative duplication of spindle poles during meiosis in Saccharomyces cerevisiae

During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.     

The fission yeast pleckstrin homology domain protein Spo7 is essential for initiation of forespore membrane assembly and spore morphogenesis

Sporulation in fission yeast represents a unique mode of cell division in which a new cell is formed within the cytoplasm of a mother cell. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores. At prophase II, the spindle pole body (SPB) forms an outer plaque, from which formation of the FSM is initiated. Several components of the SPB play an indispensable role in SPB modification, and therefore in sporulation. In this paper, we report the identification of a novel SPB component, Spo7, which has a pleckstrin homology (PH) domain. We found that Spo7 was essential for initiation of FSM assembly, but not for SPB modification. Spo7 directly bound to Meu14, a component of the leading edge of the FSM, and was essential for proper localization of Meu14. The PH domain of Spo7 had affinity for phosphatidylinositol 3-phosphate (PI3P). spo7 mutants lacking the PH domain showed aberrant spore morphology, similar to that of meu14 and phosphatidylinositol 3-kinase (pik3) mutants. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate formation of the FSM.     

Spindle pole body components are reorganized during fission yeast meiosis

During meiosis, the centrosome/spindle pole body (SPB) must be regulated in a manner distinct from that of mitosis to achieve a specialized cell division that will produce gametes. In this paper, we demonstrate that several SPB components are localized to SPBs in a meiosis-specific manner in the fission yeast Schizosaccharomyces pombe. SPB components, such as Cut12, Pcp1, and Spo15, which stay on the SPB during the mitotic cell cycle, disassociate from the SPB during meiotic prophase and then return to the SPB immediately before the onset of meiosis I. Interestingly, the polo kinase Plo1, which normally localizes to the SPB during mitosis, is excluded from them in meiotic prophase, when meiosis-specific, horse-tail nuclear movement occurs. We found that exclusion of Plo1 during this period was essential to properly remodel SPBs, because artificial targeting of Plo1 to SPBs resulted in an overduplication of SPBs. We also found that the centrin Cdc31 was required for meiotic SPB remodeling. Thus Plo1 and a centrin play central roles in the meiotic SPB remodeling, which is essential for generating the proper number of meiotic SPBs and, thereby provide unique characteristics to meiotic divisions.     

Acetate regulation of spore formation is under the control of the Ras/cyclic AMP/protein kinase A pathway and carbon dioxide in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2Δ) or carbonic anhydrase (nce103Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.     

Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex.

Yeast spindle pole bodies (SPBs) duplicate once per cell cycle by a conservative mechanism resulting in a pre-existing 'old' and a newly formed SPB. The two SPBs of yeast cells are functionally distinct. It is only the SPB that migrates into the daughter cell, the bud, which carries the Bfa1p-Bub2p GTPase-activating protein (GAP) complex, a component of the spindle positioning checkpoint. We investigated whether the functional difference of the two SPBs correlates with the time of their assembly. We describe that in unperturbed cells the 'old' SPB always migrates into the bud. However, Bfa1p localization is not determined by SPB inheritance. It is the differential interaction of cytoplasmic microtubules with the mother and bud cortex that directs the Bfa1p-Bub2p GAP to the bud-ward-localized SPB. In response to defects of cytoplasmic microtubules to interact with the cell cortex, the Bfa1p-Bub2p complex binds to both SPBs. This may provide a mechanism to delay cell cycle progression when cytoplasmic microtubules fail to orient the spindle. Thus, SPBs are able to sense cytoplasmic microtubule properties and regulate the Bfa1p-Bub2p GAP accordingly.     

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.     

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.     

Schizosaccharomyces pombe Calmodulin, Cam1, Plays a Crucial Role in Sporulation by Recruiting and Stabilizing the Spindle Pole Body Components Responsible for Assembly of the Forespore Membrane

Calmodulin in Schizosaccharomyces pombe is encoded by the cam1⁺ gene, which is indispensable for both vegetative growth and sporulation. Here, we report how Cam1 functions in spore formation. We found that Cam1 preferentially localized to the spindle pole body (SPB) during meiosis and sporulation. Formation of the forespore membrane, a precursor of the plasma membrane in spores, was blocked in a missense cam1 mutant, which was viable but unable to sporulate. Three SPB proteins necessary for the onset of forespore membrane formation, Spo2, Spo13, and Spo15, were unable to localize to the SPB in the cam1 mutant although five core SPB components that were tested were present. Recruitment of Spo2 and Spo13 is known to require the presence of Spo15 in the SPB. Notably, Spo15 was unstable in the cam1 mutant, and as a result, SPB localization of Spo2 and Spo13 was lost. Overexpression of Spo15 partially alleviated the sporulation defect in the cam1 mutant. These results indicate that calmodulin plays an essential role in forespore membrane formation by stably maintaining Spo15, and thus Spo2 and Spo13, at the SPB in meiotic cells.Calmodulin is a calcium-binding protein that is ubiquitously distributed and highly conserved among eukaryotes. It contains four EF-hand Ca²⁺-binding sites, which are required for function. Calmodulin controls a variety of cellular processes mostly related to calcium signaling. When bound to calcium, calmodulin undergoes a characteristic conformational change to an active configuration. Activated calmodulin then binds effector proteins and transmits the signal to downstream regulators.Yeast is a genetically tractable model organism suitable for studying the biological function of calmodulin, using conditional-lethal calmodulin mutants (4). In the budding yeast Saccharomyces cerevisiae, calmodulin is encoded by the CMD1 gene (5). Cmd1p is implicated in a wide variety of cellular processes, including initiation of budding and mitotic spindle formation (24). The fission yeast Schizosaccharomyces pombe has a typical calmodulin encoded by the cam1⁺ gene, which plays an indispensable role in cell proliferation, dependent on its Ca²⁺-binding activity (18, 19, 30). A green fluorescent protein (GFP)-Cam1 fusion protein localizes to sites of polarized cell growth and to the spindle pole body (SPB) in vegetative cells (19). Thus, an essential role of Cam1 might be its regulatory function in chromosome segregation (19). The role of calmodulin in the sexual cycle has been documented to a lesser extent in previous studies. A missense mutant, cam1-117, in which the Arg117 codon is changed to a Phe codon, exhibits reduced sporulation efficacy (29), suggesting that calmodulin plays a role in sporulation in fission yeast.Spore formation in fission yeast initiates with assembly of the forespore membrane (FSM), composed of double-unit membranes within the cytoplasm of a diploid zygote cell (10, 27, 28, 34). The FSM expands to encapsulate each haploid nucleus generated by meiosis and then forms a nucleated prespore. The inner bilayer of the FSM subsequently becomes the plasma membrane of the newborn spores. During meiosis II, the SPB undergoes morphological alteration from a compact single plaque to a multilayered expanded structure (10). Such modification of the SPB is a prerequisite for FSM assembly, which occurs close to the outermost layer of the modified SPB (9, 10, 20, 21).Three SPB component proteins, Spo2, Spo13, and Spo15, have been identified as essential for SPB modification and formation of the FSM (11, 23). Spo15, a large coiled-coil protein, is associated with the SPB throughout the life cycle and is indispensable for recruitment of Spo2 and Spo13 to the cytoplasmic surface of the meiotic SPB. The latter two proteins are produced only during meiosis (23). These observations imply that the SPB serves as a platform for assembly of the FSM. Cam1 has been reported to localize to the SPB during vegetative growth (19), raising the intriguing possibility that fission yeast calmodulin is involved in sporulation through proper construction of a modified meiotic SPB. To test this possibility, we report herein a detailed analysis of Cam1 localization during meiosis and the consequence of a missense mutation of cam1 on SPB modification and FSM formation.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum

Summary Changes in the spindle pole body (SPB) and meiotic nuclei from interphase I through interphase II in the hollyhock rustPuccinia malvacearum are analyzed ultrastructurally by three-dimensional reconstructions from serial sections. Interphase I nuclei undergo a coordinated migration and rotation during which the SPBs approach the convex face of the lateral promycelial wall. During the transition from interphase I to prometaphase II, the collateral disc (co-disc) apparently enlarges and fuses with the main disc of the SPB. The resulting single SPB nucleates two confluent half spindles and about 225 astral microtubules (MTs). Co-discs and middle pieces (MPs) are absent during division II. SPBs separate and form metaphase II intranuclear spindles oriented in a predictable manner. Tubular cisternae are present within the spindle at early metaphase II. The architecture of the spindle at division II is essentially identical to that reported for division I except that the spindle is about half as long. Anaphase-telophase II nuclear envelope constriction, separation of the sibling nuclei, and externalization of the SPBs is identical to that reported for division I. Genesis of the duplicated interphase II SPB apparently occurs rapidly and involves formation of the MP followed by the three-layered SPB discs. General aspects of the division II spindle are discussed. A model for the meiotic SPB cycle in a rust is presented and its phylogenetic and functional significance in relation to other basidiomycetes and ascomycetes is discussed.     

Spore number control and breeding in Saccharomyces cerevisiae: a key role for a self-organizing system

Spindle pole bodies (SPBs) provide a structural basis for genome inheritance and spore formation during meiosis in yeast. Upon carbon source limitation during sporulation, the number of haploid spores formed per cell is reduced. We show that precise spore number control (SNC) fulfills two functions. SNC maximizes the production of spores (1-4) that are formed by a single cell. This is regulated by the concentration of three structural meiotic SPB components, which is dependent on available amounts of carbon source. Using experiments and computer simulation, we show that the molecular mechanism relies on a self-organizing system, which is able to generate particular patterns (different numbers of spores) in dependency on one single stimulus (gradually increasing amounts of SPB constituents). We also show that SNC enhances intratetrad mating, whereby maximal amounts of germinated spores are able to return to a diploid lifestyle without intermediary mitotic division. This is beneficial for the immediate fitness of the population of postmeiotic cells.     

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.     

Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation in Saccharomyces cerevisiae

During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.     

ADY1, a novel gene required for prospore membrane formation at selected spindle poles in Saccharomyces cerevisiae

ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation. ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus. In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell. Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant. Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant. A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane. These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II.     

SPO21 Is Required for Meiosis-specific Modification of the Spindle Pole Body in Yeast

During meiosis II in the yeast Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body changes from a site of microtubule initiation to a site of de novo membrane formation. These membranes are required to package the haploid meiotic products into spores. This functional change in the spindle pole body involves the expansion and modification of its cytoplasmic face, termed the outer plaque. We report here that SPO21 is required for this modification. The Spo21 protein localizes to the spindle pole in meiotic cells. In the absence of SPO21 the structure of the outer plaque is abnormal, and prospore membranes do not form. Further, decreased dosage of SPO21 leaves only two of the four spindle pole bodies competent to generate membranes. Mutation of CNM67, encoding a known component of the mitotic outer plaque, also results in a meiotic outer plaque defect but does not block membrane formation, suggesting that Spo21p may play a direct role in initiating membrane formation.