Genetic Interactions among Regulators of Septin Organization

Yeast strains.The S. cerevisiae strains used for this study are listed in Table 1. All of the strains were in the BF264-15Du strain background (ade1 his2 leu2-3,112 trp1-1 ura3Δns), which has frequently been used for cell cycle studies and morphogenesis studies by the Reed, Cross, Wittenberg, Lew, and other laboratories (27). This strain grows robustly at temperatures up to 39°C. The generation of cdc42::GAL1p-CDC42::LEU2 (14), cdc42^V36T,K94E, cdc42^Y32H, and rga2::URA3 (13), rga1::TRP1 and bem3::LEU2 (4), cla4::TRP1 (3), gin4-Δ9 (24), nap1::Kan^r (25), and elm1::URA3 (6) alleles was described previously. Standard media and methods were used for yeast manipulations (16), and all multiple mutant combinations were generated by crosses between mutants in the same strain background. Plasmids pMOSB55, -56, and -57 are CEN URA3 vectors containing CDC42, cdc42^Y32H, and cdc42^V36T,K94E mutants, respectively. Expression was driven from the CDC42 promoter (26).

Microscopy.Fixation, septin staining by standard indirect immunofluorescence with commercially available anti-Cdc11p antibodies, and microscopic analysis were performed as previously described (14).

Septin phenotypes in single-mutant strains.The phenotypes of septin organization mutants in the strain background used are illustrated in Fig. 1 and quantitated in Table 2. Septin staining using anti-Cdc11p antibodies revealed a variety of defects, which were divided into four categories for the purposes of quantitation (Table 2). Cells displaying a wild-type septin hourglass at the neck were scored as “neck-normal” (Fig. 1, cells a and b), and cells that lacked all septin staining were scored as “absent” (Fig. 1, cells h and j). Cells were scored as “neck-abnormal” when septin staining was prominent at the neck but was either fainter than that of the wild type (Fig. 1, cells c, i, and k), lacked crisp boundaries (Fig. 1, cells c, d, and i), exhibited a set of parallel bars along the mother-bud axis (Fig. 1, cells i, k, and l), or displayed some combination of these features. Cells were scored as mislocalized when septin staining displayed a cap or ring (Fig. 1, cells g and l) of septin staining away from the neck (usually at or near the bud tip), even if the same cell also displayed septin staining at the neck. In addition to these variations in septin localization, cells often displayed cell morphology defects, including wide necks (Fig. 1, cells e, f, and j), elongated buds (Fig. 1, cells g, i, j, k, and l), narrow or kinked necks (Fig. 1, cells c, d, h, and k), or some combination of these features.

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FIG. 1.

Septin phenotypes in budded cells. Cells from the strains indicated below were grown to exponential phase in yeast extract-peptone-dextrose (YEPD) at 30°C, fixed, and processed for the visualization of septin localization by use of an anti-Cdc11p antibody. The illustrative cells in the figure display a normal septin hourglass (a and b; strain DLY1), irregular neck staining (c and d; strain DLY4790), wide necks (e and f; strain MOSY148), an ectopic septin hourglass in the elongated bud in addition to a faint neck hourglass (g; strain DLY4223), faint or absent septin staining and a bent or broad neck (h and j; strain DLY4859), and septin bars at aberrantly shaped necks (i and k; strain DLY4224) or displaced into the bud (l; strain DLY4224).

Some general features emerged from the analysis of these isogenic strains. Firstly, the phenotypes were heterogeneous in that the different cells of a single mutant strain could display different categories of defect. Secondly, different mutants displayed characteristic propensities to display a particular defect (e.g., gin4 mutants exhibited septin bars at a higher frequency than other mutants, whereas elm1 and cla4 mutants exhibited mislocalized septins at higher frequencies than other mutants) (Table 2). Thirdly, there was a correlation such that cells displaying mislocalized septins frequently displayed elongated buds. In addition, cells displaying aberrant neck septin phenotypes often had misshapen (aberrantly wide, narrow, and/or kinked) necks.

Initial assembly of septin ring.We also examined the morphology of the septin rings formed prior to bud emergence in a variety of mutant strains. For this analysis, cells from mostly unbudded stationary-phase cultures were inoculated into fresh medium and fixed at 4 h to enrich for cells that were about to bud (in some cases, these observations were confirmed by using synchronized cells isolated by centrifugal elutriation from growing populations). As previously reported (13), different cdc42 alleles produced either normal-looking (cdc42^V36T,K94E) or large-diameter (cdc42^Y32H) initial septin rings. Whereas single mutants lacking individual Cdc42p-directed GAPs displayed normal rings, triple-mutant rga1Δ rga2Δ bem3Δ cells displayed a spectrum of phenotypes ranging from wild-type rings to very faint or broken-looking rings (Fig. 2). gin4Δ, nap1Δ, and elm1Δ mutants formed septin rings that appeared indistinguishable from the wild type, whereas cla4Δ mutants formed appreciably larger rings (Fig. 2). These phenotypes support the hypothesis that Cdc42p, the GAPs, and Cla4p are all important for the proper assembly of the initial ring.

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FIG. 3.

Epistatic interactions among elm1, gin4, and nap1 mutants. Cells from the strains indicated below were grown to exponential phase in YEPD at 30°C, fixed, and processed for the visualization of septin localization by use of an anti-Cdc11p antibody. WT, strain DLY1; elm1Δ, strain DLY4687; gin4Δ, strain DLY4410; nap1Δ, strain DLY4790; elm1Δ gin4Δ, strain DLY4714; elm1Δ nap1Δ, strain DLY4858; gin4Δ nap1Δ, strain DLY4848; elm1Δ gin4Δ nap1Δ, strain DLY4857. Bar, 10 μm.

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FIG. 4.

Synthetic interactions among cdc42, cla4, and gin4 mutants. Cells from the strains indicated below were grown at 30°C to exponential phase in minimal medium lacking uracil (to select for plasmid retention), fixed, and visualized by differential interference contrast microscopy. WT, strain DLY3067 carrying plasmid pMOSB55; cla4Δ, strain MOSY23 carrying plasmid pMOSB55; gin4Δ, strain DLY4596 carrying plasmid pMOSB55; cdc42^V36T,K94E, strain DLY3067 carrying plasmid pMOSB57; cdc42^Y32H, strain DLY3067 carrying plasmid pMOSB56; cla4Δ cdc42^V36T,K94E, strain MOSY23 carrying plasmid pMOSB57; cla4Δ cdc42^Y32H, strain MOSY23 carrying plasmid pMOSB56; gin4Δ cdc42^V36T,K94E, strain DLY4596 carrying plasmid pMOSB57; gin4Δ cdc42^Y32H, strain DLY4596 carrying plasmid pMOSB56.

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FIG. 2.

Septin rings in unbudded cells. Cells from the strains indicated below were grown to stationary phase in YEPD at 30°C and then inoculated into fresh medium, grown for 4 h (at which point cells were entering the cell cycle), fixed, and processed for the visualization of septin localization by use of an anti-Cdc11p antibody. WT, strain DLY1; cla4Δ, strain MOSY148; elm1Δ, strain DLY4687; gin4Δ, strain DLY4410; nap1Δ, strain DLY4790; rga1Δ rga2Δ bem3Δ, strain DLY2723. Bar, 10 μm.

Genetic interactions among septin organization mutants.Previous studies examined genetic interactions among mutations in genes encoding Cdc42p-directed GAPs (9, 13, 29) and among mutations in GIN4, NAP1, and CLA4 genes (25, 34). In both cases, progressively more penetrant and severe phenotypes were observed in strains containing more mutations. We confirmed that gin4, nap1, and cla4 mutations also caused progressively more penetrant phenotypes in our strain background (Table 2). Moreover, we found that combining mutations in any of these genes with mutations in the GAP-encoding RGA1, RGA2, and BEM3 genes also yielded progressively more penetrant phenotypes (Table 2). For simplicity, we will refer to these effects as additive, although the fact that different cells in an individual strain displayed heterogeneous categories and severities of defect makes it impossible to reduce these phenotypes to a quantifiable septin defect parameter. Nevertheless, these additive genetic interactions do rule out the model that any set of these septin regulators acts in a simple linear regulatory pathway.

Genetic interactions between elm1Δ and other septin organization mutants have not been reported. We found that elm1Δ cla4Δ and elm1Δ rga1Δ strains displayed additive phenotypes (see above), growing progressively worse as rga2 and bem3 were also deleted (Table 2). In contrast, elm1Δ gin4Δ and elm1Δ nap1Δ strains appeared very similar to the elm1Δ single mutants (Fig. 3 and Table 2). Even the triple elm1Δ gin4Δ nap1Δ mutant was only marginally more severe than the elm1Δ single mutant (Fig. 3). These epistatic interactions are consistent with the view that Elm1p acts downstream of parallel pathways from Gin4p and Nap1p or that Elm1p is a key upstream activator of Gin4p and Nap1p (see Discussion).

The two cdc42 alleles that we examined were part of a collection of point mutants that had altered residues in the Cdc42p effector loop (14). From that collection, these two were unique in showing defects in septin organization but not in actin organization (13, 14). Suppression analysis showed that the septin defects could be suppressed by elevated expression of the GAP Rga1p, and biochemical analysis indicated that both mutants were mildly defective in GTP hydrolysis, suggesting that their GTP hydrolysis defect might contribute to the septin defect (13). However, the detailed biochemical defects were distinct (see Discussion), as were the septin defects (cdc42^V36T,K94E mutants had mislocalized septins, whereas cdc42^Y32H mutants had large initial septin rings and neck-abnormal septins). These mutants display dosage-sensitive phenotypes (13), and when they are expressed from CEN ARS plasmids they display only mild (cdc42^V36T,K94E) or even undetectable (cdc42^Y32H) septin defects. In assessing the effect of combining these with other mutations in septin organization genes, we found that the two cdc42 alleles displayed dramatic and allele-specific synergistic phenotypes when combined with cla4Δ or gin4Δ mutations (Fig. 4). In particular, cdc42^V36T,K94E cla4Δ mutants were synthetically lethal, whereas cdc42^V36T,K94E gin4Δ mutants were very similar to gin4Δ single mutants. In contrast, cdc42^Y32H cla4Δ mutants were very similar to cla4Δ single mutants, whereas cdc42^Y32H gin4Δ mutants exhibited a synergistic defect. The severely affected cdc42^V36T,K94E cla4Δ and cdc42^Y32H gin4Δ mutants displayed large tubular multibranched cells (Fig. 4), with only patchy septin staining detectable (data not shown). The dramatic and distinct nature of the genetic interactions exhibited by the cdc42 alleles strongly suggests that these mutants engender distinct sorts of defects in septin organization.

Pathways for septin organization?Our motivation for examining the effects of combining mutations in septin organization genes included the hope that regulatory pathways for septin organization would be revealed through epistatic interactions between mutants. However, in the large majority of cases, we observed additive effects of mutant combinations on septin ring organization, suggesting that most of the regulators act in parallel to promote septin ring integrity. The only exceptions were elm1 gin4 and elm1 nap1 mutants, which displayed phenotypes similar to that of the elm1 single mutant. It is possible that these epistatic interactions reflect a regulatory pathway in which Gin4p and Nap1p act in parallel to activate Elm1p. Alternatively, Elm1p may activate both Gin4p and Nap1p. However, Gin4p and Elm1p are recruited to the septin cortex in a manner that is sensitive to defects in septin organization (25, 33), so it may be that the perturbation of septin organization in elm1 mutants leads indirectly to a failure to recruit Gin4p to the septin cortex (or vice versa) and that Gin4p and Elm1p are thereby rendered less active. Thus, it remains possible that all of these factors act in parallel to promote septin organization and that the epistatic interactions reflect indirect effects rather than regulatory pathways linking these proteins.

We also observed two very severe (synthetic) genetic interactions between different alleles of CDC42 and gin4 or cla4 mutants. The remarkable allelic specificity of these interactions strongly suggests that the cdc42 mutants engender distinct sorts of defects in septin organization. We have reported that both alleles encode proteins with biochemical defects in GTP hydrolysis (13). However, Cdc42p^V36T,K94E displayed a defect in the intrinsic GTP hydrolysis rate which could still be stimulated by a recombinant Rga1p GAP domain, whereas Cdc42p^Y32H displayed a normal GTP hydrolysis rate which could no longer be stimulated by the GAP. It seems plausible that these biochemical differences underlie the phenotypic differences reported here. Alternatively, the alleles may impair distinct effector interactions in addition to their effect on GTP hydrolysis.

Assembly of the septin ring and septin hourglass.We examined the initial septin structures formed by unbudded cells emerging from stationary phase in a variety of mutant strains and found that gin4, nap1, and elm1 mutants all made normal-looking initial rings, whereas cla4 mutants made larger-diameter rings. In the rga1 rga2 bem3 triple mutant, we observed a heterogeneous group of structures, including large rings and broken rings in which several separate strands of septins were arranged in a roughly circular array. The simplest interpretation of these findings is that Cla4p and the Cdc42p-directed GAPs contribute to the initial assembly of the septin ring in unbudded cells, whereas Gin4p, Nap1p, and Elm1p are not required for this step. (Recent studies concluded that Elm1p also plays roles in cell cycle commitment [30] and SNF1 regulation [31], but it is not clear whether or how those roles relate to Elm1p function in septin organization.) Coincident with (or shortly after) bud emergence, the septin ring spreads to form a collar, and it was in the budded collar that gin4, nap1, and elm1 mutants displayed obvious defects. We suggest that Elm1p, Gin4p, and Nap1p act during or after bud emergence to promote the proper assembly and/or maintenance of the septin hourglass. Consistent with this view, Elm1p is only localized to the septin cortex after bud emergence (8), and even though Gin4p is recruited to the septins before bud emergence, it does not become fully active until later in the cell cycle (1).

Recent studies proposed two rather different models for the initial assembly of the septin ring. In one model, the septins are recruited directly into a ring structure (13). In the other model, septins are first recruited to a precursor cap structure which then converts to a ring (9). The latter model interprets the septin caps observed in mutant strains as a normally short-lived assembly intermediate whose conversion into a septin ring has been delayed. Our findings on elm1 mutants, which often display caps in budded cells, are particularly interesting in this context. If the caps are early assembly intermediates, we would have expected to see caps in the unbudded elm1 cells, which would eventually convert to rings after bud emergence. However, we actually observed normal septin rings in unbudded elm1 cells, and caps developed only later, generally at the bud tip in cells with elongated buds. Many cells with such caps also had septin hourglass structures (albeit often fainter than wild-type structures) at the neck. These findings support the view that caps arise as a secondary consequence of defects in the septin hourglass of budded cells and that they represent an abnormal septin structure rather than a “trapped” assembly intermediate (at least in elm1 mutants).

Relationship between septin organization and cell shape.Within the panel of strains examined for this report, there was a general and striking correlation between defects in septin organization and alterations in cell shape. Previous studies documented the elongated bud phenotype in septin mutant strains and demonstrated that this phenotype involves deregulation of the cell cycle inhibitory kinase Swe1p (2, 17, 25). However, the range of morphological aberrations observed in the present study includes wide, narrow, kinked, and stretched-looking necks as well as unusually wide, narrow, and sometimes bent buds. These phenotypes go well beyond the simple elongated bud (with relatively normal neck) phenotype of cells lacking all organized septins, raising the question of whether there is a more complex link between septins and cell morphology. Because many of the mutations we examined are known to be pleiotropic, the possibility remains that the septin and cell shape abnormalities are separate and independent products of the mutations. However, some categories of septin defect were commonly associated with specific alterations in cell shape (septin bars with stretched necks or ectopic septin rings with elongated buds), suggesting that either misorganized septins can influence cell shape (beyond the bud-elongating effects of Swe1p) or cellular geometry can influence septin organization, as previously suggested (15, 32). In work to be reported elsewhere, we present evidence suggesting that both of these are true and that the interplay between cell shape and septin organization underlies the genesis of some of the septin phenotypes.

Interpretation of septin organization phenotypes.Genetic approaches to understanding septin organization seek to identify septin regulatory genes and, through analysis of their mutant phenotypes, to elucidate how those genes impact septin organization. Several apparently distinct categories of septin defect (e.g., parallel septin bars at the neck or ectopic septin caps at the bud tip) are encountered in the mutant strains, and these phenotypes have been instrumental in generating attractive hypotheses about the roles of some regulators in septin organization. The first study to describe the septin bar phenotype focused on the gin4 strain, and Longtine et al. (24) suggested that Gin4p was important for cross-linking parallel septin filaments into a seamless collar at the neck. The localization of Gin4p to the septin collar throughout the budded phase of the cell cycle was certainly consistent with this inference (24). Support for the view that Gin4p acts as a septin cross-linker was provided by a recent study describing an increase in septin dynamics in both gin4 and cla4 mutant cells, suggesting that the role of Gin4p and Cla4p is to freeze the septins in place within the hourglass collar (11). Another recent study focused on the septin caps frequently observed in cdc42^V36G and rga1 rga2 bem3 mutants, and Caviston et al. (9) suggested that the cap was a precursor step in assembling a septin ring (described above).

The hypotheses described above sought to provide a chain of causality in which the molecular defect stemming from a genetic lesion explained the details of a particular septin phenotype: the failure to cross-link parallel septin filaments at the necks of cells lacking Gin4p caused the filaments to form bundles which are seen as bars, whereas the failure to release septins from the cap in cells with delayed GTP hydrolysis by Cdc42p allowed the persistence of what would normally be a transient assembly intermediate. Surprisingly, however, we found that there was no clear-cut correspondence between a particular genetic lesion and a specific category of septin defect: different cells of a single mutant population often displayed different categories of septin defect, and many different mutant strains contained cells displaying any specified septin defect. Although biases could be identified such that some mutants (gin4 and cdc42^Y32H) showed a higher propensity to develop septin bars while others (elm1, cla4, and cdc42^V36T,K94E) showed a higher propensity to develop septin caps, these biases were far from absolute. Moreover, a combination of two mutations that displayed only aberrant neck phenotypes, including septin bars, could yield a strain (e.g., gin4 nap1) that showed a significant frequency of mislocalized septin caps.

How can we account for the unexpectedly complex relationship between genotype and phenotype with regard to septin organization? We suggest that the phenotypes scored as different categories of septin defect reflect an intrinsic repertoire of alternate (aberrant) states of septin organization. By analogy to the alternative conformational states of individual proteins, these states might be viewed as local minima in the potential energy landscape for assemblies of septins and their partners. We suggest that specific mutations influence the probabilities that the wild-type septin hourglass in an individual cell will collapse to each of the several alternate states. Thus, a particular mutant population would display a characteristic distribution of cells exhibiting different septin states, and different mutant populations could contain cells exhibiting a specific septin state, as we observed. This state of affairs does not easily lend itself to a straightforward interpretation of the link between a mutant lesion and a specific aspect of septin organization, because the mutations influence the likelihood that septins will assume particular alternate states rather than dictating a unique outcome. Moreover, the influence of a specific mutation may include indirect effects of the mutation (e.g., through effects on cell shape or cell cycle) as well as any direct effects on septin organization. These considerations highlight the need to understand in much greater depth the genesis of particular septin phenotypes in mutant strains.