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• W2139951310 abstract "Article3 August 2006free access Nud1p, the yeast homolog of Centriolin, regulates spindle pole body inheritance in meiosis Oren Gordon Oren Gordon Department of Genetics, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Christof Taxis Christof Taxis Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Philipp J Keller Philipp J Keller Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Aleksander Benjak Aleksander Benjak Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Ernst HK Stelzer Ernst HK Stelzer Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Giora Simchen Corresponding Author Giora Simchen Department of Genetics, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Michael Knop Corresponding Author Michael Knop Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Oren Gordon Oren Gordon Department of Genetics, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Christof Taxis Christof Taxis Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Philipp J Keller Philipp J Keller Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Aleksander Benjak Aleksander Benjak Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Ernst HK Stelzer Ernst HK Stelzer Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Giora Simchen Corresponding Author Giora Simchen Department of Genetics, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Michael Knop Corresponding Author Michael Knop Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany Search for more papers by this author Author Information Oren Gordon1,‡, Christof Taxis2,‡, Philipp J Keller2, Aleksander Benjak2, Ernst HK Stelzer2, Giora Simchen 1 and Michael Knop 2 1Department of Genetics, The Hebrew University of Jerusalem, Jerusalem, Israel 2Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany ‡These authors contributed equally to this work *Corresponding authors: Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Tel.: +972 2 658 5106; Fax: +972 2 658 6975; E-mail: [email protected] Biology and Biophysics Unit, EMBL, Meyerhofstrasse, Heidelberg 69117, Germany. Tel.: +49 6221 387631; Fax: +49 6221 387512; E-mail: [email protected] The EMBO Journal (2006)25:3856-3868https://doi.org/10.1038/sj.emboj.7601254 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Diploid cells of the yeast Saccharomyces cerevisiae cultured in the absence of nitrogen and in the presence of a non-fermentable carbon source undergo meiosis and form four haploid spores. Both meiotic divisions occur within a single, continuous nuclear envelope, in which the spindle pole bodies (SPBs), the functional equivalents of centrosomes in higher eukaryotes, are embedded (Moens and Rapport, 1971). Regulation of SPB function during meiosis is critical for spore formation. During sporulation, the primary role of the cytoplasmic face of each SPB, the outer plaque (OP), changes from the anchoring of cytoplasmic microtubules to the initiation of prospore membrane (PSM) formation. This leads to the encapsulation of the post meiotic nuclei into individual compartments inside the boundaries of the mother cell and to the formation of spores. Thereby the SPBs are required for PSM synthesis by assisting with the docking and fusion of secretory vesicles and continuous anchorage of the nucleus to the growing membrane during cellularization (Moens and Rapport, 1971; Davidow et al, 1980; Okamoto and Iino, 1982; Neiman, 1998; Knop and Strasser, 2000; Knop et al, 2005). The functional shift of the SPB during sporulation results from a change in the molecular composition of the OP. Spc72p is a mitotic component of the OP that recruits the γ-tubulin complex to the SPB and is required for cytoplasmic microtubule nucleation and anchorage (Knop and Schiebel, 1998). Spc72p binds to two different sites at the SPB, to Nud1p at the OP and to Kar1p at the SPB half-bridge (Pereira et al, 1999; Gruneberg et al, 2000). The half-bridge is required for SPB duplication and organizes microtubules during karyogamy and in the G1 phase of the cell cycle (Byers and Goetsch, 1975; Pereira et al, 1999). These cytoplasmic microtubules are specifically required for nuclear congression during mating (Rose and Fink, 1987; Brachat et al, 1998; Pereira et al, 1999). In meiosis, cytoplasmic microtubules are observed until late meiosis I. The function of cytoplasmic microtubules in meiosis is unknown and neither is it known whether Spc72p interacts with Nud1p or with Kar1p or both. Spc72p is present at the SPB throughout meiosis I and disappears with the onset of meiosis II (Knop and Strasser, 2000; Nickas et al, 2004). It is replaced by the three essential meiosis-specific components Mpc54p, Mpc70p/Spo21p and Spo74p along with the non-essential protein Ady4p (Knop and Strasser, 2000; Bajgier et al, 2001; Nickas et al, 2003). These proteins form the meiotic plaque (MP). It has been proposed that Mpc54p, Mpc70p and Spo74p make up a paracrystalline protein matrix (Taxis et al, 2005), analogous to the central crystal of the SPB, which is composed of Spc42p (Bullitt et al, 1997). Cnm67p, a coiled-coil protein (Schaerer et al, 2001), and Nud1p (Adams and Kilmartin, 1999; Gruneberg et al, 2000) are constitutive components of the SPBs. Both proteins are thought to bring the meiosis-specific components of the MP into proximity to each other and anchor them to the SPB (Knop and Strasser, 2000; Bajgier et al, 2001; Nickas et al, 2003). SPBs are duplicated twice during meiosis in a conservative manner. Thus, the four meiotic SPBs can be categorized into three different generations: the oldest SPB (present upon entry of the cell into meiosis), second oldest (formed during meiosis I) and two new SPBs (formed during meiosis II). S. cerevisiae is able to limit the number of formed spores to the amount of available external energy resources (e.g. acetate) (Davidow et al, 1980). It has been shown that MPs are assembled in higher probability on newer SPBs, in the case that less than four MPs are formed (Nickas et al, 2004; Taxis et al, 2005). The number of MPs that are formed, which determines the number of spores that are formed, is regulated via the amounts of MP components available. This allows a cell to precisely specify the number of spores it can afford to form. This system ensures that in asci containing only two spores (dyads), the spores contain genomes that derive from different meiosis II spindles (non-sister genomes). Because of the relatively tight genetic linkage of the mating type locus (MAT) to a centromere, the two spores in a dyad are mostly of opposite mating type (MATa and MATα) and able to mate with each other upon germination. This provides a selective advantage associated with preservation of heterozygosity (Taxis et al, 2005). SPB inheritance is also subject to control in mitotically dividing yeast cells, where it has been shown that the old SPB is preferentially inherited into the bud (Pereira et al, 2001). Also in Schizosaccharomyces pombe, inheritance of SPBs in mitosis is subject to regulation (Grallert et al, 2004). Different fates have furthermore been reported for centrosomes and centrioles of different generations in higher organisms (Piel et al, 2001; Uetake et al, 2002). At present, the molecular basis for discrimination of SPBs from different generations is not clear, nor is it known whether the molecular mechanism is evolutionary conserved. Nud1p is the only core component of the SPB, apart from calmodulin/Cmd1p, with recognizable homologies to proteins from other species. It is a constitutive component of the OP of the SPB in mitosis and meiosis (Adams and Kilmartin, 1999; Knop and Strasser, 2000). It shares approximately 120 aa of homology with the human protein Centriolin, which functions in cytoplasmic microtubule organization and in late stages of cytokinesis (Gromley et al, 2003). Recently, it was found that the Nud1p homology domain of Centriolin interacts with Sec15, a member of the exocyst complex, and that Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory vesicle-mediated abscission of the cells at the end of cytokinesis (Gromley et al, 2005). Cdc11p, the Nud1p homolog in the yeast S. pombe, is a scaffold protein of the SPB and its N-terminal domain is essential for the proper function of the septation initiation network (SIN; Krapp et al, 2004). In S. cerevisiae, Nud1p functions as a structural component of the SPB that binds the γ-tubulin receptor protein Spc72p to the OP. Furthermore, Nud1p is required in mitotic exit where it functions as a scaffold for Bub2p and Bfa1p that constitute a dual component GAP complex for the small GTPase Tem1p (Gruneberg et al, 2000; Pereira et al, 2001). In this work, we report that Nud1p has different roles during meiosis, one during spore wall maturation and another during the selection of specific SPBs for the assembly of MPs, such that only non-sister dyads will form. Our results indicate that this process requires the binding of Spc72p to the SPB. We furthermore provide indication that two different mechanisms act to specify differences between all four SPBs present in cells in meiosis II. Results The nud1-2 mutation reduces spore formation in a KAR1-dependent manner To examine the role of NUD1 in meiosis, we assayed the frequency of ascus formation in a temperature-sensitive nud1-2 mutant (Gruneberg et al, 2000). We chose this mutant allele of NUD1 because of its relatively low restrictive temperature of 34°C, at which vegetative cell division is completely prevented. Temperature-sensitive mutants with restrictive temperatures above 35°C are not useful for meiotic analysis, because meiosis in S. cerevisiae (as well as in many other species) is intrinsically temperature sensitive (Byers and Goetsch, 1982). When counting the number of cells that formed spores (asci) in the nud1-2 mutant at 34°C, we found that conduction of meiosis was not impaired (Figure 1A). Sporulating yeast cells often form asci containing a variable number of spores due to selective abortion of haploid genomes after meiosis II (Davidow et al, 1980; Okamoto and Iino, 1981). We therefore analyzed the number of spores present in the asci of the nud1-2 mutant compared to the wild-type strain. This revealed that asci containing fewer spores (one or two) were more frequent in the nud1-2 mutant at 34°C but not at 23°C (Figure 1B). This indicates that NUD1 is required to form wild-type number of spores per ascus. Nevertheless, the nud1-2 mutant is still able to form spores at 34°C, whereas vegetative cell division is completely abolished (data not shown), indicating that the functions of Nud1p that are impaired in the nud1-2 mutant are not essential for sporulation. We also tested germination of nud1-2 spores from sporulations at 23 or 34°C, and found no difference to the wild-type strain (data not shown). Figure 1.The nud1-2 mutation leads to reduced spore production under restrictive temperature in a KAR1-dependent manner. (A) Wild-type (WT), nud1-2, kar1Δ15 and nud1-2 kar1Δ15 double mutant cells (strains YKS32 (WT), YOG23, YOG52 and YOG66, respectively) were sporulated at the permissive (23°C) and restrictive (34°C) temperatures in standard acetate concentration (0.3%) for 1–2 days. At least 200 cells per strain and temperature were counted (4–13 experiments per strain; except only two for kar1Δ15 at 34°C). (B) Formation of ascus types with 1–2 or 3–4 spores in sporulated cultures of the strains from (A). At least 200 cells per strain and temperature were counted. (C) WT, nud1-2 kar1Δ15 double mutant cells, double mutant cells transformed with a KAR1 plasmid (pMR77) and a homozygous Δspc72 deletion strain expressing the fusion protein N-Spc72p-ΔN-Kar1p (YCT883-2) were sporulated at 23 and 34°C on sporulation medium plates for 3 days. The sporulation efficiency (the sum of spores divided by four times the sum of cells; 100% efficiency means 100% tetrads) was determined for at least 200 cells per strain and temperature. The means and standard errors from three to six experiments are shown. Download figure Download PowerPoint Nud1p exhibits two reported functions during vegetative cell division. First, it is required for anchorage of cytoplasmic microtubules via the γ-tubulin receptor protein Spc72p to the OP of the SPB (Knop and Schiebel, 1998; Elliott et al, 1999; Gruneberg et al, 2000). Second, it serves as a scaffold for components that function in the mitotic exit network (MEN) (Gruneberg et al, 2000; Bardin and Amon, 2001; Luca et al, 2001). In vegetative cells, not only the OP but also the half-bridge of the SPB binds Spc72p in order to organize cytoplasmic microtubules in a cell cycle-dependent manner. Binding of Spc72p to the half-bridge requires Kar1p but not Nud1p (Byers and Goetsch, 1975; Pereira et al, 1999). In order to investigate the consequences of Spc72p binding to the SPB on the abortion of spores, we combined the nud1-2 mutation with the kar1Δ15 mutation. kar1Δ15 encodes for an N-terminally deletion variant of Kar1p that is selectively impaired in binding of Spc72p but not in the other functions of Kar1p (e.g. in SPB duplication) (Vallen et al, 1992; Pereira et al, 1999). We found that the kar1Δ15 mutation did not significantly affect ascus (Figure 1A) or spore formation (Figure 1B). Surprisingly, spore formation at 34°C in the double mutant nud1-2 kar1Δ15 was comparable with wild-type levels (Figure 1B). This means that the kar1Δ15 mutation rescued the spore abortion phenotype of the nud1-2 mutation. Plasmid encoded KAR1 could restore spore abortion in the nud1-2 kar1Δ15 mutant, indicating the consistency of the result (Figure 1C). This suggests that the interaction of Spc72p with Kar1p, which is specifically affected by the karΔ15 mutation, leads to reduced spore formation in the nud1-2 mutant. To explore this further, we used a fusion of Spc72p to Kar1p (Spc72p1−272-Kar1p), which leads to constitutive localization of Spc72p at the half-bridge of the SPB. This fusion complements for loss of Spc72p at the OP of the SPB (Gruneberg et al, 2000). When we examined spore formation in the Δspc72 Spc72p1−272-Kar1p strain, we noticed that it exhibits significant lower sporulation efficiency than the wild type (Figure 1C). This is the opposite effect to the one seen in the kar1Δ15 mutant (Figure 1B), where Spc72p is not able to interact with the half-bridge. This indicates that Spc72p at the half-bridge may be responsible for the lower level of spore formation in the nud1-2 mutant. The nud1-2 mutation leads to kar115-dependent defects in spore wall maturation Reduced spore formation may reflect either a deficiency during initiation and formation of the PSMs or during the maturation of the spore wall. We examined the reduced spore formation phenotype in the nud1-2 strain, using immunofluorescence microscopy of PSMs in meiotic cells. We counted the total number of PSMs that were present in the cells at different time points during a sporulation time-course experiment (Figure 2A) and compared this with the frequencies of spores formed at the end of sporulation (after 36 h) (Figure 2B). This experimental approach counts slightly more PSMs than spores for the wild-type strain. This is owing to a systematic error that is made in the quantification of PSM formation, because meiotic progression of sporulating cells is not perfectly synchronous, and cells forming more PSMs (three or four) are usually performing sporulation faster than the ones forming less PSMs (one or two) (Taxis et al, 2005). The comparison revealed that at 34°C the nud1-2 mutant formed approximately 15–20% fewer spores, when compared with the wild type, the kar1Δ15 and the nud1-2 kar1Δ15 mutants (Figure 2C). However, the number of PSMs formed did not show significant differences. This suggests that the spore abortion phenotype in the nud1-2 mutant at 34°C is due to a late defect, after the formation of PSMs. Figure 2.The nud1-2 mutation leads to PSM loss at the restrictive temperature. (A) Colocalization of Ady3, tubulin and DNA in cells in meiosis II using immunofluorescence microscopy. The figure shows examples of cells with different numbers of PSMs. Ady3 localizes to the leading edge of the PSM; hence, a bar, vertically aligned to a meiosis II spindle, formed by the flattened ring-like opening of the PSM indicates a forming PSM. (B) Examples of asci containing 1–4 spores. Phase-contrast microscopy was used to obtain these pictures. (C) Wild-type (WT), nud1-2, kar1Δ15 and nud1-2 kar1Δ15 mutant cells (strains YKS32 (WT), YOG23, YOG52 and YOG66, respectively) were sporulated at the permissive (23°C) and restrictive (34°C) temperatures in liquid cultures. Aliquots of cells were taken after 5, 6, 7, 8 and 9 h following induction of meiosis. The number of PSMs per cell in anaphase of meiosis II (two elongated spindles) was determined (PSM count) (∼150 cells per strain and temperature were counted). After 36 h, the number of spores per ascus (monads, dyads, triad and tetrads) was assayed (spore count). By integration of the PSM count over sporulation time, the percent of PSM per ascus was determined (100% means that 100% of the cells produced four PSMs). The same was carried out for the spore count (∼200 cells per strain and temperature were counted). One experiment was carried out for each strain at each temperature, except for nud1-2, in which case the average of two experiments is shown (4% difference between the two experiments). (D) Electron microscopy of examples of sporulated cells from the WT and the nud1-2 strains used in (C). The section of the WT cell shows two mature spores, whereas the nud1-2 section shows two immature spores. Arrows point to the spore walls of the WT and the mutant spores. The immature spores are surrounded by black dots of unclear origin (they might consist of vesicular structures that contain dityrosin, a component of the outermost layer of the spore wall; Coluccio et al, 2004). (E) Quantification of spore maturation in sporulated cells using electron microscopy (24 h after induction of sporulation). For each strain, the ratio of immature versus mature spores as visible by electron microscopy (150–200 spore containing cells per strain) is shown. Download figure Download PowerPoint To analyze directly the spore formation defect in the nud1-2 mutant, we used electron microscopy to investigate the cells after completion of sporulation (24 h after induction). In the nud1-2 mutant, at 34°C, we detected an increased number of asci containing immature spores in comparison to wild type, kar1Δ15 and nud1-2 kar1Δ15 mutants (Figure 2D and E). At 23°C, no significant difference between the different strains was observed (Figure 2E). This indicates that the nud1-2 mutant exhibits a partial defect during maturation of PSMs to spores at 34°C, a late step in spore formation, and that this defect can be rescued by the kar1Δ15 mutation, in which the binding of Spc72p to the half-bridge is abolished. The nud1-2 mutation leads to random encapsulation of genomes present in the two spores of dyads It has previously been shown that the haploid spores in dyads contain genomes that originate from a different meiosis II spindle. This is owing to regulation of SPB inheritance. Thereby, in dyads, only one SPB per meiosis II spindle, namely the new one formed during meiosis II, becomes involved in spore formation (Davidow et al, 1980; Okamoto and Iino, 1981; Nickas et al, 2004). As the chromatids present in the two spores are thus of non-sister origin in centromere-linked regions (each derived from a different homologous chromosome), the dyads are called non-sister dyads. We examined the fidelity of formation of non-sister dyads in the nud1-2, kar1Δ15 and nud1-2 kar1Δ15 homozygous strains. For this, we used spore-autonomously expressed red and green fluorescent proteins (FPs) that are expressed from loci next to the centromeres of the two homologous chromosomes V (CENV-FP assay; illustrated in Figure 3A). This assay enables quantitative measurement of non-sister dyad formation using fluorescence microscopy (Figure 3B). We found 4% of dyads to be sister dyads in the wild-type strain and only slightly elevated levels in the kar1Δ15 mutant. In the nud1-2 and nud1-2 kar1Δ15 mutants, the frequency of sister dyads was almost exactly 33% (Figure 3C). This value is expected when regulation of genome inheritance into the spores in dyads is completely abolished. No difference between the two temperatures used (23 and 34°C) was observed. At 34°C, the production of random dyads in the nud1-2 strain may be caused by abortion of PSMs. However, the randomization of genome inheritance in dyads is also seen in the nud1-2 strains at 23°C. In addition, the kar1Δ15 mutation does rescue spore abortion at 34°C, but not faithful non-sister dyad formation (Figures 1B, 2 and 3C). These results strongly indicate that nud1-2 is impaired in faithful regulation of SPB inheritance, and that this function is genetically distinct from the function of Nud1p in promoting spore formation. Figure 3.The nud1-2 mutation leads to random encapsulation of genomes in dyads. (A) Cartoon illustrating the CENV-FP assay (see text). (B) Samples of non-sister and sister dyads. Merged fluorescence pictures and phase-contrast images are shown. (C) Wild-type (WT) nud1-2, kar1Δ15 and nud1-2, kar1Δ15 double mutant cells (strains YCT716, YOG26, YOG72 and YOG73, respectively) were sporulated at the permissive (23°C) and restrictive (34°C) temperatures. Dyad formation was induced by the presence of limited amounts of acetate (0.01%) in the sporulation medium. The segregation of centromere-linked spore markers (CENV-linked FP markers, either heterozygote CENV-GFP/CENV-RedStar or heterozygote CENV-GFP/no marker, each under the control of a spore autonomous promoter) was assayed in approximately 200 dyads per strain and temperature. Different labelling of the spores in dyads (red/green or green/dark) indicates the presence of non-sister genomes (genomes segregated in meiosis I). Random incorporation of genomes in dyads would give rise to 2/3 dyads of spores with different labelling (non-sister dyads) and 1/3 with equal labelling (sister dyads). The picture shows examples for the three different possibilities of dyads from the WT strain containing CENV-GFP/CENV-RedStar (phase-contrast pictures and fluorescence images are shown). The error for this experiment (estimated in independent experiments and by ‘dyad’ dissection) was found to be less than 10%. Download figure Download PowerPoint The nud1-2 mutation leads to randomization of SPB selection during MP assembly The results so far indicate that randomization of genome inheritance in dyads in the nud1-2 strain is not caused by abortion of spore formation on the level of PSMs or spore maturation. Therefore, randomization of genome inheritance must originate from a defect earlier in the process of spore formation. The assembly of a functional MP is the earliest directly observable feature of SPBs that form a PSM. The most likely defect would be randomization of MPs assembly such that older SPBs from the same meiosis II spindle rather than only new ones become also involved in spore formation in dyads. To address this possibility, we used the previously established reporter assay that allows the differentiation of SPBs based on their age by using a slow-maturing red fluorescent protein (RFP) fused to the integral SPB component Spc42p (Pereira et al, 2001; Nickas et al, 2004; Taxis et al, 2005). The t1/2 maturation rate of the chromophore of the particular RFP used (RedStar) (Knop et al, 2002) is approximately 3–5 h and thus matches the time it requires for a cell to progress through meiosis (approximately 5–7 h). This allowed us to distinguish between SPBs from the three different generations present in a cell in meiosis II, based on the brightness of the SPB signal (Figure 4A, see cartoon). Assembly of MPs was visualized using Mpc54p-YFP or GFP at 23 and 34°C (Figure 4B and C). Figure 4.The nud1-2 mutation leads to randomization of MPs assembly. Wild-type (WT) and nud1-2 strains harboring MPC54-GFP (nud1-2) or MPC54-YFP (WT) and SPC42-RedStar (strains YCT806-1 and YOG84) were sporulated at the permissive (23°C) and restrictive (34°C) temperatures. The configuration of MPs assembly on old and new SPBs was determined by imaging green and red fluorescence in cells in meiosis II. The cartoon (A) outlines the relationship of the SPBs and the FP labels used to distinguish SPBs from three different generations. Arrows indicate duplication of the SPBs during meiosis I (dashed arrow) and meiosis II (normal arrow). MPs were visualized using Mpc54p-GFP (green signals); old and new SPBs were discriminated based on the signal brightness of a fluorescent timer (RedStar/RFP; Knop et al, 2002) fused to the integral SPB component Spc42p (Donaldson and Kilmartin, 1996). This allows correlating the age of SPBs with the assembly of MPs. The oldest SPB is marked with the brightest RFP signal, and the second oldest SPB with the second brightest RFP signal. Statistical evaluation of ∼30 cells showing two MPs (B) and of ∼100 cells showing three MPs (C). The fluorescence images in (B) and (C) provide examples for the different possible constellations. The associated cartoons illustrate the different categories that the examples belong to and that can be distinguished. Formation of cells with two and three MPs was induced by the presence of limited amounts of acetate (0.01%) in the sporulation medium. Download figure Download PowerPoint We first investigated cells with two MPs. In wild type at 23°C, we observed a fidelity of 87% for assembly of the MPs preferentially at the new SPBs, whereas in 13% of the cases one new and the second oldest SPB contained an MP (Figure 4B). Interestingly, the corresponding values were 65 and 35% at 34°C. We also performed this experiment using in addition Spc110p-CFP to assess the position of all SPBs. This allows distinguishing situations where two SPBs are aligned behind each other. In this strain, the fidelity for the involvement of the second oldest SPB instead of its associated new SPB is 4% at 30°C (Taxis et al, 2005). At 34°C, we found again a temperature dependency for the distinction between the second oldest and its associated new SPB in this strain (data not shown). As the fidelity of non-sister dyad formation in the wild-type strain is not compromised at 34°C at all (Figure 3), the logical explanation for the above observation is that the distinction between the second oldest SPB and its direct daughter is compromised under elevated temperatures, whereas the daughter SPB of the oldest one is still the most likely one to assemble an MP. In the nud1-2 strain, a much higher degree of randomization was observed. At 23°C, only 35% of the cells involved the two new SPBs for MP assembly, whereas in 65%, the second oldest SPB and one new SPB were involved. As the nud1-2 mutant shows compromised non-sister dyad formation at 23°C, this suggests that cells are no longer able to distinguish between the two new SPBs in relation to their origin from the oldest or second oldest SPB. Under elevated temperature, the distinction between the two old SPBs was also compromised (Figure 4B). We intended to perform this experiment also with the additional Spc110p-CFP marker as well. However, nud1-2 combined with Spc42p-RFP and Spc110p-CFP (or Spc29p-CFP or Cnm67p-CFP) was inviable (data not shown). When looking at cells with three MPs, the results were similar (Figure 4C). Wild-type cells show a clear preference not to assemble MPs on the oldest SPB. In this case also, a weak temperature dependency was observed. The nud1-2 strain exhibited significant randomization of SPB selection for MP assembly already at 23°C. This was enhanced further at 34°C. Together, these results suggest that the nud1-2 mutant is compromised in directing" @default.
• W2139951310 created "2016-06-24" @default.
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• W2139951310 date "2006-08-03" @default.
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• W2139951310 title "Nud1p, the yeast homolog of Centriolin, regulates spindle pole body inheritance in meiosis" @default.
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