Article |
Address correspondence to Peter Sudbery, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN UK. Tel.: 44-114-222-6186. Fax: 44-114-272-8697. email: psudbery{at}shef.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: basal septin band; germ tube; hypha; septin ring; SWE1
The online version of this article contains supplemental material.
Steven Bates's present address is University of Aberdeen, Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD UK.
Abbreviations used in this paper:
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C. albicans yeast cells can be induced to form hyphae or pseudohyphae by a variety of environmental signals such as the presence of serum, 3537°C growth temperature, and neutral pH (Odds, 1985; Sudbery, 2001). The presence of serum at a growth temperature of 35°C, pH 6, results in a culture consisting almost entirely of the hyphal form, whereas in the absence of serum at 35°C, pH 6, cells develop as long pseudohyphae (Sudbery, 2001). Although pseudohyphae superficially resemble true hyphae, it is becoming increasingly clear that there are fundamental differences between the two forms in the organization of the cell cycle and the septin cytoskeleton (Sudbery, 2001). In the development of pseudohyphae, a septin ring forms at the junction between the mother cell and the daughter cell. The first mitosis and formation of the primary septum take place across the plane of this septin ring. Thus, the daughter cell behaves as a bud, modified by hyperpolarized growth. In hyphal development, germ tube evagination occurs before the cell cycle has initiated in the mother cell (Hazan et al., 2002). A band of longitudinal septin bars forms at the base of the germ tube, which later becomes faint and disorganized (Sudbery, 2001). A cap of septin is also present at the germ tube tip (Warenda and Konopka, 2002). A septin ring then appears within the germ tube, 1015 µm from the mother cell, probably marking the initiation of the cell cycle. The nucleus migrates out of the mother cell and the first mitosis takes place across the plane of this septin ring. After mitosis, the septin ring organizes the formation of the primary septum (for review see Berman and Sudbery, 2002). Throughout this paper the band of septin at the base of the germ tube will be referred to as the "basal septin band" and the ring within the germ tube, which organizes the formation of the primary septum, will be referred to as the "septin ring".
In S. cerevisiae, septin rings at the bud neck provide a scaffold for proteins that are required for cytokinesis (for review see Longtine and Bi, 2003). The proper organization and function of the septin ring requires Gin4, a homologue of the Nim1 kinase of Schizosaccharomyces pombe (Russell and Nurse, 1987). In a gin4 mutant, septin is deposited in a series of longitudinal bars around the bud neck instead of a continuous ring (Longtine et al., 1998a, 2000). Gin4 localizes to the bud neck and this localization is septin dependent (Okuzaki et al., 1997; Longtine et al., 1998b; Barral et al., 1999). Therefore, the function and localization of septins and Gin4 are mutually interdependent. Mutations that disrupt septin organization cause an impairment of cytokinesis and bud elongation. This is due to the induction of the morphogenesis checkpoint that delays mitosis and prevents a switch from polarized to isometric growth of the bud (Lew and Reed, 1993, 1995a). The morphogenesis checkpoint is dependent on Swe1, the S. cerevisiae homologue of the S. pombe Wee1 kinase (Booher et al., 1993), which inhibits Cdc28 by phosphorylation of tyrosine 19 (Sia et al., 1996). A swe1
mutation abrogates the morphogenesis checkpoint in a temperature-sensitive cdc3 mutant and consequently exacerbates its growth defect (Barral et al., 1999). The morphogenesis checkpoint operates through control of Swe1 stabilization. When first synthesized in G1, Swe1 accumulates in the nucleus. After bud formation it is targeted to the mother side of the bud neck and it is degraded in the G2/M phase (McMillan et al., 1999). Swe1 also exerts a cell size checkpoint over the onset of mitosis (Harvey and Kellogg, 2003). This observation has given rise to an alternative model to the morphogenesis checkpoint in which it is posited that abnormally prolonged bud growth occurs in cells lacking Gin4 because the signal indicating that cell size has passed the critical size threshold is not transmitted to Swe1. Apart from Gin4, the S. cerevisiae genome encodes two other Nim1-kinase homologues, Hsl1 and Kcc4. These also localize to the bud neck in a septin-dependent fashion (Barral et al., 1999). However, septin organization is not dependent on either kinase (Longtine et al., 2000), although Hsl1 is required for the transmission of the checkpoint signal to Swe1.
The C. albicans genome contains a single homologue of the S. cerevisiae SWE1 gene and two Nim1-kinases homologous to S. cerevisiae GIN4 and HSL1, respectively. In this paper, we have investigated the role of these genes in the organization of the basal septin band and the septin ring in hyphal germ tubes of C. albicans. We show that Gin4 is required for the organization of the septin ring but not the basal septin band. Thus, the difference in organization between these two structures may be due to the action of Gin4. Hsl1 is not required for the organization of either structure. Unexpectedly, we found that both gin4 and hsl1
mutants constitutively form pseudohyphae and that these pseudohyphae are unable to form hyphae when challenged with serum. Furthermore, as wild-type cells develop pseudohyphae, the level of Gin4 declines and they also become unable to form hyphae upon serum challenge. However, high levels of Gin4, ectopically expressed from the MET3 promoter, overcome this block. Thus, in addition to organizing the septin ring, Gin4 appears to determine the developmental potential of pseudohyphae.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The gin4 mutant was viable, but grew in chains of elongated cells indicative of a severe defect in cytokinesis (Fig. 2 a). The failure to undergo cytokinesis resulted in a change in colony morphology from the smooth appearance of the wild-type colonies to a crenulated appearance in gin4
mutants characteristic of pseudohyphal formation in wild-type cells (Fig. 2, b and c). After the addition of serum, these cells were unable to form hyphae, which are defined by narrow parallel sides without constrictions (Fig. 2 d). Calcofluor white staining showed that the primary septum, composed of chitin, failed to form (Fig. 2 e). An identical phenotype was seen in the MET3-GIN4 strain when the MET3 promoter was turned off (Fig. 2, fh). Calcofluor staining showed that primary septa fail to form (Fig. 2 g). Furthermore, immunocytofluorescence, using polyclonal antisera raised against S. cerevisiae Cdc11, showed that septin rings failed to form properly (Fig. 2 h). Cells were indistinguishable from wild type when the MET3 promoter was turned on (Fig. 2, ik), indicating that the gin4
phenotype is due to loss of Gin4.
|
|
|
|
The pseudohyphal phenotype of gin4 cells is at least partially independent of Swe1
The pseudohyphal phenotype of the C. albicans gin4 strain could be due to the action of Swe1 acting to either exert a morphogenesis or cell size checkpoint. To address this issue, we constructed a MET3-GIN4 swe1
strain. When Gin4 was depleted in a swe1
mutant, cells were slightly less elongated than SWE1 cells (Table I), suggesting that some of the elongation was due to the action of Swe1. Further evidence for the operation of a Swe1-dependent checkpoint was a growth defect when Gin4 expression was turned off in swe1
MET3-GIN4 cells, that was not evident when Gin4 expression was turned off in SWE1 cells (Fig. 5 c). Together, these observations suggest that depletion of Gin4 triggers a Swe1-dependent checkpoint. However, SWE1 is not entirely responsible for the pseudohyphal phenotype observed because cells remained significantly elongated when GIN4 expression is turned off in swe1
strains. Furthermore, swe1
GIN4-off cells remained capable of agar invasion (Fig. 5 d). Thus, the pseudohyphal phenotype observed in gin4
cells is partly independent of a Swe1-dependent checkpoint.
|
|
To confirm that levels of Gin4 are elevated when ectopically expressed from the induced MET3 promoter, we tagged Gin4 with GFP in the MET3-GIN4 strain (MET3-GIN4-GFP) and also in the wild-type strain so that GIN4-GFP expression was under the control of the GIN4 promoter (GIN4-GFP). The capacity of these strains to form hyphae, after incubation as pseudohyphae, was similar to that observed in the previous experiment (Table S2, available at http://www.jcb.org/cgi/content/full/jcb.200307176/DC1). Cellular Gin4-GFP levels were monitored by Western blots using a mixture of two mAbs to GFP. During incubation as pseudohyphae, Gin4-GFP levels were much higher in the induced MET3-GIN4-GFP culture compared with the GIN4-GFP culture (Fig. 6 d). Thus, the continued capacity to form hyphae after incubation as pseudohyphae is associated with a high level of Gin4. The repressed MET3-GIN4-GFP culture also showed a rise in Gin4-GFP levels after 2 h, but by 6 h the levels had declined to that seen in the GIN4-GFP culture. Interestingly, Fig. 6 c shows that there is delay in this culture before the ability to form hyphae is lost, providing further confirmation that there is a correlation between Gin4 levels and the ability of pseudohyphae to form hyphae. Gin4 protein levels also show variation with growth form in cells expressing the protein from the native promoter. Gin4-GFP levels were lower in GIN4-GFP cells grown as pseudohyphae compared with the same cells grown as yeast (Fig. 6 d). Finally, we observed that Gin4 appeared to be phosphorylated in cells grown as yeast and cells overexpressing Gin4 from the MET3 promoter because there was a band shift in these samples, which disappeared after treatment with phosphatase (shown in Fig. 6 d for the yeast culture). We interpret these experiments as showing that high levels of Gin4 and/or phosphorylation are required for the transition from pseudohyphae to hyphae. Because Gin4 levels are low in pseudohyphae and Gin4 is not phosphorylated, the pseudohypha to hypha transition is normally blocked.
Gin4 is required for the formation of the septin ring but not the basal septin band
Hyphal germ tubes that formed in Gin4-depleted cells induced from the yeast state were examined to determine the pattern of septin ring formation by immunocytofluorescence using polyclonal antisera to S. cerevisiae Cdc11 (Cdc11; Sudbery, 2001). The appearance of basal septin bands and septin rings in representative cells is shown in Fig. 7 (ae) and quantified in Fig. 7 (h and i). The location of nuclei is also shown in Fig. 7 (ae). By 90 min, a septin ring had formed in 30% of germ tubes regardless of whether Gin4 was turned on or off (Fig. 7 i). In GIN4-off cells, the proportion declined to 10% by 180 min and all septin rings had completely disappeared by 210 min (Fig. 7 i) and primary septa failed to form (Fig. 7 f). In contrast, when GIN4 was turned on, the proportion of cells with a ring in the germ tube had increased to nearly 90% by 180 min (Fig. 7 i) and primary septa formed normally (Fig. 7 g). The first mitosis occurred at the normal time and in the normal position (Fig. 7 c) in GIN4-off cells. Thus, although in wild-type cells mitosis takes place across the plane of the septin rings (Fig. 7 a), the septin rings are not required for mitosis and do not determine the position at which mitosis will occur.
|
To monitor the level of residual Gin4 in MET3-GIN4-off cells, we repeated the experiment using the MET3-GIN4-GFP strain and monitored Gin4-GFP levels by Western blotting as described above. Hyphal germ tubes evaginated at an identical rate regardless of whether the MET3 promoter was turned on or off (Fig. 8 a). When the MET3 promoter was turned on, the Gin4GFP fusion caused a delay in the appearance of septin rings as they appeared between 240 and 270 min (compare Fig. 7 i with Fig. 8 b). Nevertheless, basal septin bands were apparent in newly evaginated germ tubes (Fig. 8 c) and persisted longer than in cells containing the untagged MET3-GIN4 (compare Fig. 8 c with Fig. 7 h). Thus, although septin ring formation was delayed by the Gin4GFP fusion, the appearance of basal septin bands was not affected. Again, nuclear migration and mitosis were not delayed and took place in the absence of septin rings (Fig. 8 d).
|
Gin4 localizes to the septin ring but not to the basal septin band
To further test the hypothesis that Gin4 is required for the germ tube ring, but not the basal septin band, we examined the localization of Gin4-GFP in yeast, pseudohyphae, and hyphal germ tubes. We found that it localizes to the bud neck of pseudohyphae (Fig. 9 a), yeast (Fig. 9 b), and to a ring in the germ tube consistent with the position of the septin ring (Fig. 9 e). In striking contrast, we never observed any Gin4 localization to the base of developing hyphae (Fig. 9 c) where the basal septin band forms (Fig. 9 d).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several observations suggest that gin4 mutants may form genuine pseudohyphae. First, gin4
cells vigorously invaded the agar substratum under conditions that do not stimulate invasive growth in wild-type cells. The extent of the invasion was at least as great as that observed during hypha and pseudohypha formation in wild-type cells, although the morphology of the invading structures was different. A cytokinesis defect was reported recently to result from mutations in genes encoding septins (Warenda and Konopka, 2002). These septin mutants are not constitutively invasive, in fact, they are defective in invasive growth (Warenda and Konopka, 2002). Therefore, invasiveness is not simply a consequence of the chains of cells that result from a failure of cytokinesis. Second, chains of elongated cells still formed in a swe1
mutant depleted of Gin4, although the extent of cell elongation was reduced. This observation suggests that although a gin4
mutation induces Swe1-dependent elongation, approximately half of the cell elongation is independent of Swe1. Moreover, these cells still invaded agar, thus agar invasiveness is also not dependent on Swe1. Third, gin4
mutants were unable to form true hyphae in response to serum. Again, this contrasts with septin mutants that formed germ tubes, although there were some abnormalities in their shape. Therefore, the failure to complete cytokinesis cannot explain the inability to respond to serum. Fourth, like gin4
mutants, hsl1
mutants also have a constitutive pseudohyphal phenotype, although septin ring organization is unaffected and septum formation takes place normally. Indeed, the hsl1
mutants were more convincingly pseudohyphal than gin4
cells, possibly because there was no additional complication of a failure to properly organize the septin ring and septate. Together, these results suggest that cells depleted of either Nim1 kinase become constitutively pseudohyphal and therefore that Nim1-kinases, Gin4 and Hsl1, act as negative regulators of pseudohyphal development.
Pseudohyphae are in a developmental state that precludes the formation of hyphae
Pseudohyphae that form because of either Gin4 or Hsl1 depletion cannot form hyphae. However, using the conditional MET3-GIN4 allele, we showed that when yeast cells are produced in the presence of Gin4, they are able to form hyphae when Gin4 is repressed during the serum challenge. One explanation of this observation is that the pseudohyphal state blocks the formation of hyphae. We tested this hypothesis using the conditional MET3-GIN4 allele. Yeast cells formed in the presence of Gin4 were incubated in pseudohyphae-inducing conditions for various times before being challenged to make hyphae by the addition of serum. Wild-type and MET3-GIN4 off cells lost the ability to develop into hyphae. This suggests that it is the pseudohyphal state that prevents Gin4-depleted cells from forming hyphae. However, MET3-GIN4 cells cultured in derepressing conditions retained the ability to form germ tubes. We used a GIN4GFP fusion to follow Gin4 levels during this experiment. The results of a Western blot using mAbs to GFP showed that these cells expressed high levels of Gin4 in a phosphorylated form. Thus, high levels of Gin4 and/or Gin4 phosphorylation overcomes the block that normally prevents pseudohyphae from forming germ tubes. This block may result from the low levels of Gin4 observed in wild-type pseudohyphae. The continued hyphal growth after Gin4 depletion in the MET3-GIN4 off culture shows that Gin4 is not required for the maintenance of the hyphal state. When wild-type cells are challenged to make hyphae in the yeast state when Gin4 levels are low, germ tubes quickly emerge, suggesting that high Gin4 levels are not required for the yeast-hyphal transition.
Gin4 is required for the organization of septin rings but not basal septin bands
Multiple lines of evidence strongly suggest that Gin4 is not involved in the formation of the basal septin band. First, basal bands resemble the striated appearance of the septin ring in S. cerevisiae gin4 mutants. Second, when GIN4 expression is turned off using the MET3 promoter, basal septin bands persist after there is insufficient Gin4 to maintain septin rings. The slow kinetics of Gin4 depletion do not allow us to completely rule out the possibility that Gin4 is required for the organization of the basal septin band on the basis of this experiment. However, as Gin4 was depleted, septin rings disappeared but basal septin bands persisted. Moreover, by 300 min Gin4 was undetectable, but second germ tubes formed that displayed a basal septin band, but no septin rings. Third, Gin4-GFP locates to septin rings, but not basal septin bands. Fourth, a Gin4-GFP allele delays the appearance of septin rings but not basal septin bands.
Septin rings are not required for nuclear migration and mitosis
Nuclear migration and mitosis occurred normally in MET3-GIN4-GFP cells and MET3-GIN4-off cells. In the latter case, mitosis also occurred normally in the second germ tube that formed in the mother cell when the MET3 promoter was turned off. None of the germ tubes displayed a septin ring when mitosis occurred. Thus, the septin ring is not required for mitosis and the position where mitosis occurs cannot be determined by the position of the septin ring. Importantly, the location of the septin ring cannot be determined by the position of mitosis, because in wild-type cells, the septin ring forms in the germ tube before nuclear migration commences (Sudbery, 2001). Therefore, the location of the septin ring and the position where mitosis occurs must be specified by independent markers.
Hyphae and pseudohyphae do not result from induction of the Swe1-dependent morphogenesis checkpoint
In S. cerevisiae, the Swe1-dependent morphogenesis checkpoint, which delays mitosis and prevents the switch from polarized to isotropic growth. This has led to the suggestion that the operation of the morphogenesis checkpoint is the mechanism by which hyphae and/or pseudohyphae form (Kron and Gow, 1995). We have tested this hypothesis by constructing a swe1 mutant. There was evidence that Swe1 is acting to trigger a morphogenesis checkpoint in C. albicans, because cells are longer in a gin4
mutant compared with a gin4
swe1
mutant. Moreover, swe1
mutants accumulate a small fraction of cells with two nuclei, which are never observed in wild-type cells. However, all aspects of hyphal and pseudohyphal formation are normal in a swe1
mutant and we conclude that the morphogenesis checkpoint is not important for hyphal or pseudohyphal growth. This is consistent with the recent report that there is no change in the amount or timing of tyrosine phosphorylation of Cdc28p in developing hyphae compared with yeast (Hazan et al., 2002). Furthermore, pseudohyphae form normally in an S. cerevisiae swe1
mutant (Ahn et al., 1999).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Western blot analysis of Gin4-GFP
Details of protein extraction procedures and antibodies used for Western blotting are provided in the supplemental material.
Microscopy
Immunocytofluorescence using Cdc11 (Santa Cruz Biotechnology, Inc.) was performed as described previously (Sudbery, 2001). Chitin was stained with calcofluor white (Sigma-Aldrich) as described previously (Sudbery, 2001). DNA was stained with DAPI. Cells were examined either with a fluorescence microscope (model DMLB; Leica) or a microscope (model DeltaVision Linux 4; Applied Precision Instruments). Digital images were captured by a CCD camera (model RTE; Princeton Instruments) controlled by an Apple Macintosh G4 computer running Open Lab software version 2.2.5 (Improvision). Images were exported as TIFF files and edited in Adobe Photoshop version 5.5. Images from the DeltaVision microscope were captured, deconvolved, and three-dimensional images were reconstructed using the Softworx software suite supplied with the microscope. Composite figures were assembled using Microsoft PowerPoint 2000.
Axial ratio measurements
Cell images were captured using a microscope (model DMLB; Leica) using DIC optics and the 100x objective. Cell length was determined by measuring the distance between the constrictions using Open Lab Version 2.2.5 software (Improvision). The width was determined at the widest part of the cell. The axial ratio is defined as length divided by width. In yeast cells, the axial ratio is reported for mother cells only as buds may still be growing in an apical fashion. In cells forming pseudohyphae in MET3-repressing conditions, only the penultimate cell in a chain of four or more cells was measured as these have completed their apical growth.
Cell size determination
The long and short axes of mother cells were measured as described in the previous paragraph. Cell volume was calculated according to the formula 4/3pab2 where a is the radius of the long axis and b is the radius of the short axis (Sudbery et al., 1980).
Online supplemental material
Supplemental material provides details of strain construction, and methods and antibodies used in Western blots. Table S1 specifies sequence of oligonucleotides. Table S2 provides quantitation of the proportion of each culture producing germ tubes in the experiment described in Fig. 6 d. Fig. S1 provides a Clustal alignment of the C. albicans and S. cerevisiae Nim1 kinases. Fig. S2 provides a Clustal alignment of the C. albicans and S. cerevisiae Swe1 kinases. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200307176/DC1.
![]() |
Acknowledgments |
---|
This work was funded by a project grant from the Wellcome Trust.
Submitted: 28 July 2003
Accepted: 10 December 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahn, S.H., A. Acurio, and S.J. Kron. 1999. Regulation of G2/M progression by the STE mitogen-activated protein kinase pathway in budding yeast filamentous growth. Mol. Biol. Cell. 10:33013316.
Altman, R., and D. Kellogg. 1997. Control of mitotic events by Nap1 and the Gin4 kinase. J. Cell Biol. 138:119130.
Barral, Y., M. Parra, S. Bidlingmaier, and M. Snyder. 1999. Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast. Genes Dev. 13:176187.
Berman, J., and P.E. Sudbery. 2002. Candida albicans: A molecular revolution built on lessons from budding yeast. Nat. Rev. Genet. 3:918930.[CrossRef][Medline]
Booher, R.N., R.J. Deschenes, and M. Kirschner. 1993. Properties of Saccharomyces cerevisiae wee1 and its differential regulation in response to G1 and G2 cyclins. EMBO J. 12:34173428.[Abstract]
Brown, A.J.P. 2002. Morphogenic signalling pathways in Candida albicans. Candida and Candidiasis. R. Calderone, editor. American Society for Microbiology, Washington, DC. 95106.
Brown, A.J.P., and N.A.R. Gow. 1999. Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol. 7:333338.[CrossRef][Medline]
Care, R.A., J. Trevethick, K.M. Binley, and P.E. Sudbery. 1999. The MET3 promoter: a new tool for Candida albicans molecular genetics. Mol. Microbiol. 34:792798.[CrossRef][Medline]
Gerami-Nejad, M., J. Berman, and C. Gale. 2001. Cassettes for PCR-mediated construction of green, yellow and cyan fluorescent protein fusions in Candida albicans. Yeast. 18:859880.[CrossRef][Medline]
Gow, N.A.R. 1997. Germ tube growth of Candida albicans. Curr. Top. Med. Mycol. 8:4355.[Medline]
Harvey, S.L., and D.R. Kellogg. 2003. Conservation of mechanisms controlling entry into mitosis: budding yeast Wee1 delays entry into mitosis and is required for cell size control. Curr. Biol. 13:264275.[CrossRef][Medline]
Hazan, I., M. Sepulveda-Becerra, and H.P. Liu. 2002. Hyphal elongation is regulated independently of cell cycle in Candida albicans. Mol. Biol. Cell. 13:134145.
Kron, S.J., and N.A.R. Gow. 1995. Budding yeast morphogenesis: signaling, cytoskeleton and cell-cycle. Curr. Opin. Cell Biol. 7:845855.[CrossRef][Medline]
Lew, D.J., and S.I. Reed. 1993. Morphogenesis in the yeast cell cycle: Regulation by Cdc28 and cyclins. J. Cell Biol. 120:13051320.[Abstract]
Lew, D.J., and S.I. Reed. 1995a. A cell-cycle checkpoint monitors cell morphogenesis in budding yeast. J. Cell Biol. 129:739749.[Abstract]
Lew, D.J., and S.I. Reed. 1995b. Cell cycle control of morphogenesis in budding yeast. Curr. Opin. Genet. Dev. 5:1723.[Medline]
Liu, H.P. 2001. Transcriptional control of dimorphism in Candida albicans. Curr. Opin. Microbiol. 4:728735.[CrossRef][Medline]
Lo, H.J., J.R. Kohler, B. DiDomenico, D. Loebenberg, A. Cacciapuoti, and G.R. Fink. 1997. Nonfilamentous C. albicans mutants are avirulent. Cell. 90:939949.[Medline]
Longtine, M.S., and E.F. Bi. 2003. Regulation of septin organization and function in yeast. Trends Cell Biol. 13:403409.[CrossRef][Medline]
Longtine, M.S., H. Fares, and J.R. Pringle. 1998a. Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function. J. Cell Biol. 143:719736.
Longtine, M.S., A. Mckenzie, D.J. Demarini, N.G. Shah, A. Wach, A. Brachat, P. Philippsen, and J. Pringle. 1998b. Additional modules for versatile and economical PCR-based gene deletions and modification in Sacharomyces cerevisiae. Yeast. 14:953961.[CrossRef][Medline]
Longtine, M.S., C.L. Theesefield, J.N. McMillan, E. Weaver, J.R. Pringle, and D.J. Lew. 2000. Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae. Mol. Cell. Biol. 20:40494061.
Martínez-Anaya, C., J.R. Dickinson, and P.E. Sudbery. 2003. In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint. J. Cell Sci. 116:34233431.
McMillan, J.N., M.S. Longtine, R.A.L. Sia, C.L. Theesfeld, E.S.G. Bardes, J.R. Pringle, and D.J. Lew. 1999. The morphogenesis checkpoint in Saccharomyces cerevisiae: Cell cycle control of Swe1p degradation by Hsl1p and Hsl7p. Mol. Cell. Biol. 19:69296939.
Merson-Davies, L.A., and F.C. Odds. 1989. A morphology index for cell shape in Candida albicans. J. Gen. Microbiol. 135:31433152.[Medline]
Odds, F.C. 1985. Morphogenesis in Candida albicans. Crit. Rev. Microbiol. 12:4593.[Medline]
Okuzaki, D., S. Tanaka, H. Kanazawa, and H. Nojima. 1997. Gin4 of S. cerevisiae is a bud neck protein that interacts with the Cdc28 complex. Genes Cells. 2:753770.
Okuzaki, D., T. Watanabe, and H. Nojima. 2003. The Saccharomyces cerevisiae bud neck proteins Kcc4 and Gin4 have distinct, but partially overlapping cellular functions. Genes Genet. Syst. 78:113126.[CrossRef][Medline]
Russell, P., and P. Nurse. 1987. The mitotic inducer nim1+ functions in a regulatory network of protein-kinase homologs controlling the initiation of mitosis. Cell. 49:569576.[Medline]
Sia, R.A.L., H.A. Herald, and D.J. Lew. 1996. Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast. Mol. Biol. Cell. 7:16571666.[Abstract]
Sudbery, P.E. 2001. The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localisation. Mol. Microbiol. 41:1931.[CrossRef][Medline]
Sudbery, P.E., A.R. Goodey, and B.L.C. Carter. 1980. Genes that control cell proliferation in the yeast Saccharomyces cerevisiae. Nature. 288:401404.[Medline]
Warenda, A.J., and J.B. Konopka. 2002. Septin function in Candida albicans morphogenesis. Mol. Biol. Cell. 13:27322746.
Wilson, B., D. Davis, and A.P. Mitchell. 1999. Rapid hypothesis testing in Candida albicans through gene disruption with short homology regions. J. Bacteriol. 181:18681874.
Zhang, J., C. Schneider, L. Ottmers, R. Rodriguez, A. Day, J. Markwardt, and B.L. Schneider. 2002. Genomic scale mutant hunt identifies cell size homeostasis genes in S. cerevisiae. Curr. Biol. 12:19922001.[CrossRef][Medline]