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• W2100256768 abstract "Article2 March 1998free access A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina Véronique Berteaux-Lecellier Véronique Berteaux-Lecellier Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Denise Zickler Denise Zickler Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Robert Debuchy Robert Debuchy Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Arlette Panvier-Adoutte Arlette Panvier-Adoutte Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Catherine Thompson-Coffe Catherine Thompson-Coffe Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Marguerite Picard Corresponding Author Marguerite Picard Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Véronique Berteaux-Lecellier Véronique Berteaux-Lecellier Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Denise Zickler Denise Zickler Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Robert Debuchy Robert Debuchy Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Arlette Panvier-Adoutte Arlette Panvier-Adoutte Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Catherine Thompson-Coffe Catherine Thompson-Coffe Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Marguerite Picard Corresponding Author Marguerite Picard Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France Search for more papers by this author Author Information Véronique Berteaux-Lecellier1, Denise Zickler1, Robert Debuchy1, Arlette Panvier-Adoutte1, Catherine Thompson-Coffe1 and Marguerite Picard 1 1Institut de Génétique et Microbiologie de l' Université Paris-Sud, CNRS-URA 2225, Bâtiment 400, F-91405 Orsay, cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1248-1258https://doi.org/10.1093/emboj/17.5.1248 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1-1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1-1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1-1 also shows defects in ascospore germination and growth rate. GFP-tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating-type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus. Introduction The transition from unicellular forms to pluricellular organization was certainly a key step in the evolutionary history of eukaryotes. At least two non-exclusive scenarios could result in this transition. In the first scenario, a pluricellular state was obtained through the aggregation of individual cells, as is observed in the life cycle of lower eukaryotes such as the slime mould Dictyostelium discoideum (reviewed in Gross, 1994). In the second scenario, pluricellular organization arose from a syncytial plurinucleate state. This type of transition is encountered in the early development of insects, the paradigm of which being Drosophila (see Schejter and Wieschaus, 1993 for a review). The genes which control this syncytial/cellular switch are of considerable interest from both the developmental and the evolutionary points of view. In addition to Drosophila, simpler organisms such as the filamentous ascomycetes Neurospora crassa and Podospora anserina also show a transition between a syncytial and a true cellular state. This switch is required for proper sexual development and can thus present an interesting complementary model system to understand such transitions. In these fungi, as shown in Figure 1, fertilization does not alter the syncytial (vegetative) state since both reproductive nuclei (carrying different mating types) divide in a common cytoplasm, without undergoing cytokinesis. The true cells, which arise from this plurinucleate heterokaryotic syncytium, contain one nucleus of each mating type (mat+ and mat− in P.anserina). In these hook-shaped crozier cells, coordination between nuclear and cellular division ensures the formation of three cells: a dikaryotic upper cell which can differentiate into an ascus in which meiosis will take place, and two uninucleate cells which, after fusion, will give rise to a new dikaryotic cell (see Figure 1). It is noteworthy that this dikaryotic phase is the only part of the fungal life cycle in which a septum is formed between two daughter nuclei after each mitosis. This is, in fact, a prerequisite for a true cellular organization and its maintenance. Figure 1.Sexual cycle of wild-type Podospora anserina and homozygous cro1-1 mutant with emphasis on crozier and ascus formation. Note that in the mutant strain, multinucleate croziers can form by two different mechanisms. Either two nuclei are isolated as in wild-type dikaryons but they divide without septum formation (top), or several nuclei migrate in the crozier (below). Download figure Download PowerPoint In our systematic investigation of the sexual cycle of P.anserina, we have shown previously that the mating-type genes control the proper assortment of one mat+ and one mat− nucleus in the dikaryotic cells. However, these genes are not involved in the syncytial/cellular switch (Zickler et al., 1995). Among the mutants identified during a systematic search for impaired sexual development (Simonet and Zickler, 1978), the cro1-1 mutant showed abnormal cellularization. The cro1-1 mutation leads, when homozygous, to croziers that can contain up to several dozens of nuclei instead of the two normally seen in wild-type croziers. Cytological analyses of fruiting body development, meiotic nuclear divisions and cytoskeleton components, led to the hypothesis that the primary defect of the mutant is its inability to switch from the syncytial to the cellular state. The cloning and sequencing of the cro1 gene revealed a significant similarity between the P.anserina CRO1 protein and the Saccharomyces cerevisiae SHE4 protein (Jansen et al., 1996). In yeast, the SHE4 protein is required, at the time of cell division, for asymmetric mating-type switching. Contrary to the yeast she4 mutants (Wendland et al., 1996), the P.anserina cro1-1 mutant does not show internalization defects as revealed by the FM4-64 endocytic probe (Vida and Emr, 1995). The CRO1 protein is localized in the cytosol and displays peaks of expression at developmental stages which are coincident with the mutant defects, i.e. at the beginning of the dikaryotic stage and during ascospore maturation. Results The cro1-1 mutation affects sexual development mainly during the dikaryotic phase In Podospora, the sexual cycle is initiated when a female structure (the ascogonium) is fertilized by a male cell of opposite mating type. Fertilized female organs develop into fruiting bodies (perithecia), within which meiotic cells (asci) form from successively developing dikaryons. After meiosis and a postmeiotic mitosis, each ascus produces four dikaryotic ascospores (Figure 1). Fertilization occurs normally in a cro1-1×cro1-1 cross, but perithecium development is defective at several stages. First, compared with the 160–200 asci observed in each wild-type perithecium, only 3–30 asci are formed per perithecium (50 analysed) in the homozygous mutant crosses, and most ascospores are abnormal in shape and size. Second, all perithecia are filled with giant, highly multinucleate cells which are more or less hook-shaped. This is never observed in wild-type, in which hook-shaped croziers always contain two nuclei of opposite mating type. After a coordinated mitosis, septa form on each side of the crook, resulting in three cells: an upper binucleate ascus-mother cell, flanked by a basal and a lateral uninucleate cell (Figure 2A). In cro1-1 crosses, a few normal croziers are also observed, and the small number of wild-type asci and ascospores probably result from such croziers. All other croziers are plurinucleate and of varying morphology and size. Some are aseptate (Figure 2B); in the others, despite formation of septa, all three cells contain varying numbers of nuclei (Figure 2C). These multinucleate cells exhibit, however, the characteristics of crozier cells. Their nuclei are larger than the vegetative and ascogonal nuclei and, more strikingly, all mitoses remain synchronous whatever the number of nuclei (from three to over 100) contained in a cell (see below). In wild-type perithecia, several ‘trees’ of asci are formed from successively developing croziers. In the mutant perithecia, abnormal croziers also form ‘trees’, but although interconnected, they are not affected in the same way: croziers with few nuclei and normal septa are connected to aseptate and multinucleate giant croziers (Figure 2B); ‘normal’ binucleate croziers can also form on deformed cells containing over 100 nuclei (Figure 2D). Figure 2.Crozier and ascus phenotypes in a cro1-1 mutant. (A) Wild-type binucleate croziers. Crozier on the right side contains two nuclei while crozier on the left side shows four nuclei separated by two septa. (B) A group of five croziers showing on the left side, a large plurinucleate crozier (arrow) in which no septa are formed, while the croziers at the right side show septa and fewer nuclei. (C) Croziers with septa, separating plurinucleate cells. (D) A giant plurinucleate crozier on which a normal crozier (arrow) has developed with an upper binucleate cell and a lateral plus a basal uninucleate cell. (E) Karyogamy of several haploid nuclei: compare the size of the nucleus and nucleolus (nu) with the size of the crozier nuclei seen in (A). (F) A giant crozier with three nuclei of intermediate sizes between haploid and polyploid nuclei. (G) Bifurcated ascus with degenerating giant polyploid nucleus. (H) P.anserina has seven chromosomes. This spread early meiotic prophase (leptotene) nucleus shows clearly that more than two nuclei were fused. (I) Wild-type pachytene with seven paired homologues. (J) Pachytene in a probable tetraploid nucleus (compare nucleolar size with the diploid nucleolus of (I). Note that the four homologues of each set of chromosomes are paired; (I) and (J) were taken and printed at the same magnification. (K) Abnormal spores with excluded nucleus (arrow). (L) In this triploid postmeiotic mitosis, both SPBs are large [compare with the haploid metaphase plates of (M)]. (M) Normal postmeiotic mitotic spindles with clear SPBs (arrow on one of the four SPBs); note that (L) and (M) were taken and printed with the same magnification. Scale bars represent 5 μm. Download figure Download PowerPoint Third, karyogamy can take place—whatever the number of nuclei present—in the upper cell of the crozier or in the giant cells with mislocalized septa. Nuclear size being correlated with the number of nucleoli visible in these large nuclei, these latter presumably arise from nuclear fusion (Figure 2E). This assertion is based on the fact that young croziers often contain groups of nuclei which could correspond to the divisions of the four nuclei issued from the first mitosis while older croziers contain groups of nuclei with a different level of ploidy (Figure 2F). Although we cannot exclude that some polyploid nuclei result from re-replication events, those containing an uneven set of chromosomes (3n, 5n, …) support the karyogamy assumption. However, karyogamy does not occur in all croziers and most abnormal cells degenerate. Abnormal croziers result in polyploid nuclei with large spindle pole bodies, but asci can proceed through meiosis and sporulation The wild-type diploid nucleus isolated in the upper cell of the crozier enters meiosis as the ascus mother cell begins to elongate (Figure 1). Asci reach their full length by the end of the first meiotic division. This developmental pattern is similar to that of a cro1-1 cross except that, probably due to polyploidy, asci are always larger than in wild-type crosses and are sometimes bifurcate (Figure 2G). In asci proceeding through both meiotic divisions, polyploidy ranges from triploid to octoploid, larger nuclei aborting at early prophase stages (Figure 2H). Nuclear evolution during prophase I (nucleolar increase from leptotene to diplotene and chromatin condensation) is normal when compared with wild-type (Figure 2I). In addition, at pachytene, all homologues (from three to eight) pair along their entire length, as clearly seen in Figure 2J. In the occasional postmeiotic mitoses, ploidy does not exceed 4n. Ascospore formation is mainly abnormal (Figure 2K) and, occasionally, all nuclei in an ascus are delimited within a single giant spore. The spindle pole body (SPB) of filamentous ascomycetes is composed of a plaque structure apposed to the nuclear envelope (Zickler, 1970). As in yeast, where diploid SPBs' increase in size and nucleating capacity are compared with those of haploid SPBs (for a review see Rout and Kilmartin, 1990), SPB size appears to be a function of ploidy in the cro1-1 mutant (compare Figure 2L and M). The SPBs of wild-type strains alter in size, nucleating capacity and orientation over the sexual cycle (Zickler, 1970; Thompson-Coffe and Zickler, 1994). Similar ultrastructural differentiation processes are observed in the giant SPBs (data not shown). Cytoskeleton organization in the cro1-1 sexual cycle Analysis of the distribution of microtubules and actin microfilaments during crozier and ascus development was performed by anti-tubulin and anti-actin immunofluorescence. In wild-type croziers, cytoplasmic microtubules are relatively sparse, bundled, and appear to originate mostly on or near the nuclear envelope (Figure 3A). These microtubules are disassembled during the coordinate mitosis. Immediately after division, all four nuclei display large asters with open ends towards the septal sites (Thompson-Coffe and Zickler, 1994). The wild-type arrangement is observed in the few normal cro1-1 croziers. However, in the giant multinucleate croziers, microtubule organization differs significantly from that of wild-type. Microtubules are abundant, curving, and appear mainly cortical (Figure 3B and C). As in wild-type, these non-nuclear microtubules are no longer visible during nuclear divisions and mitoses remain strictly coordinate (Figure 3D and E). However, the spindles are randomly oriented and few astral microtubules are visible, while in wild-type the astral microtubules are quite long and apposed to the plasma membrane. Figure 3.Cytoskeleton organization in the mutant croziers. (A) Anti-tubulin of wild-type croziers. (B) Anti-tubulin of a cro1-1 giant crozier with numerous microtubules spanning the gap between the nuclei [note that (A) and (B) were printed at the same magnification]. (C) DAPI of the same crozier. (D) Synchronous mitoses in one giant crozier. Note that the cortical microtubules seen in (B) are all gone and that microtubules are only in the spindles. (E) DAPI of the same crozier showing that the dividing nuclei were in anaphase. (F) Anti-actin of an aseptate giant crozier with broad cortical microfilaments. (G) DAPI of the same crozier. (H) Anti-actin of a septate giant crozier with actin-plaques and no cortical microfilaments. Microfilaments form belts (arrows), but contrary to what is observed in wild type, they do not correspond to the metaphase plates as shown by the disposition of the corresponding nuclei seen in (I) DAPI staining. Scale bars represent 5 μm. Download figure Download PowerPoint Actin is present as both plaques and fibres in wild-type and mutant cells, but its distribution differs between the two. In premeiotic wild-type croziers, filaments are relatively sparse, mainly seen in the crook area and between the nuclei (Thompson-Coffe and Zickler, 1993). The microfilaments of giant cro1-1 croziers are abundant and, like the microtubules, appear to be mostly cortical with little evident orientation respective to the nuclei (Figure 3F and G). Wild-type actin belts assemble as future septa sites in anaphase of crozier mitosis and later disassemble (Thompson-Coffe and Zickler, 1993). In cro1–1 croziers, such belts may form between more or less properly oriented nuclei (Figure 3H and I) or may not be seen at all. After development of the ascus, actin is organized as a cortical array of longitudinal microfilaments with associated plaques, parallel to the cortical microtubule array, in both wild-type and mutant strains (not shown). The cro1-1 mutant also displays a vegetative phenotype The cro1-1 mutant grows slowly and in waves: first flat, the mycelium becomes increasingly dense before initiating a new flat surface, the band size and period varying during radial growth. However, septa and nuclear distribution are similar to what is observed in wild-type mycelia (data not shown). The longevity of the cro1-1 strain is just slightly shorter than the wild-type lifespan (Rossignol and Silar, 1996). The second vegetative defect of cro1-1 concerns ascospore germination. This process, which takes a few hours for wild-type ascospores (at 27°C), can take several days or even several weeks for the mutant. This defect is less pronounced at 20°C. However, at both temperatures, the mycelium issued from the mutant ascospores is first very spindly and then displays a normal thickness. Both vegetative characteristics and the sporulation defect of the mutant co-segregate in crosses and appear recessive. The CRO1 protein shows similarity with the yeast SHE4 protein The cro1 gene was cloned by complementation and SIB selection (Akins and Lambowitz, 1985) using the cro1-1 strain as the recipient. The cosmid library used contained, as selectable marker, the bacterial hygromycin resistance gene (see Materials and methods). Hygromycin-resistant transformants displaying a wild-type growth phenotype were crossed with the cro1-1 mutant to test their sporulation ability. Some showed a completely wild-type phenotype while others conserved a sporulation defect. One possible explanation is that, in some cases, the integration site of the cosmid carrying the cro1 gene led to a low expression of the gene. This expression might be sufficient for growth, but not for restoration of the wild-type ability to sporulate. In any case, the relevant cosmid was subcloned and a 2.3 kb DNA fragment, which complemented all the phenotypic defects of the cro1-1 mutant, was sequenced. Analysis of the nucleotide sequence identified two open reading frames. A putative intron was found by consensus sequences for the 5′, 3′ splice sites and for the lariat formation site (Ballance, 1986). In addition, the cDNA corresponding to the cro1 gene was obtained by reverse transcription and PCR amplification of the cro1 mRNA. This experiment was performed on RNA extracted from wild-type mycelia and perithecia (see Materials and methods). The gene was found to be expressed in both vegetative and sexual stages of the Podospora life cycle. However, amplification of the cro1 mRNA was always difficult to obtain and Northern blot analysis provided no signal despite positive controls (data not shown). Furthermore, the cro1 gene displays a low codon usage bias (data not shown). Taken together, these results suggest a low expression of the cro1 gene. The cro1 cDNA analysis confirmed the presence of the intron (see star in Figure 4). Figure 4.Comparison of the amino acid sequences deduced from the P.anserina cro1-1 gene and the S.cerevisiae SHE4 gene. P.anserina0cro1-1 gene upper line; yeast SHE4 gene, lower line. Similar and identical amino acids are boxed; identical amino acids are stippled. The localization of the frameshift in the cro1-1 mutant is designated by an arrow. The star above the sequence shows the intron position. The alignment was obtained with the GAP algorithm, with a gap penality of 3.000 and a length penality of 0.100. The DDBJ/EMBL/GenBank accession number for the cro1 nucleotide sequence is Y16261. Download figure Download PowerPoint The gene encodes a putative protein of 702 amino acids. Analysis of the protein sequence reveals several interesting points. First, there are several regions rich in positively charged residues, for instance between amino acids 162 and 169 (RRKWKSRK) which might be nuclear localization signals (for a review see Garcia-Bustos et al., 1991). Second, a putative zinc finger motif (Coleman, 1992) of the C2H2 family was found between amino acids 71 and 94 (CX5CX12HX3C). Third, several regions are rich enough in proline, negatively charged and hydroxylated amino acids, to resemble PEST motifs, especially since they are bordered by positively charged residues (for a review see Rogers et al., 1986). Fourth, the carboxy-terminal part of the protein is rich in valine residues (among 57 valines, 26 are contained within the last 160 amino acids of the protein). Furthermore, these valines are generally located in pairs or triplets. Finally, the CRO1 protein shows (between amino acids 231 and 235) the sequence RRHSL, which is one of the motifs for a phosphorylation site by the cAMP-dependent protein kinase, PKA (consensus: RRXS/TY with X being any residue and Y a hydrophobic residue; for review see Taylor and Radzio-Andzelm, 1994). A search of the Saccharomyces Genome Database with the FASTA program revealed a similarity between the putative CRO1 protein and the budding yeast SHE4 protein required for asymmetric mating-type switching in haploid cell divisions (Jansen et al., 1996). As shown in Figure 4, the yeast and the fungal proteins display 21% identity and 40% similarity in a 702 amino acid overlap. The yeast and Podospora sequences were compared by the BESTFIT program (data not shown). The parameter termed ‘quality of the alignment’ was compared with the average quality of 100 alignments of random permutations. The reduced deviation (Z parameter), calculated as (cognate quality–average quality/standard deviation of quality of random permutations), was 14. This value is highly significant (Slonimski and Brouillet, 1993). Another search was performed at the NCBI using the BLAST network service and revealed a low, but significant similarity of the CRO1 protein with a 993 amino acid protein of Caenorhabditis elegans encoded by a gene of chromosome III (EMBL accession number U29096): the two proteins show 24% identity and 48% similarity; the Z parameter is 10.2. A tentative alignment was performed between SHE4 and the C.elegans protein: the Z parameter being very low (4.8), the similarity found was statistically not significant. To confirm that the cro1 gene was actually cloned and to identify the nature of the mutation, the cro1-1 mutant gene was sequenced. In the mutant allele a cytosine is missing 40 bases downstream of the ATG. This frameshift leads to a premature chain termination, 45 codons after the start codon (the localization of the frameshift is designated by an arrow in Figure 4). Thus, cro1-1 is a null mutation of the cro1 gene. CRO1 is a cytosolic protein mainly expressed in young croziers and at the time of ascospore maturation To elucidate the localization of the CRO1 protein, fusions were constructed between the cro1 gene and the green fluorescent protein (EGFP) sequence (see Materials and methods). Fourteen cro1-1 strains carrying the transgene encoding the CRO1–GFP fusion protein and exhibiting a wild-type phenotype, were first screened for GFP expression during vegetative growth. Green fluorescence was observed in the cytoplasm of the living mycelium whatever the age and size of the hyphae (Figure 5A). However, GFP expression varied among the 14 strains: three showed a bright uniform distribution of the green fluorescence in all compartments of the growing mycelium, the ascogonia but not the microconidia; five gave a weaker signal; and six showed no fluorescence (however, a weak emission signal cannot be excluded, the mycelium being observed with conventional fluorescence microscopy and not with a high-sensitivity video camera). In comparison, a transformant expressing GFP under the control of the strong Aspergillus nidulans GPD promoter (Punt et al., 1987; see Materials and methods) used as a control, showed also GFP fluorescence in the cytoplasm, but with a much brighter intensity, including in the microconidia. Strains expressing GFP–CRO1 grew and sporulated at a rate indistinguishable from that of strains expressing wild-type CRO1. Figure 5.Expression of the green fluorescent protein (EGFP) in P.anserina strains carrying the transgene encoding the CRO1–GFP fusion protein. (A) All hyphae, whatever their thickness, are labelled over their entire surface. (B) In this young fruiting body, only the croziers (arrow points to one) are brightly fluorescent. The surrounding asci (arrowheads) are hardly visible. (C) Two asci with four ascospores. The upper ascus is older and shows also a brighter fluorescence than the ascus below, which is younger (compare the size of the ascospores). Scale bars represent 5 μm. Download figure Download PowerPoint In contrast to what is seen in the mycelium, there was no difference in GFP expression during the sexual cycle of different strains. In all crosses between mat+ and mat− strains issued from the same primary transformant (see Materials and methods), the CRO1–GFP was highly expressed in the croziers and very young asci as well as in older asci with maturating ascospores (Figure 5B and C). During meiosis, postmeiotic mitosis and ascospore delimitation, the cytoplasm of the asci was only slightly green (Figure 5C). No fluorescence was observed in the hypha-like plurinucleate paraphyses which are formed between the asci. In contrast, the control GPD::GFP cross showed a strong GFP fluorescence only in the paraphyses and the ascospores. Prior to sporulation, the SPB alters in size, nucleating capacity and orientation (Thompson-Coffe and Zickler, 1994). This SPB also incorporates GFP–CRO1 and could be identified as a bright fluorescent body, similar in size to what is observed in fixed cells by the mitotic phosphoprotein antibody MPM-2 (Davis et al., 1983; Thompson-Coffe and Zickler, 1994), while the SPBs present on the vegetative and meiotic nuclei could not be visualized (data not shown). Cro1-1 is able to internalize FM4-64 as efficiently as the wild-type strain Identification of a mutant allele of the budding yeast SHE4 gene by a screen for mutants defective in endocytosis (Wendland et al., 1996), prompted us to follow the bulk internalization of plasma membrane in both the wild-type and the cro1-1 mutant strains with the endocytic tracer FM4-64 (Vida and Emr, 1995; Wendland et al., 1996; see Materials and methods). As the internalization defect of the she4 mutants was shown to be temperature-dependent, the experiments were performed at 27°C (normal growth temperature) and 34°C (highest temperature before cellular death). They led to three conclusions. First, the fluorescent dye FM4-64 is clearly a useful marker to observe vacuolar membranes in the filamentous fungus P.anserina (Figure 6). Second, the number, distribution and size of the vacuoles were similar in the wild-type and cro1-1 (Figure 6) mycelia. Third, in both strains, the size and number of vacuoles were clearly increased at high temperature. Thus, in contrast to the she4 mutants, the cro1-1 mutant does not show internalization defects, at least when the process is investigated with FM4-64 as an endocytic tracer. Figure 6.FM4-64 labelling of cro1-1 mutant mycelium at 34°C. Vacuoles are clearly visible due to their high fluorescent intensity. As in wild" @default.
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• W2100256768 title "A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina" @default.
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• W2100256768 doi "https://doi.org/10.1093/emboj/17.5.1248" @default.
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