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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Early pseudohyphal growth of Saccharomyces cerevisiae is well described, and is known to be subject to a complex web of developmental regulation. In maturing filaments, young cells differ significantly from their pseudohyphal progenitors, in their shape, and in their timing and direction of cell division. The changes that occur during filament maturation result in round and oval cells surrounding and covering the pseudohyphal filament. In a screen for mutants that affect this process, a vacuolar protein sorting gene, MOS10 (VPS60), and a gene encoding an α subunit of the proteasome core, PRE9, were isolated. Characterization of the mos10/mos10 phenotype showed that the process of filament maturation is regulated differently from early filamentous growth, and that the requirement for Mos10 is limited to the maturation stage of pseudohyphal development. The mos10/mos10 phenotype is unlikely to be an unspecific effect of disruption of endocytosis or vacuolar protein sorting, because it is not recapitulated by mutants in other genes required for these processes. Disruption of homologues of MOS10, which act as components of the ESCRT-III complex in targeting proteins for vacuolar degradation, results in abnormal early pseudohyphal growth, not in the filament maturation defect seen in mos10/mos10. Thus, Mos10 may function in targeting of specific cargo proteins for degradation, under conditions particular to maturing filaments.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Filamentous growth of Saccharomyces cerevisiae has provided an important model both of multicellular eukaryotic development and of morphogenesis of pathogenic fungi. Diploid yeast cells placed on filamentation medium produce daughter cells with dramatically different characteristics: cell shape changes from round or oval to elongated; duration of the G2 phase of the cell cycle increases; cells bud unipolarly and remain attached after division, forming chains; filaments composed of cell chains invade the agar; and cell growth is directed away from the mass of cells at the centre of the colony (Gimeno et al., 1992; Kron et al., 1994).

Study of the regulation of this process has led to findings with wider implications for eukaryotic development. A single MAP kinase cascade was found to signal for development of different cell types – filamentous differentiation of diploid cells and mating of haploid cells (Liu et al., 1993) – as well as for different developmental steps of the same cell type, agar invasion and mating of haploid cells (Roberts and Fink, 1994). In the analysis of how two distinct outputs are generated from a signal travelling through the same module, the MAP kinases Kss1 and Fus3 were found to exert both an inhibitory and an inducing effect on a developmental output, depending on their activation state (Cook et al., 1997; Madhani et al., 1997). Connections between cyclicAMP-dependent kinase and MAP kinase signalling, were found, when Ras2 was shown to signal through both pathways for filamentous growth (Mösch et al., 1996; 1999; Kübler et al., 1997; Lorenz and Heitman, 1997).

Discovery of regulatory pathways of filamentous growth in S. cerevisae has also greatly accelerated the study of genetically less tractable pathogenic fungi, such as the human opportunist Candida albicans. Candida albicans also switches from yeast-form growth to filamentous growth under certain nutritional conditions, although its morphogenetic repertoire is broader, as it forms both pseudohyphae, like S. cerevisiae, and true hyphae like other filamentous fungi. The two main filamentation signalling modules known from S. cerevisiae– the mating and filamentous MAP kinase cascade and the cyclic AMP dependent kinase cascade – also signal for filamentous growth in C. albicans (reviewed in Lengeler et al., 2000). As in S. cerevisiae, cell cycle regulation also affects filamentous growth in C. albicans, as the mutant in the mitotic cyclin CaCLN1 has a hyphal growth defect (Loeb et al., 1999). Studies of C. albicans morphogenesis, often focused on homologues of known regulatory genes in S. cerevisiae, showed that the ability to switch between growth as yeast and growth as filaments is important for Candida's ability to cause invasive disease (Braun and Johnson, 1997; Lo et al., 1997; Gale et al., 1998).

During filamentous growth, C. albicans produces large amounts of blastoconidia, round or oval cells which are budded off from filaments as the filaments mature. Most likely, they fulfill the same function as the conidia of other well-studied filamentous fungi such as Aspergillus nidulans: dispersal to new substrates beyond the reach of the filamentous mycelium. The developmental step that produces them, however, differs from that of A. nidulans and many other filamentous ascomycetes, in that C. albicans produces blastoconidia directly on its hyphae, whereas the Aspergillus species elaborate specialized structures, conidiophores, from which conidia are produced (Larone, 2002).

Maturing S. cerevisiae pseudohyphae also produce round or oval cells directly on filaments, which eventually cover the entire filament (Gimeno et al., 1992). In this work, maturation of Saccharomyces filaments was followed by long-interval time-lapse microscopy, to define the steps in which these lateral yeast cells are produced. A transposon-mutagenised pool of homozygous diploids was then created by a novel protocol, to find genes whose disruption affects this process. Two genes were found, which represent the two major protein turnover pathways in the cell: the proteasomal and the vacuolar pathway. One of the genes isolated, MOS10 (VPS60), encodes a class E vacuolar sorting protein (Kranz et al., 2001).

Mutants in vacuolar protein sorting were originally identified because they secrete the vacuolar glycoprotein carboxypeptidase Y, instead of trafficking it to the vacuole (Bankaitis et al., 1986; Rothman and Stevens, 1986). Based on vacuolar morphology and acidification, these mutants were classified into six groups (Banta et al., 1988; Raymond et al., 1992). Class E comprises the mutants whose vacuoles look normal, but which have an abnormal organelle adjacent to the vacuole, the class E compartment, which contains markers of both the late Golgi and the vacuole (Raymond et al., 1992). Subsequent work has shown that the class E compartment represents an enlarged late endosome, whose sorting function for vacuolar lumenal versus vacuolar membrane proteins is impaired. Cargo proteins in the class E compartment – newly synthesised vacuolar enzymes as well as endocytosed proteins targeted for vacuolar degradation – are stalled before reaching the vacuole (reviewed in Lemmon and Traub, 2000), see also (Shaw et al., 2001; Babst et al., 2002).

The defect in filament maturation seen in the mos10/mos10 mutant is not shared by mutants in other proteins important for this process, including the structural homologues of Mos10. In addition, the mos10/mos10 phenotype is restricted to conditions of filament maturation. The simplest model is that Mos10 is required for endosomal sorting of specific proteins which must be degraded in order for filament maturation to proceed normally.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Steps of filament maturation

The development of young filamentous colonies is well described in the literature (Gimeno et al., 1992; Kron et al., 1994). When a yeast-form cell is placed on filamentation-inducing medium, its progeny form chains of elongated cells growing radially on the surface of the agar (Kron et al., 1994). In the first day of growth, the young filamentous microcolony is covered with a dome of cells that forms a circular smooth edge on the agar surface. Invading the agar beneath the dome of cells, chains of elongated, unipolarly budding cells protrude beyond the edge of the surface colony between days one and three (Gimeno et al., 1992) (Fig. 1A). The growth of young invasive filaments from a colony is directed radially to the mass of cells; from a patch, it is directed perpendicularly to the mass of cells.

image

Figure 1. Maturation of wild-type filaments on SLAD28 in 35 mm plates, photographed at the indicated times. Bars are 20 µm. A. First panel: young invasive filament. Mother cell budding suppression: the first cell of the filament (1), has not budded again, whereas its daughter, the second cell (2), has budded. The tip cell of the young invasive filament (C) is elongated. Fourth panel: mature invasive filament. Cells nearer the base of the filament (star pointer) are dividing, whereas cells at the tip (dot pointer) are not dividing. The tip cell of the mature filament (D) is oval. B. Mature invasive filament. Mother cell budding suppression is lifted. Mother cell has divided two times (small arrowhead) or three times (large arrowhead) before one of its daughters divides. C and D. Cartoon of tip cells in first and fourth panels of A respectively E and F. Cartoon of cells in triangled sections of B.

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By following the development of single filaments at 12 and 24 h intervals, the developmental steps of filament maturation were delineated. Changes characteristic of filament maturation begin at the base of the filament where cell density is highest. Whereas cells at the tip of the growing filament continue to bud elongated daughters, mother cells at the base of the filament begin to bud oval or round cells. These daughters in turn give rise to non-elongated cells. During maturation of the filament, cells are never displaced in the agar. Each cell can be observed in its previous position in the course of 12- or 24-hourly photomicrographs, with the only change over time being the emergence and growth of new buds. Thus, it is the younger, oval or round, cells which continue to proliferate and not the older filamentous cells in the centre of the pseudohypha.

Cell elongation is preserved longest at the tip of the filament, but eventually tip cells become oval too (Fig. 1A). This transition occurs at different times in different filaments.

Young superficial filaments on the agar surface have been shown to differ dramatically from young colonies growing in yeast form, in that daughter cells bud simultaneously with their mothers (Kron et al., 1994). This difference from yeast-form growth also pertains to young invasive filaments; though in young invasive filaments, most daughters bud before mothers (Fig. 1A).

The suppression of budding of mother cells is necessary to create the geometry of a filamentous projection: per number of cell divisions, the projection will extend farther if daughters bud before mother cells. Conversely, when mother cells bud before daughters, the distance spanned by the projection per number of cell divisions becomes shorter and more cells accumulate at the base of the filament. This, in fact, is the case on maturing filaments: mothers often bud once, and occasionally twice, before their daughters (Fig. 1B). When the suppression of mother budding is lifted, filaments not only become shorter and thicker, but their growth is also no longer directed away from the centre of the colony. Instead, growth direction becomes random.

In young invasive filaments the tip is the most rapidly dividing portion. In some older filaments, cells at the tip divide more slowly than cells closer to the base of the filament (Fig. 1A). This contributes to the randomization of growth direction of older filaments.

In young filaments, budding is unipolar (Gimeno et al., 1992). Mature filaments retain unipolar growth: of 100 budding events scored in the last two days of 21 filaments followed, 92 occurred on the end of the cell opposite the birth end; six occurred on the birth end; and two occurred in the middle of the mother cell. This is still in accordance with a pseudohyphal budding pattern, as analysed in (Gimeno et al., 1992), where 10% of second buds of pseudohyphal cells were seen at the birth end.

The main elements of filament maturation observable in the microscope are thus: shortening of new cells, first on the older part of the filament then at its tip; lifting of the suppression of mothers’ budding; and randomization of growth direction. The resulting growth resembles blastoconidia of filamentous fungi such as Candida albicans. Because the developmental switch from hyphal filament to blastoconidium in C. albicans is more profound than the switch from pseudohyphal filament to non-directional yeast-form growth in S. cerevisiae, I chose a different term. Oval or round cells on filaments, whose mothers bud before daughters, and whose growth is not directed away from the mass of cells, were termed lateral yeast cells. Growth of lateral yeast cells on filaments continued for weeks to months on filamentation medium (Supplementary material, Fig. S1).

Lateral yeast cells on filaments, but not on the colony centre, increase over time

The fraction of round (length-to-width ratio = 1), oval (length-to-width ratio between 1 and 2) and long cells (length-to-width ratio = or greater than 2) was determined on filaments of colonies growing on 100 mm plates of my filamentation medium SLAD28 (see Experimental procedures), at 1, 2, 3 and 4 weeks. Over this time period, as lateral yeast growth occurred on the maturing filaments, the percentage of long cells decreased whereas that of round cells increased (Fig. 2).

image

Figure 2. Proportion of cell types in maturing colonies. Wild type (JKY573<pRS316>), mos10/mos10 (JKY228<pRS316>), mos10/mos10 complemented with wild-type MOS10 (JKY228<pJK164>) and wild type expressing Ras2Val19 (JKY573<B3414>), were grown in 100 mm plates on SLAD28. Cells of invasive filaments were counted as long, oval or round. Between four and 12 colonies of each strain were analysed, and 200 cells per colony were counted. Error bars show standard deviation. A. Per cent long cells. B. Per cent oval cells. C. Per cent round cells. *P for round cells of wild type and mos10/mos10 as well as Ras2Val19 at 3 weeks < 0.01.

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As a marker for the proliferation of lateral yeast cells on filaments, the percentage of round cells composing a filament was chosen. Though they comprised the smallest proportion of total cells, they most clearly represented lateral yeast growth, because oval cells can be derived from a filament, or from lateral growth.

To investigate the possibility that round cells counted in maturing filaments were contaminants from the dome of cells on the surface, at the centre of the colony, the composition of those cells was counted. Surprisingly, the colony centre surface was found to contain less than 5% round cells, throughout the 4 week duration of the experiment, arguing against contamination as a source of the round cells.

A mutant in MOS10 is defective in lateral yeast growth on maturing filaments

Because proliferaton of lateral yeast cells is a typical feature of maturing wild-type filaments, mutants defective in this process were sought, in order to gain insight into its mechanisms.

Filamentous growth is a developmental process that is limited to diploids. Therefore, a pool of homozygous diploid mutants had to be constructed. A novel protocol was developed for this purpose, with which a pool of haploid yeast, mutagenised by transposon insertion (Burns et al., 1994), was diploidised using the Ho endonuclease (see Experimental procedures). Of the mutants that had undergone this procedure,  2% remained haploid, and  1.6% were heterozygotes, so that the final pool consisted of  96% homozygous diploids.

From the total homozygous diploid mutant pool of 5 × 104 clones, 7.8 × 104 colonies were visually screened under the microscope. Insertions in two genes were isolated: one in MOS10 (VPS60), and nine clones representing seven independent insertions in different portions of PRE9, which encodes the S. cerevisiae Y13 component of the 20S proteasome core (Emori et al., 1991). Lateral yeast growth on pseudohyphae was completely absent only in one of the PRE9 insertion isolates, but the phenotype was found not to be due to the transposon insertion alone (unpublished data). Lateral yeast cells on filaments were reduced, but not absent, in an insertion mutant in the MOS10 gene, whose mutation has been described to result in a class E vacuolar protein sorting phenotype, and whose product has localized together with an endosomal marker (Kranz et al., 2001).

A complete deletion of MOS10 was constructed in the haploid and diploid, and the resulting strains were used for further characterization of the phenotype. The deletion mutant mos10/mos10 formed colonies of normal size on SLAD28, and filaments appeared wild type in early stages of their growth (Fig. 3G and H). By 3 weeks of incubation on 100 mm SLAD28 plates, the defect of lateral yeast growth on mos10/mos10 filaments was apparent (Fig. 3I and J).

image

Figure 3. Filament maturation of mos10/mos10 compared to wild type. A. Wild type (JKY482) and (B) mos10/mos10 (JKY223) prototrophs on SLAD28 in 35 mm plates, in the same experiment. Bar is 20 µm. C–F. Wild type and (G–J) mos10/mos10 prototrophs on SLAD28 100 mm plates. The right panel in each row shows the same colony as the left panel, at higher magnification. Both bars are 100 µm.

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Single mos10/mos10 filaments followed over time on 35 mm plates, were able to form some lateral yeast cells (Fig. 3B). The same changes occurred as in wild type: cell shape became oval or round, mother cell budding suppression was lifted, and growth direction became random. However, there was less lateral yeast growth than on wild-type filaments. Initiation of lateral yeast growth in the mos10/mos10 strain typically began at some distance from the base of the filament; as opposed to wild type, in which it began at the base, where cell density is highest. As mos10/mos10 filaments matured, they were composed of fewer round cells than those of wild type (Fig. 2). This difference became apparent at 2 weeks on 100 mm plates, and was statistically significant at 3 weeks (Fig. 2C).

To test whether the defect in lateral yeast growth on filaments of the mos10/mos10 strain results only from the deletion of MOS10, the wild-type gene was introduced into the mutant on a centromeric vector. Filaments of the mutant rescued by MOS10 matured like those of wild type, with normal growth of lateral yeast cells (Fig. 2C). The phenotype of wild type expressing MOS10 from a high copy vector was not distinguishable from wild type carrying the empty 2µ vector (not shown).

The mos10/mos10 defect in producing yeast-form cells is limited to the context of maturing filaments

The question arose, whether mos10/mos10 cells, once having switched to filamentous growth, are unable to produce non-filamentous cells under all conditions. To answer this question, the response of cells in a filamentous growth state to conditions inducing yeast-form growth was tested. Young filamentous cells were placed into rich nutrient conditions on solid and liquid YPD, and into liquid synthetic complete minus uracil (sc – ura) medium, by extracting the invasive filaments of two day old pseudohyphal colonies from filamentation medium, fragmenting them into single cells, and placing them in the yeast-form inducing conditions (see Experimental procedures).

When taken from SLAD28 agar and placed onto YPD agar plates, both wild-type and mos10/mos10 filamentous cells continued to bud unipolarly for several generations, but by 20 h, both strains formed round colonies (not shown). When taken from SLAD28 agar and placed into liquid YPD or synthetic rich medium, both wild-type and mos10/mos10 cells switched from elongated to oval or round growth at the first cell division. I compared the switch from filamentous to non-filamentous growth in rich medium, between wild type and mos10/mos10 carrying vector, and wild type carrying the hyperfilamentous alleles STE11-4 and Ras2Val19 (Mösch et al., 1996). All strains switched efficiently from filamentous growth to yeast-form growth under these conditions of replete nutrients (Tables 1 and 2; Fig. 4).

Table 1. . Length-to-width ratio of invasive cells grown in SLAD28 agar for 2 days.
Strain (n cells)Average length- to-width ratioStandard deviationP for difference to wild-type<vector>
  • a

    . JKY573<pRS316>.

  • b

    . JKY228<pRS316>.

  • c

    . JKY573<pSL1509>.

  • d

    . JKY573<B3414>.

Wild type<vector>a (284)1.870.42 
mos10/mos10<vector>b (249)1.860.38 0.73
Wild type<STE11-4>c (178)2.040.57<0.01
Wild type<Ras2Val19>d (194)1.940.46 0.13
Table 2.  Length-to-width ratio of first daughters of cells grown in sc-uracil for 6.5 h, after invasive growth in SLAD28 agar for 2 days.
Strain (n cells)Average length -to-width ratioStandard deviation
  • a

    . JKY573<pRS316>.

  • b

    . JKY228<pRS316>.

  • c

    . JKY573<pSL1509>.

  • d

    . JKY573<B3414>.

Wild type<vector>a (125)1.130.08
mos10/mos10<vector>b (121)1.120.08
Wild type<STE11-4>c (104)1.150.11
Wild type<Ras2Val19>d (90)1.130.08
image

Figure 4. Switch from early invasive filamentous growth to yeast form growth. Filaments were cut out of 2 day old washed SLAD28 plates, fragmented into single cells, placed in 35 mm dishes containing liquid synthetic complete medium without uracil (sc – ura), and grown on a shaker. Cells were photographed at the time of placement in sc – ura (0 h), and at 6.5 hours. Both bars are 20 µm. A. JKY573<pRS316>. B. JKY228<pRS316>. C. JKY573< pSL1509>. D. JKY573<B3414>.

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Early filamentous growth of mutants in MOS10 is distinct from that of hyper filamentous strains

Though the mos10/mos10 strain does not appear morphologically hyperfilamentous, one might postulate that its filament maturation defect may be a variant of a hyperfilamentous state. To address this issue, growth characteristics of mutants in MOS10 were compared with known alleles and mutants of genes, which result in hyperfilamentous growth in diploid cells or in hyperinvasive growth in haploids.

Haploid invasive growth, which has been found to parallel diploid invasive growth in known mutants, is most clearly scored on YPD, not on selective synthetic medium. For this reason, plasmids carrying hyperfilamentous alleles were avoided in investigating this phenotype; instead, haploid invasive growth of mos10 was compared with known hyper- and hypoinvasive mutants. Loss of the cell surface protein Flo11 is known to result in severely deficient haploid invasive growth, whereas loss of the transcriptional repressor Sfl1 results in increased haploid invasive growth (Robertson and Fink, 1998). The mos10 mutant was compared with these two mutants, and found to have a subtle defect in haploid invasive growth (Fig. 5).

image

Figure 5. Deletion of MOS10 does not mimic hyperinvasive or hyperfilamentous states. (A–H) Haploid invasive growth. Five µl cell suspensions at an OD600=1, were spotted on YPD and incubated for 5 days. Plates were washed by rotating the flooded plate gently on a shaker. Spots were photographed before washing (A, C, E and G), and after washing (B, D, F and H). Bar is 5 mm. (A, B) wild type (10560-5A), (C, D) mos10 (JKY1027), (E, F) flo11(L6947) and (G, H) sfl1 (L6930). I and J. Cells of invasive filaments were counted as long, oval or round. Four colonies of each ura3/ura3 strain, grown on 100 mm SLAD28 plates, were analysed. Two hundred cells per colony were counted. Shown here is the proportion of round cells in invasive filaments over four weeks. I. Wild type<2µ<vector> is JKY573<pRS426> (diamond), mos10/mos10<2µ vector> is JKY228<pRS426> (square), wild type<STE12 2µ> is JKY573<B2553> (triangle) and wild type<TEC1 2µ> is JKY573<BHM256> (cross). J. Wild type is JKY573<pRS316> (diamond), mos10/mos10<CEN vector> is JKY228<pRS316> (square), wild type<STE11-4> is JKY573<pSL1509> (triangle) and wild type<Ras2Val19> is JKY573<B3414> (cross). The Ras2Val19 curve is the same as in Fig. 2, and shown again only for comparison.

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The gain-of-function MAP kinase kinase kinase allele STE11-4 and the constitutively active GTPase allele Ras2Val19 induce hyperfilamentous growth when expressed from a centromeric vector in wild-type cells (Gimeno et al., 1992; Mösch et al., 1996); as do the transcription factors TEC1 and STE12 when overexpressed from a high copy vector (Gavrias et al., 1996). Early filamentous growth and diploid invasive growth of wild-type cells carrying these hyperfilamentous alleles was compared with that of wild-type and mos10/mos10 cells carrying the respective vectors. All strains were grown on SLAD28 agar for 2 days, and colony morphologies were compared before and after washing. The mos10/mos10 colonies were not hyperfilamentous or hyperinvasive, as compared with wild-type and the hyperfilamentous alleles (not shown).

Defective filament maturation is not a general feature of hyperfilamentous strains

The converse question, whether hyperfilamentous strains behave like mos10/mos10 in filament maturation, was addressed by comparing lateral yeast growth on filaments of the four hyperfilamentous strains, with wild type and mos10/mos10 transformed with the respective vectors. Whereas the hyperfilamentous strains differed distinctively from wild type and from mos10/mos10 early in filament development, three of the four increasingly approached wild-type filament composition during their maturation. The exception was the strain carrying Ras2Val19, whose filaments consisted of < 5% round cells for the duration of this experiment (Fig. 5J).

Hyperfilamentous alleles were expressed in mos10/mos10 to test whether they further decreased lateral yeast growth on filaments. Except for the strain containing Ras2Val19, none of the resultant strains had a further defect of lateral yeast growth, as assessed by comparison of colony morphologies with mos/mos10 and with wild type carrying the respective vectors (not shown).

The mutant in MOS10 thus did not behave like hyperfilamentous strains in haploid invasive growth, or in early filamentous growth. Conversely, the hyperfilamentous strains tested did not behave like mos10/mos10 in filament maturation. The mos10/mos10 mutant's filament maturation defect is therefore a distinct, novel phenotype. Ras2Val19 confers a filament composition phenotype that differs both from the other hyperfilamentous alleles, and from mos10/mos10. The Ras2Val19 phenotype is remarkable for the large proportion of long cells, and small proportion of round cells, both in young and in maturing filaments.

Mos10 function is distinct from the PKA and MAP kinase filamentation signalling pathways

Ras2 is known to regulate multiple aspects of cell physiology beside filamentous growth. On SLAD28 medium, colonies containing Ras2Val19 were significantly smaller than those of wild type or of mos10/mos10. The question arose, whether aspects of Ras2 signalling that regulate filamentation, can be isolated from its other effects on the cell. Thus, components of a known filamentation signalling pathway which is regulated by Ras2, the cAMP dependent protein kinase pathway, were investigated as to their filament maturation phenotype and for genetic interaction with MOS10.

Double mutants were constructed with TPK2, the subunit of protein kinase A which is required for filamentous growth, and with SFL1, a transcriptional repressor regulated by TPK2 (Robertson and Fink, 1998; Pan and Heitman, 1999). The tpk2/tpk2 mutant is known to be defective in filamentous growth. On SLAD28, tpk2/tpk2 colonies initiated few filaments, but those that were initiated, grew to normal length and morphology. The sfl1/sfl1 mutant is known to be hyperfilamentous: it produced high-domed, rough colonies with large numbers of filaments on SLAD28.

Pseudohyphae of both tpk2/tpk2 and sfl1/sfl1 matured normally, both morphologically and by quantification of round cells on filaments. When these strains were further deleted in MOS10, an additive phenotype was seen. Initiation of filaments was as sparse in tpk2/tpk2 mos10/mos10 as it was in tpk2/tpk2, and exuberant both in the single mutant for SFL1 and its double mutant with MOS10. The filaments of both double mutants showed the mos10/mos10 maturation defect. The conclusion was, that the effect of RAS2 on filament maturation is not mediated by the protein kinase A pathway nor does MOS10 have an epistatic relationship to the tested components of the pathway.

The MAP kinase filamentation pathway regulates early filamentous growth (Liu et al., 1993). Defects in kinases of this pathway partially abrogate filament formation (Lo et al., 1997). A double mutant of MOS10 with STE7, which encodes the MAP kinase kinase of the pathway, was constructed. Deletion of MOS10 did not suppress the pseudohyphal growth defect of the ste7/ste7 mutant, which at three weeks had short stubby filaments (not shown). There was thus no evidence for genetic interaction between MOS10 and the filamentation MAP kinase kinase for early filamentous growth. Maturation could not be reliably assessed in the defective filaments.

mos10/mos10 filaments give rise to new foci of filament formation

Whereas in young filamentous colonies of the mos10/mos10 mutant, as in those of wild type, growth of filaments was directed radially outward from the mass of cells at the colony centre, some maturing filaments of the mutant gave rise to new centres of filament formation. New foci differed from the other filaments of the colony, in that their growth was directed toward, as well as away from, the mass of cells in the colony (Supplementary material, Fig. S2).

In order to test whether a new mutation had arisen in the new foci, cells from mos10/mos10 foci, from remaining filaments without foci, and from the colony centres, as well as from wild-type filaments and colony centres, were excised from agar. They were grown up in YPD, transformed with MOS10 on a centromeric vector, MOS10 on a high-copy vector, and empty vectors, and tested for filament formation and formation of new foci. In mos10/mos10 transformants carrying empty vector, there was no morphological difference between colonies derived from new foci, colonies derived from filaments without new foci and colonies derived from colony centres. In particular, there was no difference in the frequency of formation of new filamentous foci. In mos10/mos10 transformants carrying MOS10 on a low- or high-copy vector, filaments appeared wild type, without formation of new foci. Thus, the formation of new foci could not be shown to result from a second mutation.

The mos10/mos10 phenotype is distinct from other mutants in endocytosis and vacuolar protein sorting

As Mos10p localizes to the late endosome, the question arose whether proteins required for early steps in endocytosis, such as End3 and End4 (Raths et al., 1993; Benedetti et al., 1994), or at the intersection of endocytosis and vacuolar protein sorting, such as Vps21 (Horazdovsky et al., 1994) are required for normal filament maturation.

End3 is part of a protein complex required for endocytosis and for normal cortical actin patch formation (Benedetti et al., 1994; Tang et al., 1997; Tang et al., 2000). End4 is also required for the internalization step of endocytosis, as well as for polarization of the cortical actin cytoskeleton (Holtzman et al., 1993) and actin nucleation (Li et al., 1995). Mutants in END3 and END4 were deficient in early filament formation: very few, short filaments were made; but the short projections formed, were able to invade the agar (Fig. 7E–H). Filament maturation could not be assessed in these defective filaments.

image

Figure 7. Defective early filamentous growth of mutants in END3 and END4, genes required for endocytosis. Strains were streaked on 100 mm plates of SLAD28, and photographed at one week. The same colonies are shown for each strain in left and right images, at different magnifications. Both bars are 100 µm. A and B. JKY573<pRS316>. C and D. JKY228<pRS316>. E and F. JKY989<pRS316>. G and H. JKY991<pRS316>.

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The GTPase VPS21 is required for vesicle fusion at the intersection of the late endocytic, and the vacuolar protein sorting pathways (Horazdovsky et al., 1996; Gerrard et al., 2000). A deletion mutant in VPS21 was constructed. Young vps21/vps21 colonies appeared hyperfilamentous, but lateral yeast growth occurrred on vps21/vps21 maturing pseudohyphae as on wild type. A mutant in the ubiquitin isopeptidase DOA4, which is required for ubiquitin homeostasis (Swaminathan et al., 1999; Amerik et al., 2000; Dupre and Haguenauer-Tsapis, 2001) and therefore for vacuolar protein sorting by the ESCRT-III complex (Babst et al., 2002), had a barely discernible defect in filament maturation, which in the round cell quantification assay did not reach statistical significance (not shown).

General disruption of endocytosis or vacuolar protein sorting thus does not result in the filament maturation defect seen in the mos10/mos10 mutant.

The mos10/mos10 phenotype is unique among its family of homologues

The MOS10 gene was first described as stabilising the normally short-lived ABC transporter Ste6, when overexpressed. The deletion gives rise to a class E vacuolar protein sorting phenotype (Kranz et al., 2001). The Mos10 protein co-fractionates with the endosomal marker Pep12p, suggesting an endosomal localization of Mos10p. The authors identified two homologues which share the class E vacuolar protein sorting phenotype, and share coiled coil domains in the predicted protein structure: SNF7 and VPS20 (Kranz et al., 2001). These three proteins, Mos10, Snf7 and Vps20, form one of the two branches of a phylogenetic tree comprising the yeast homologues of human CHMP4, CHMP5 and CHMP6 (CHarged Multivesicular body Protein) (Howard et al., 2001). The other branch is formed by Did2, Did4 and Vps 24 (Babst et al., 1998; Amerik et al., 2000). Vps20 and Snf7 have been shown to form a subcomplex within the ESCRT-III protein complex, which completes the final step of sorting proteins destined for the vacuolar lumen into the vesicles of the multivesicular body (Babst et al., 2002). These authors suggest Mos10 and Did2 may have a partially redundant, or a regulatory role, in ESCRT-III activity.

To determine whether the filamentation phenotype of mos10/mos10 is shared by its homologues, deletion mutants of SNF7 and VPS20 were constructed and tested for filamentous growth. The snf7/snf7 mutant was severely defective in early filament formation (Fig. 8E and F). The vps20/vps20 mutant formed some filaments at the surface of SLAD28 agar, but only very few filaments invaded the agar (Fig. 8G and H). Each of these three members of this family of homologues thus had a distinct phenotype of filamentous growth.

image

Figure 8. Filament maturation phenotype of mutants in the MOS10 homologues VPS20 and SNF7. Strains were streaked on SLAD28 medium and photographed at three weeks. The same colonies are shown for each strain in left and right images, at different magnifications. Both bars are 100 µm. A and B. JKY482. C and D. JKY223. E and F. JKY763<pRS316>. G and H. JKY897.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Maturation of filaments, like early filamentous growth, has characteristic features

Early invasive filamentous growth of S. cerevisiae is a characteristic process involving multiple developmental changes, which were delineated in (Gimeno et al., 1992). Budding is unipolar, permitting cells to form chains. Chains of cells grow into the agar as well as on its surface, expanding the accessible nutrient sources into three dimensions. Cells are elongated, increasing chain length per cell division. Growth is directional, away from the mass of cells at the centre of the colony giving the organism access to new sources of nutrients. Mother cell division is delayed or suppressed, increasing projection length per number of cell divisions.

Filament maturation consists in reversal of some, but not all, of the components of the filamentous developmental programme. Elongation, directional growth, and mother cell budding suppression are reversed, whereas budding remains unipolar and invasive growth continues. Like the filamentous growth programme, the programme of filament maturation is not executed uniformly throughout all filaments of an inoculum but its features are just as regular. Growth of oval or round cells on an invasive filament, whose mothers bud before daughters and whose growth direction is random, are here termed lateral yeast growth; in (Gimeno et al., 1992), this growth was already noted and called ‘production of blastospore-like cells by . . . pseudohyphal cells’.

Lateral yeast cells on maturing filaments may be the functional equivalent of conidia

Filamentous fungi as evolutionarily distant as the Zygomycotina and the Ascomycotina produce non-filamentous cells, some directly on their filaments, like Trichophyton mentagrophytes and C. albicans, and many on specialized sporangia or conidiophores, like the Mucor and Aspergillus spp. Commonly, fungi use these round or oval cells to facilitate long-distance travel. Whereas the filamentous mycelium explores a substrate and provides a large surface area for absorption of nutrients, airborne sporangiospores or conidia are easily dispersed, and can provide access to completely new substrates for the organism. In the case of C. albicans, the mycelium is suited to penetrating the intestinal lining of an immunocompromised host, whereas the yeast forms growing on these hyphae are more easily carried to distant organs by the bloodstream, e.g. to produce hepatosplenic candidiasis.

I speculate that the lateral yeast growth on S. cerevisiae filaments fulfills similar functions: as wine yeast is sufficiently widespread on fruit in nature, to immediately begin fermenting crushed fruit or juice without being intentionally inoculated, S. cerevisiae must have dispersal forms that permit its ubiquitous spread onto potential substrates. Whereas the pseudohyphal mycelium is likely the form by which the organism penetrates semi-solid sources of nutrition such as ripe fruit, the lateral yeast cells produced from these filaments may be the form by which, in air or rainwater, it is dispersed to new substrates.

A screen for filament maturation mutants yields mutants in the two major cellular pathways of protein turnover

Insight into the regulation of filament maturation was sought by screening for mutants which fail to cover their pseudohyphae with lateral yeast. Pseudohyphal growth does not occur in the S288c strain background (Liu et al., 1996), which was used to create a collection of deletion strains for all open reading frames (Winzeler et al., 1999). Therefore, a protocol was developed to create a pool of homozygous diploid mutants in the Σ1278b strain background, utilizing a Tn3 transposon mutagenised genomic library (Burns et al., 1994), and the Ho endonuclease. One isolate with the transposon insertion in MOS10, and nine isolates with seven insertions in different sites of the PRE9 open reading frame, were identified.

In early filamentous growth, none of the known single mutations in regulatory pathways – the filamentation MAP kinase pathway, the PKA pathway and the pathway that signals to PHD1– completely abolishes filamentation. Similarly in this work, single-gene mutants were found whose lateral yeast growth on filaments is reduced, but not completely abolished. This suggests multiple layers of regulation of these complex developmental programs.

Unlike in investigations of early filamentous growth, in this work genes were identified whose products function in the two major cellular pathways of protein trafficking and turnover, the vacuolar and the proteasomal pathway. Pre9 is an α-subunit of the 20S proteasome core (Emori et al., 1991). A functional link between Mos10 and Pre9 in the regulation of filament maturation might be deduced from an analogy to mammalian receptor downregulation via endocytosis and degradation of activated receptors. This process is critical to return cells to their basal, unstimulated state after ligand binding. In yeast, degradation of surface proteins, such as the α factor receptor Ste2, to date is only known to occur in the vacuole (reviewed in Rotin et al., 2000). In mammalian cells, both the lysosome, equivalent to the vacuole, and the proteasome have been implicated in downregulation of activated receptors. For example, there is evidence for proteasomal degradation of the growth hormone receptor cytosolic tail, and lysosomal degradation of its lumenal portion (van Kerkhof and Strous, 2001). A speculation resulting from this work might be, that in a pseudohyphal colony, a molecule that responds to conditions specific to young filaments, needs to be degraded in order for maturation to occur; the vacuole and the proteasome may both be involved in this process.

mos10/mos10 represents a novel phenotype: defective filament maturation

The mos10/mos10 phenotype could due to disruption of a developmental programme of filament maturation. Alternative models are, that this mutant is locked into a filamentous state, or that it is a variant of hyperfilamentous mutants.

The prototype for a mutant locked into a filamentous form, is the C. albicans tup1/tup1 mutant, which forms no normal yeast cells under any growth conditions tested (Braun and Johnson, 1997). Seeking to determine whether the mos10/mos10 mutant is similarly incapable of responding to standard signals for yeast-form growth, I found, to the contrary, that it readily and immediately produces oval to round cells when placed into appropriate conditions of ample nutrients (Fig. 4).

In order to test whether the mos10/mos10 filament maturation phenotype is due to a hyperfilamentous state, I compared its growth features with those of known hyperfilamentous mutants and alleles. Neither in haploid invasive growth, nor in early diploid pseudohyphal growth, does the mutant in MOS10 resemble hyperfilamentous strains (Fig. 2A and Fig. 5). Conversely, maturing filaments of hyperfilamentous strains do not resemble mos10/mos10. Round cell growth on filaments of the hyperfilamentous strains approaches wild type by four weeks, in three of the four strains tested (Fig. 5I and J). The exception is wild type expressing Ras2Val19: it has almost no lateral yeast growth on filaments for four weeks. The Ras2Val19 strain appears to represent the case of a consistently hyperfilamentous state at every time point; however, its filament maturation phenotype must be mediated through as yet unrecognized molecules, because mutations in proteins known to function downstream of Ras2 in filamentous growth, Tpk2 and Sfl1, do not affect filament maturation (Fig. 6).

image

Figure 6. Maturation of filaments of mutants in SFL1 and TPK2, and their double mutants with MOS10. Prototrophic strains were grown on SLAD28 100 mm plates for three weeks. The same colonies are shown for each strain in the left and right images, at different magnifications. Both bars are 100 µm. A and B. JKY573<pRS316>. C and D. JKY228<pRS316>. E and F. LRY829 <pRS315, pRS316>. G and H. JKY803<pRS316>. I and J. LRY624 <pRS316>. K and L. JKY805<pRS316>. M. The proportion of round cells on invasive filaments of the strains shown in A–L was quantified at three weeks of growth. Four colonies per strain were analysed, and 200 cells per colony were counted. Error bars show standard deviation.

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The mos10/mos10 filament maturation defect cannot be attributed to general disruption of endosomal or vacuolar trafficking

As Mos10 may function in the sorting of cargo into incipient vesicles of the multivesicular body, various steps of endocytosis and vacuolar protein sorting were investigated to determine whether the mos10/mos10 defect is due to non-specific perturbation of these processes.

End3 and End4 are required for the internalization step of early endocytosis. End3 is part of an ‘EH domain’ complex involved in regulation of endocytosis, which through another member of the complex, Pan1, binds to the actin-regulating Arp2/3 complex (Raths et al., 1993; Wendland and Emr, 1998). End4 and an activator of the Arp2/3 complex have redundant functions in endocytosis (Wesp et al., 1997); thus the function of both these proteins in endocytosis is linked to the actin cytoskeleton. Deletion mutants in these two genes resemble each other and differ dramatically from mos10/mos10. They form very sparse, short filaments which, however, are able to invade the agar (Fig. 7E–H).

A mutant in the small GTPase VPS21, which is required for vesicle fusion and whose loss of function severely affects vacuolar targeting of the model cargoes carboxypeptidase Y and proteinase A (Horazdovsky et al., 1994) is slightly hyperfilamentous but the filaments mature normally. The deubiquitinating enzyme Doa4 plays a central role in endocytosis, multivesicular body formation, and proteasomal degradation, because it recycles ubiquitin moieties that target proteins for these processes, and thereby maintains free ubiquitin levels for these processes to continue (Swaminathan et al., 1999; Amerik et al., 2000; Katzmann et al., 2001; Dupre and Haguenauer-Tsapis, 2001; Losko et al., 2001). Deletion of DOA4 has a subtle effect on filament maturation, which does not reach statistical significance in the assay of round cell growth on filaments (not shown).

The effect of loss of MOS10 is therefore due neither to a general defect in vacuolar protein sorting, nor to a general disruption of endocytosis. The specificity of the filament maturation defect to the mos10/mos10 mutant is consistent with specific cargo of Mos10 associated vesicles, required for normal filament directional growth and maturation.

The mos10/mos10 filament maturation defect is unique among its homologues

MOS10 belongs to a family of six proteins, which are structurally related, and whose mutants have class E vacuolar protein sorting phenotypes. The members of this protein family all have human homologues, CHMP1–6, and comprise two divergent subgroups (Howard et al., 2001). Together with Mos10, the two other members of its subgroup, Snf7 and Vps20, were shown by Kranz and colleagues to be required for normal trafficking of Ste6p from the plasma membrane through the endocytic pathway to the vacuole (Kranz et al., 2001). However, these authors also noted differing growth characteristics of the three homologues: vps20 and snf7 mutants are unable to grow at 37°C, and grow poorly on raffinose as a carbon source, whereas mos10 cells grow at 37°C like wild type, and utilize raffinose normally. Concurrently, Forsberg and colleagues found a mutation in VPS20 to suppress the effect of amino acid uptake disruption in mutants of the amino acid sensors SSY1 and PTR3 (Forsberg et al., 2001). The suppressors isolated by these authors included other genes involved in ubiquitin-dependent endocytosis, such as RSP5 and BUL1 (Yashiroda et al., 1996; reviewed in Rotin et al., 2000), and in late endosomal protein sorting, such as DOA4 and VPS36 (Luo and Chang, 1997), but only one member of the MOS10 subfamily: VPS20. Incomplete saturation of the screen could of course account for VPS20, but not MOS10 and SNF7 having been isolated by these authors. Alternatively, Vps20, but not Mos10 or Snf7, may be required for normal turnover of the amino acid permeases involved in suppressing the ssy1 and ptr3 mutations, consistent with the idea that the sorting function of the Mos10 subfamily members is partially specific to distinct cargo proteins. My results agree with the findings of these two groups, in that the filamentation phenotypes of mutants in the two other subfamily members, SNF7 and VPS20, differ drastically from that of MOS10: mutants in SNF7 and VPS20 make only very short filaments which barely invade the agar (Fig. 8E–H).

Snf7 and Vps20 have been characterized in detail as components of the ESCRT-III complex, which sorts endosomal cargo into budding vesicles of the multivesicular body, for eventual discharge into the vacuolar lumen, and degradation (Babst et al., 2002). These authors also found that mos10 (vps60) mutants differ from mutants in SNF7 and VPS20: general trafficking defects are less severe in the former, and membrane association of the ESCRT-III complex is not blocked. Babst et al. (2002) discuss the possibility that Mos10 has a regulatory role in ESCRT-III activity. In agreement with this concept, one could speculate that Mos10 differs from Snf7 and Vps20, in that it acts on specific cargoes that are directed into the multivesicular body for vacuolar degradation only under certain conditions of development, which prevail during filament maturation. Such a role for Mos10 would have remained inaccessible to investigation under the conditions of yeast-form growth and replete nutrients, conventionally used to study endosomal protein sorting.

mos10/mos10 filaments do not respond appropriately to their context

Unlike that of the Ras2Val19 strain, the mos10/mos10 mutant's morphogenesis differs from wild type only in a circumscribed developmental stage: in filament maturation. In particular, the normal response to high cell density appears to be impaired.

On wild-type pseudohyphae, production of lateral yeast cells begins in the area of highest cell density, at the filament base. In contrast, production of lateral yeast cells on mos10/mos10 pseudohyphae is not only quantitatively decreased, but is also mislocalized, because most lateral yeast growth occurs near the filament tip (Fig. 3B). Whereas wild-type pseudohyphae direct their growth away from the mass of cells until growth direction becomes randomized during filament maturation (Supplementary material, Fig. S1), mos10/mos10 pseudohyphae initiate new foci of filaments which grow towards, as well as away from, the mass of cells (Supplementary material, Fig. S2). This work showed no evidence for a second mutation in the new foci, so that perhaps Mos10p itself acts in the normal response to high cell density.

A simple model is that maintenance of the early filamentous growth programme of a young pseudohypha could require a cell surface receptor, which is endocytically downregulated in the context of high cell density. In this model, mos10/mos10 pseudohyphae are unable to respond to high cell density with formation of lateral yeast cells and consistent direction of growth away from the colony centre, because downregulation of the filamentation signalling programme is backed up at the step of receptor targeting for degradation. A signal for such a receptor could be nutrient depletion, a metabolic waste product (Lorenz et al., 2000), or an autocrine molecule like bacterial quorum sensing compounds (reviewed in Bassler, 2002). Downregulation of such a receptor may involve several layers of regulation, so that the lack of Mos10 can only result in a partial defect. A significant limitation of this study is that no molecules were identified which might play the role of such a receptor. Possible reasons for this include, that the screen was not saturated, that insertion of the Tn3 transposon used is non-random (Vidan and Snyder, 2001), or that there are two or more molecules with partially overlapping functions, which play such a role. Identification of a cell surface molecule, whose endosomal sorting requires Mos10, and which is downregulated on maturing filaments, will be critical toward testing this model.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Media and strains

Standard genetic methods and yeast media were used as described in (Guthrie and Fink, 1991). Incubation temperature was 30°C.

For analysis of the development of filamentous colonies, of the mos10/mos10 phenotype and for its comparison with other strains, a modified low nitrogen medium was used, which enhances early filamentous growth and slightly accelerates filament development. Called SLAD28, this medium differs from previously described SLAD (Gimeno et al., 1992) in that it contains 0.5% glucose and 12.5 mM (NH4)2SO4.

For daily photomicrography, over 3 weeks, of colony growth on 100 mm diameter agar plates, SLAS27 medium was used, which is identical to SLAD28 except that sucrose is substituted for glucose. On this medium, filaments are spaced out more at colony rims so that it is easier to distinguish single filaments.

Yeast strains are congenic to the Σ1278b background. Deletion mutants were constructed by replacement of the open reading frames with selectable markers (Lorenz et al., 1995; Longtine et al., 1998), and were confirmed by PCR at the 5′ and 3′ ends of the open reading frames. The MOS10 gene was cloned by colony hybridization from a S. cerevisiae genomic library (Thompson et al., 1993), and cloned into the KpnI/SacII sites of pRS316 and pRS426 (Sikorski and Hieter, 1989); these plasmids contain the genomic fragment from 273 bp upstream of the MOS10 start, to 1297 bp downstream of the MOS10 stop codon. Yeast strains and plasmids used in this study are shown in Tables 3 and 4.

Table 3. . Yeast strains used in this study.
StrainGenotypeReference/Source
10512-3CMATa ura3-52 leu2::hisG his3::hisGFink laboratory
10560-5AMATaFink laboratory
L6947MATa flo11::HIS3 his3::hisGGuo et al. (2000)
L6930MATa sfl1::HIS3 his3::hisGRobertson and Fink (1998)
JKY1027MATa mos10::HIS3 his3::hisGThis work
JKY482MATa/αThis work
JKY223MATa/α mos10::HIS3/mos10::HIS3 his3::hisG/his3::hisGThis work
JKY573MATa/α ura3Δ/ ura3ΔThis work
JKY228MATa/αmos10 ::HIS3/mos10::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisGThis work
LRY829MATa/α sfl1::HIS3/sfl1::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG leu2::hisG/leu2::hisGRobertson and Fink (1998)
LRY624MATa/α tpk2::TRP1/tpk2::TRP1 ura3-52/ura3-52 trp1::hisG/trp1::hisGRobertson and Fink (1998)
JKY803MATa/α mos10::LEU2/mos10::LEU2 sfl1::HIS3/sfl1::HIS3 ura3-52/ura3-52 his3::hisG/ his3::hisG leu2::hisG/leu2::hisGThis work
JKY805MATa/α tpk2::HIS3/tpk2::HIS mos10::LEU2/mos10::LEU2 ura3-52/ura3-52 his3::hisG/ his3::hisG leu2::hisG/leu2::hisGThis work
JKY763MATa/α snf7::his5MX6/snf7:: his5MX6 ura3Δ/ura3Δhis3::hisG/ηισ3::ηισΓ 
JKY897MATa/α vps20::his5MX6/vps20::his5MX6 his3::hisG/his3::hisGThis work
JKY902MATa/α doa4::kanRMX6/doa4::kanRMX6This work
JKY904MATa/α vps21::kanRMX6/vps21::kanRMX6This work
JKY989MATa/α end3::kanRMX6/end3::kanRMX6 ura3::hisG/ura3::hisGThis work
JKY991MATa/α end4::kanRMX6/end4::kanRMX6 ura3::hisG/ura3::hisGThis work
Table 4. . Plasmids used in this study.
PlasmidCharacteristicsReference/source
pRS315LEU2, CEN6, ARS H4, ampRSikorski and Hieter (1989)
pRS316URA3, CEN6, ARS H4, ampRSikorski and Hieter (1989)
pRS426URA3, 2µ, ampRChristianson et al. (1992)
YCp50-HOHO under its own promoter, URA3, CENRussell et al. (1986)
B2553STE12, URA3, 2µ, ampRFink laboratory
BHM256TEC1, URA3, 2µ, ampRMadhani and Fink (1997)
pSL1509STE11-4 (Thr596->Ile), URA3, CEN6, ampRStevenson et al. (1992)
B3414Ras2Val19, URA3, CEN6, ampRFink laboratory
pJK164MOS10, URA3, CEN6, ampRThis work
pJK165MOS10, URA3, 2µ, ampRThis work

Haploid invasive growth

Strains were grown over night in liquid YPD and diluted to a density of OD600 = 1. Five µl of each cell suspension were spotted on YPD. Plates were grown at 30°C for five days. They were then gently flooded with water, and placed on a shaker at 80 r.p.m. for one hour. Photomicrographs were taken before and after washing on a Leica MZFL III dissecting microscope with a Nikon Cool Pix 995 camera.

Development of filamentous colonies

Pseudohyphal development was followed with photomicrographs every 12 or every 24 h, on a Nikon Eclipse TE300 inverted microscope using phase-contrast optics. On 100 mm diameter SLAS27 plates, containing 30 ml medium, strains were streaked for single colonies, no more than four streaks per plate. Normal colony morphology can be observed in this manner, but abundant filaments, in many planes of the agar, quickly obscure each other from view. This difficulty is ameliorated only slightly on SLAS27 medium.

In order to follow the growth of single filaments, 35 mm diameter plates containing 1 ml SLAD28 agar medium were used. On these plates, whose bottom is partially replaced by a coverslip for better optical properties (MatTek Corporation, Ashland, MA), nutrient limitation results in filaments spaced singly, which can be followed individually over time; and in acceleration of the maturation process, so that characteristics of older filaments, which on 100 mm plates arise at three or four weeks of incubation, can be observed within days. Single cells on filaments can be followed over the course of several budding events, thus yielding information about budding patterns (budding patterns cannot be distinguished by calcofluor staining, because the days or weeks old cells of maturing filaments take up the dye diffusely, thus not permitting sufficient visualization of bud scars). Forty filaments in 35 mm plates were followed by 12- or 24-hourly photomicrographs over periods of between 6 and 22 days, on a Nikon Eclipse TE300 inverted microscope using DIC optics. 21 filaments were analysed in detail for growth characteristics.

Construction of a pool of homozygous diploid mutants

The strategy to create a pool of homozygous diploid mutants was to allow cells in isolated colonies of haploid mutants to switch mating type, having been transformed with the HO endonuclease. They then mated with their neighbouring cells, which were isogenic, provided neighbouring colonies did not touch. HO was then eliminated from the cells, so that further mating type switching could not occur.

Strain 10512–3C (MATa ura3–52 leu2::hisG his3::hisG) in the Σ1278b background was transformed with a transposon-mutagenised genomic library (Burns et al., 1994). 2 × 106 transformants were pooled. These haploid insertional mutants were transformed with plasmid YCp50-HO (HO under its own promoter CEN URA3), and four 10-fold dilutions of the transformants were plated on sc – ura plates to determine the optimal density of colonies, such that neighbouring colonies did not touch. The transformed cells were held on ice for 36 h until the optimal dilution, for a density of  500 colonies per large (150 mm) plate, could be read. They were then plated at that dilution.

During growth for 2.5 days, cells in colonies of haploids transformed with HO had time to switch mating type and to mate. Colonies were then replica-plated to sc – ura plates, in order to dilute out the untransformed background. After overnight incubation, they were replicaplated to sc + 5-fluoro-orotic acid, in order to select for loss of the HO-containing plasmid. After one day,  5 × 104 colonies were pooled and frozen.

The quality of the homozygous diploid transposon insertion pool was assessed by testing for diploids, capable of filamentous growth, before cells transformed with HO were plated; and by testing for haploids in the final pool.

I wanted to determine the percentage of transformants that had switched mating type and mated in the 36 h period on ice, between transformation and plating, thus leading to heterozygotes in the final pool because they could mate with neighbouring cells in the suspension. For this purpose, 2000 colonies were plated on SLAD from the transformant suspension. 1.6% of them grew as filamentous colonies. They had thus become diploid in the transformant suspension, and were assumed to be heterozygous. In order to estimate the fraction of haploid cells in the final pool, 2000 colonies were tested for the ability to mate: 2% of these mated with tester strains and had therefore remained haploid.

Screen for mutants defective in proliferation of lateral yeast cells on filaments

On SLAD plates supplemented with histidine and uracil, colonies of the mutant pool plated to a density of 200–500 colonies per 100 mm plate were incubated for 3 weeks. They were then screened visually under the microscope for colonies which had grown to average size and had normal length filaments, but had less lateral yeast cells covering the filaments. Colonies with growth defects were not picked, because their mutations were assumed to have pleiotropic effects. Isolates were streaked on SLAD + his + ura, together with a wild-type control, and were saved if they continued to show the phenotype at that point. A total of  78 000 colonies were screened.

In order to determine whether the phenotype was due to the transposon insertion, the insertion site was identified by random-primed PCR or by vectorette PCR (Botstein Laboratory Web Page). An exact deletion mutant of the open reading frame into which the transposon had inserted, was then constructed, and tested for the phenotype.

Phenotype of filament maturation

For comparison of maturing filaments of wild-type and mutant strains, and strains expressing hyperfilamentous alleles, strains were streaked on SLAD28 agar, not more than four streaks per plate. At appropriate times of incubation, representative filamentous colonies were photographed at 100- and 400-fold magnification on a Zeiss Telaval 31 inverted microscope with a 35-mm camera. Slides were scanned as Photoshop files, and processed in PHOTOSHOP.

Quantification of round, oval and long cells composing a filament, and composing the colony on the agar surface

Wild-type and mutant prototrophic strains, or strains carrying the same vector type, were streaked on SLAD28 plates, at four strains per plate, so that wild type and mutants were on the same plates. In order to determine the composition of filaments at various stages of maturation, filaments were cut out of SLAD28 agar with a hypodermic needle, from their base at the rim of their colony, to their tip, under a dissecting microscope. The resulting agar fragment was placed into a drop of 1 M sorbitol on a glass slide and chopped, so that the filament was broken into single cells and small fragments. Single cells, and the mother cells of mother-daughter pairs, were counted according to (Merson-Davies and Odds, 1989; Mösch and Fink, 1997). For several fields, photomicrographs were taken, and cell types were confirmed by measurement. For each strain, filaments from at least four colonies were analysed; for each colony, 200 cells were counted. At 3 and 4 week time points, cells with irregular or polygonal shapes appeared; these were not counted.

To determine cell types in the colony centre, on the agar surface, filaments were cut out as described above. When only the centre of the colony remained standing, it was sliced off as a ∼ 1 mm slice with the hypodermic needle, and surface cells were suspended in a drop of 1 M sorbitol on a glass slide, by gently touching the slice to the liquid (Supplementary material, Fig. S3).

A high-copy URA3 vector decreased lateral yeast growth on filaments. Therefore, in each quantification experiment, the filament composition of the wild-type strain, complemented with the same vector type, from the same experiment, was used as the control.

Inducing the switch from filamentous to lateral yeast growth in rich medium

Two day old colonies on SLAD agar were washed to remove surface cells. Entire colonies were then excised from the medium with a needle, fragmented into single cells, and placed into liquid YPD or sc − ura medium or onto YPD agar plates. Photomicrographs were taken on a Nikon Eclipse TE300 inverted microscope immediately after placing the cells into rich liquid medium, and at 6.5 h of growth, at which time most daughters of the original filamentous mother had budded once.

Length and width of wild-type and mos10/mos10 cells were measured as pixels in Openlab 2.2.5 software (Improvision), for two cell types: filamentous cells immediately after isolation from SLAD28; and first buds of filamentous mothers, which had budded once themselves, at 6.5 h growth in rich medium.

Statistics

Statistical calculations for cell measurements, and for quantification of cell types in filaments, were performed in Microsoft Excel. Values for probability of difference being due to chance, were obtained by two-tailed t-test, assuming unequal variance.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

I thank David D. Miller for support and encouragement. I thank Gerald R. Fink and the members of the Fink Laboratory for many helpful discussions. Thanks also to Carlos Gimeno, Todd Milne and Steve Helliwell. I thank Mike Lorenz, Simon Dove, Martin Dünnwald, Ifat Rubin-Bejerano, Tim Galitski, Todd Reynolds and Reeta Prusty for critical readings of the manuscript. I am grateful to Rick Young for the generous gift of the genomic S. cerevisiae library, and to Todd Milne for yeast strains of the Sigma 2000 series in the Σ1278b background. Micrographs of the development of filaments, and the switch to yeast form growth in liquid medium, were taken utilising the W. M. Keck Foundation Biological Imaging Facility at the Whitehead Institute; thanks to Nicki Watson and Barry Alpert for suggestions on photomicrography. This work was supported by NIH K08 A101528.

Supplementary material

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Fig. S1. Development of filamentous colony. Wild-type and mos10/mos10 strains were streaked for single colonies on a 100 mm diameter SLAS27 plate. A colony from each strain was photographed every 24 h on an inverted microscope. The figure shows a colony of JKY 482 (wild type).

Fig. S2. Development of filamentous colony. Wild type and mos10/mos10 strains were streaked for single colonies on a 100 mm diameter SLAS27 plate. A colony from each strain was photographed every 24 h on an inverted microscope. The figure shows a colony of JKY 223 (mos10/mos10).

Fig. S3. Process of cutting out filaments for cell type quantitation. Invasive filaments of a 3 week old colony of JKY223 (mos10/mos10) on SLAD28 were cut from the agar with a hypodermic needle under a dissecting microscope. Finally, the centre of the colony, where cells lie on the agar surface, was cut off. The agar surface was then touched into a drop of sorbitol to release the cells lying on it.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Fig. S1. Development of filamentous colony. Wild-type and mos10/mos10 strains were streaked for single colonies on a 100 mm diameter SLAS27 plate. A colony from each strain was photographed every 24 h on an inverted microscope. The figure shows a colony of JKY 482 (wild type).

Fig. S2. Development of filamentous colony. Wild type and mos10/mos10 strains were streaked for single colonies on a 100 mm diameter SLAS27 plate. A colony from each strain was photographed every 24 h on an inverted microscope. The figure shows a colony of JKY 223 (mos10/mos10).

Fig. S3. Process of cutting out filaments for cell type quantitation. Invasive filaments of a 3 week old colony of JKY223 (mos10/mos10) on SLAD28 were cut from the agar with a hypodermic needle under a dissecting microscope. Finally, the centre of the colony, where cells lie on the agar surface, was cut off. The agar surface was then touched into a drop of sorbitol to release the cells lying on it.

FilenameFormatSizeDescription
MMI_3556_sm_FigS1.ppt1344KSupporting info item
MMI_3556_sm_FigS2.ppt1494KSupporting info item
MMI_3556_sm_FigS3.tif45473KSupporting info item

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