Department of Biology and Program in
Molecular Biology and Biotechnology, University of North Carolina,
Chapel Hill, North Carolina 27599-3280,1 and
Department of Pharmacology and Cancer Biology, Duke
University Medical Center, Durham, North Carolina
277102
Received 22 December 1999/Returned for modification 16 February
2000/Accepted 15 March 2000
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INTRODUCTION |
The septins are a family of
conserved filament-forming proteins that appear to form scaffolds for
the localized assembly of various other proteins at the cell surface
(21, 39, 40, 42). In Saccharomyces cerevisiae,
the septins encoded by CDC3, CDC10,
CDC11, and CDC12 are localized to a band at the
cytoplasmic face of the plasma membrane in the mother bud neck
(20, 25, 31, 39), to which they recruit proteins involved in
bud site selection (14, 56), asymmetric chitin deposition
(17), and cytokinesis (9, 18, 38).
Temperature-sensitive mutations in any of these genes cause the
complete disassembly of the septin-based scaffold at the restrictive
temperature, resulting in severe pleiotropic defects in all of the
aforementioned processes (20, 25, 26, 31). A striking
perturbation of the septin scaffold also results from deletion of
GIN4 (40), which encodes one of three S. cerevisiae protein kinases related to Nim1p (a mitotic inducer in
Schizosaccharomyces pombe [55]). Gin4p
localizes to the neck in a septin-dependent manner (40, 48),
and in gin4
cells, the septins and associated proteins
are frequently found as a set of five to eight bars that traverse the
neck. However, these cells display only mild defects in the various
septin-dependent processes (40).
Cells containing septin or gin4 mutations also display an
elongated-bud morphology associated with a hyperpolarization of the
actin cytoskeleton towards the tip of the bud (1, 26, 40).
In wild-type cells, bud formation involves an initial period of apical
growth, during which new cell wall is inserted primarily at the bud
tip, followed by a longer period of isotropic growth during which new
cell wall is inserted all over the bud surface (19, 34). The
shape of the bud reflects the relative times spent in the apical and
isotropic growth stages, and the switch from one to the other is
triggered by the cell cycle-regulatory kinase Cdc28p in association
with the B-type cyclins (primarily Clb2p) (34). Inactivation
of Clb-Cdc28p complexes results in prolonged apical growth and the
formation of elongated buds (34). Therefore, the elongated
buds of septin mutants and gin4 mutants could arise from a
delay in the activation of Clb-Cdc28p complexes, from a defect in the
ability to execute the switch to isotropic growth in response to
Clb-Cdc28p activation, or from a combination of these mechanisms.
Activation of Clb-Cdc28p complexes is subject to multiple layers of
regulation, including the inhibitory phosphorylation of Cdc28p at Tyr
19 by the kinase Swe1p (35, 47). Swe1p is dispensable for
normal cell cycle progression in unperturbed cells, but it is essential
for the morphogenesis checkpoint response (11, 33, 60). This
checkpoint is triggered by perturbations that disrupt the process of
bud formation, and it introduces a G2 delay in the nuclear
cycle that provides time for further bud growth prior to nuclear
division (33, 46). In unperturbed cells, Swe1p is stable
during G1 and S phases but becomes quite unstable during
G2/M (59). Rapid degradation of Swe1p requires a
second Nim1p family kinase, Hsl1p (or Nik1p), the conserved protein
Hsl7p, and the ubiquitination complex SCFMet30 (28,
43, 44, 58). Overexpression of Swe1p results in G2
arrest accompanied by the formation of highly elongated buds (11). Thus, one hypothesis to explain the elongated buds in septin mutants and gin4 mutants is that Swe1p accumulates
and/or is activated in these mutants (6).
A different hypothesis has been proposed by Kellogg and colleagues
(2, 13, 62), who have suggested that Clb-Cdc28p complexes
trigger the switch from apical to isotropic bud growth through a signal
transduction cascade that involves Gin4p, the septins, and at least two
other proteins, Nap1p and Cla4p. Nap1p was identified as a protein that
binds to the cyclin Clb2p (30), whereas Cla4p is a member of
the p21-activated kinase family that bind to and are activated by
Cdc42p-type small GTPases in the GTP-bound form (5, 16). In
this hypothesis, the septins contribute to bud morphogenesis by helping
Clb-Cdc28p complexes to activate Gin4p (and possibly other components
of the signaling pathway), leading ultimately to the switch to
isotropic growth.
In this paper, we report studies of the link between septin
organization and cell cycle control. Our results suggest that perturbations of septin organization do indeed result in a
Swe1p-dependent G2 delay associated with a delayed switch
from apical to isotropic bud growth and hence an elongated-bud
phenotype. Gin4p, Cla4p, and Nap1p make partially redundant
contributions to normal septin organization, which is a prerequisite
for the hierarchical assembly of a cell cycle-regulatory module
involving Hsl1p, Hsl7p, and Swe1p at the daughter side of the
mother-bud neck. Release of this module from the neck is associated
with, but cannot entirely account for, the Swe1p-dependent
G2 delay in the mutants with perturbed septin organization.
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MATERIALS AND METHODS |
Strains, plasmids, and PCR manipulations.
Escherichia
coli strain DH12S (Life Technologies, Gaithersburg, Md.) and
standard media and methods (3) were used for plasmid manipulations. High Fidelity Expand DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.) was used for the PCR synthesis of DNA
fragments for cloning and for PCR-mediated epitope tagging; Taq DNA polymerase (Promega, Madison, Wis.) was used in
other PCR applications. Oligonucleotide primers were obtained from
Integrated DNA Technologies (Coralville, Iowa). Transformation of yeast
was performed as described previously (22), and other yeast
genetic manipulations were performed by standard procedures
(24).
The S. cerevisiae strains used in this study are listed in
Table 1. Construction of deletion and
tagged alleles in the YEF473 background was carried out by first
modifying the target gene in the diploid strain YEF473. All other
strains were obtained by crosses among segregants derived from these
original transformants. The successful deletion or tagging of target
genes was verified by PCR with genomic DNA from transformants as
template and the oligonucleotide primers shown in Table
2, as well as by demonstrating that the
marker and any associated phenotype segregated 2:2 after sporulation.
Plasmids containing the swe1::LEU2,
mih1::LEU2, and
hsl11::URA3 null alleles have been described
previously (11, 43, 54). To replace the wild-type alleles
with these mutant alleles, yeast cells were transformed with
appropriate fragments from these plasmids. The chromosomal
cla4::HIS3, nap1::HIS3,
and hsl7::kan alleles were constructed by the
PCR method (7) with plasmid pRS303 (61) or
pFA6a-kanMX6 (41) as template and oligonucleotide primers as
described in Table 2. The PCR method was also used to generate alleles
of GIN4, HSL1, and HSL7 encoding
proteins tagged at their C termini with GFP(S65T), 13 tandem myc
epitopes, or 3 tandem hemagglutinin (HA) epitopes. The plasmids
described previously (41) were used as templates, and the
primers used are listed in Table 2. Gin4p-GFP appeared to be fully
functional by the criterion that the GIN4-GFP:kan
strains had normal cell morphology at 37°C (unpublished results), in
contrast to the abnormal morphology of gin4 strains
(40). Western blotting showed that Hsl1p-13myc and Hsl7p-3HA
migrated near the predicted sizes of ~190 kDa and ~100 kDa,
respectively (unpublished results). Moreover, Hsl1p-13myc, Hsl1p-GFP,
and Hsl7p-3HA appeared to be fully functional by the criteria that
HSL1-13myc mih1, HSL1-GFP mih1, and
HSL7-3HA mih1 strains were all viable and had normal cell
morphology (unpublished results), in contrast to hsl1
mih1 and hsl7 mih1 strains, which are inviable
and arrest in G2 with extremely elongated buds (44). Construction of the chromosomal
SWE1myc:URA3, SWE1myc:TRP1, SWE1myc:HIS2, and
SWE1myc:TRP1(3x) alleles has been described previously (44, 59). The myc-tagged Swe1p appeared to be
fully functional by the criterion that it provided a morphogenesis
checkpoint-dependent G2 delay in a cdc24 mutant
(59).
To introduce the cdc12-6 allele (1) into
different strain backgrounds, primers ML277 and ML278 (Table 2) were
used to amplify the region encoding the cdc12-6 C terminus
with DNA isolated from the cdc12-6 strain M-238
(40) as template. The PCR product was digested with
MfeI and PstI (sites in the CDC12
region sequences) and ligated into
EcoRI/PstI-digested YIplac128 (23),
resulting in plasmid YIplac128/cdc12-6. DNA sequencing verified that
the cloned insert contains the cdc12-6 mutation, which is an
insertion of an adenine into a stretch of seven adenines spanning
nucleotides 1167 to 1173 of the CDC12 open reading frame
(B. K. Haarer and J. R. Pringle, unpublished data). Strains
were then transformed with YIplac128/cdc12-6 after digestion at the
unique HpaI site (upstream of the cdc12-6
mutation), yielding Leu+ transformants. Dissection of
tetrads from these transformants yielded
2Ts+:2Ts
segregants, and the Ts
lethality was rescued by introduction of a low-copy-number
CDC12 plasmid.
Growth conditions and synchronization.
Yeast media (YM-P and
YPD rich media, synthetic complete medium [SC] lacking specific
nutrients, and sporulation medium) have been described previously
(24, 37). Cells were synchronized by 
-factor
arrest-release or by centrifugal elutriation as described previously
(34, 45). Latrunculin-A (Molecular Probes, Eugene, Oreg.)
was added from a 20 mM stock solution in dimethyl sulfoxide that was
stored at 
20°C.
Protein analysis.
Yeast proteins were isolated by
resuspending whole cells in 2× Laemmli sample buffer (32)
and boiling for 5 min. Western blot analysis was performed by standard
procedures (3) with mouse monoclonal anti-myc antibodies
(9E10; Santa Cruz Biotechnology, Santa Cruz, Calif.), mouse monoclonal
anti-HA antibodies (HA.11; Berkeley Antibody Co., Richmond, Calif.),
and the ECL chemiluminescence detection system (Amersham, Arlington
Heights, Ill.).
Immunofluorescence and other microscopic analysis.
Except as
noted below, all microscopy was performed using a Nikon Mikrophot SA
microscope. Overall cell morphologies were examined by differential
interference contrast (DIC) microscopy, and cells were stained with
4',6'-diamidino-2-phenylindole (DAPI; Sigma Chemical Co., St. Louis,
Mo.) to visualize DNA or with Calcofluor (Sigma) to visualize chitin
(50). Green fluorescent protein-tagged proteins were
visualized using the fluorescein isothiocyanate filter set. To
determine whether cells had completed cytokinesis (52),
cells were fixed by adding formaldehyde directly to the growth medium
to a 3.7% final concentration and incubated for 2 h at the growth
temperature. After being washed three times with phosphate-buffered
saline (PBS), cells were suspended in PBS with 0.1%
-mercaptoethanol and digested with 0.2 mg of Lyticase (ICN
Pharmaceuticals, Costa Mesa, Calif.) per ml for 1 h at room temperature with continuous gentle rolling.
Immunofluorescence localization of Cdc11p, Hsl1p-13myc, and Hsl7p-3HA
was performed essentially as described previously (51), using bisBenzamide (Sigma) in the mounting medium to stain DNA. Anti-Cdc11p antibodies were purified as previously described
(20) and used at a 1:10 dilution. Mouse anti-myc antibodies,
mouse anti-HA antibodies, and rat monoclonal anti-HA antibodies (3F10; Boehringer Mannheim) were all used at a 1:300 dilution. Fluorescein isothiocyanate- or Cy3-labeled secondary antibodies were purchased from
Jackson ImmunoResearch (West Grove, Pa.), and Alexa-labeled secondary
antibodies were purchased from Molecular Probes; both were used at a
1:200 dilution.
Immunofluorescence detection of Swe1p in a majority of the cells
required the use of a four-antibody sandwich protocol. Strains carrying
two or four integrated copies of SWE1myc were grown to 5 × 106 cells/ml in YPD medium, fixed by adding
formaldehyde to the growth medium to a 4.5% final concentration, and
incubated for 45 min at 23°C. Following a 30-min treatment with 12.6 µg of Zymolyase (ICN) per ml at 30°C, the samples were washed with
PBS, pH 7.5, and affixed to polylysine-coated slides (51).
Cells were then incubated successively with the following antibodies at
the indicated dilutions: 9E10 mouse anti-myc, 1:10; rabbit
anti-mouse-IgG (Jackson ImmunoResearch), 1:100; mouse anti-rabbit IgG
(Jackson ImmunoResearch), 1:100; and Cy3-labeled goat anti-mouse IgG
(Jackson ImmunoResearch), 1:25. All antibodies were diluted in PBS
containing 1 mg of bovine serum albumin (BSA) per ml (BSA-PBS) and
incubated with the cells for 1 h at 23°C in a dark, humid
chamber. Between incubations, cells were washed 10 times with 10 µl
of BSA-PBS. DNA was visualized by including DAPI in the mounting medium
(51). Microscopy was performed with a Zeiss Axioskop with
standard fluorescence optics. Images were captured with a cooled
charge-coupled device camera (Princeton Instruments, Princeton, N.J.)
interfaced with Metamorph software (Universal Imaging Corp.,
Silver Spring, Md.).
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RESULTS |
Swe1p-dependent cell cycle delay in septin mutants.
Temperature-sensitive septin mutants arrest as multibudded,
multinucleate cells after prolonged incubation at restrictive temperature, indicating that cell cycle progression continues in these
mutants (26, 39). However, it seemed possible that there
might be a small cell cycle delay that could contribute to the
characteristic elongated-bud morphology (1, 26) of septin
mutants. Indeed, in agreement with others (6), we found that
nuclear division was delayed by 30 to 45 min in septin mutant strains
as compared to isogenic wild-type strains (unpublished results). The
delay was eliminated by deletion of SWE1
(6; unpublished results), indicating that it
depended on Swe1p. Similar results were observed with
cdc12-6 and cdc10-1 mutants and by using either
-factor or centrifugal elutriation to obtain synchronized cells
(unpublished results).
To ask if the Swe1p-dependent cell cycle delay contributes to the
elongated-bud phenotype, we examined the morphologies of septin mutants
that contained or lacked Swe1p or Mih1p (the phosphatase that reverses
the Swe1p-catalyzed phosphorylation of Cdc28p [54]). In agreement with others (6), we found that deletion of
SWE1 largely eliminated the elongated-bud phenotype of a
septin mutant at restrictive temperature (Fig.
1, panels 2 and 4). In contrast, deletion
of MIH1 exacerbated the elongated-bud phenotype (Fig. 1,
panel 6), and these cells arrested permanently with a single nucleus
(unpublished results). The cdc11-6 mih1 double-mutant cells even displayed a slight elongated-bud phenotype at permissive temperature (Fig. 1, panel 5). In contrast to its effect on bud morphology, deletion of SWE1 neither restored septin
localization nor rescued the cytokinesis defect in the septin mutant
cells, as judged by immunofluorescence (unpublished results) and by the accumulation of multiple buds (Fig. 1, panel 4) that did not detach upon Lyticase treatment of fixed cells (unpublished results) (see Materials and Methods). Introduction of the nonphosphorylatable CDC28Y19F allele (using plasmid pJM1046
[45]) into septin mutant strains also restored nearly
normal bud morphology (unpublished results), indicating that the effect
of Swe1p was mediated largely, at least, by phosphorylation of Cdc28p
at Tyr 19, as expected.
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FIG. 1.
Role of Swe1p-dependent inhibition of Cdc28p in the
elongated-bud morphology of septin mutants. Homozygous diploid strains
M-905 (cdc11-6), M-1207 (cdc11-6 swe1), and
M-1208 (cdc11-6 mih1) were grown to exponential phase at
23°C in YM-P medium and examined by DIC microscopy before (panels 1, 3, and 5) and after (panels 2, 4, and 6) a shift to 37°C for 6 h. Arrows indicate multibudded cells (panel 4) or elongated buds (panel
5), as discussed in the text.
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Taken together, the data suggest that septin disassembly triggers a
Swe1p-dependent inhibition of Cdc28p that leads to a cell cycle delay
associated with continued apical bud growth. This response appears to
be functionally important: when we dissected tetrads from the doubly
heterozygous strains M-990 and M-991, we observed synthetic lethality
between the cdc11 mutation (21) and either
swe1 or mih1.
Swe1p-dependent cell cycle delay in gin4,
cla4, and nap1 mutants.
Mutation of
GIN4 leads to an alteration of septin organization at the
mother-bud neck associated with modest effects on septin function (see
Fig. 3A and B) (40). In addition, gin4 mutant cells display an elongated-bud phenotype (Fig. 2D) particularly at
elevated temperatures (40).
Deletion of SWE1 largely eliminated, and deletion of
MIH1 greatly exacerbated, the elongated-bud phenotype of
gin4 mutants (Fig. 2E and F; for quantitation of the bud
morphology and other phenotypes of these and other mutant strains, see
Table 3). Immunofluorescence analysis and
staining of chitin showed that septin organization and function were
similar in the gin4 and gin4 swe1
strains and only slightly more abnormal in the gin4
mih1 strain (Table 3), indicating that the effects of deleting
SWE1 or MIH1 on bud morphology did not simply
reflect effects on septin organization. The nonphosphorylatable
CDC28Y19F allele also restored nearly normal bud
morphology to gin4 cells (unpublished results),
indicating that the elongated-bud phenotype of gin4 mutants,
like that of septin mutants, is due largely to Swe1p-dependent
inhibition of Cdc28p.
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FIG. 2.
Role of Swe1p-dependent inhibition of Cdc28p in the
elongated-bud morphology of gin4, cla4, and
nap1 mutants. The indicated strains were grown to
exponential phase in YM-P medium at 30°C and examined by DIC
microscopy. (A) YEF473; (B) M-1077; (C) M-600; (D) M-272; (E) M-825;
(F) M-829; (G) M-515; (H) M-522; (I) M-1025; (J) M-546; (K) M-544; (L)
M-976.
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Cla4p and Nap1p have been suggested to function in a common pathway
with Gin4p, based on both genetic data (similar phenotypes were
observed for cells lacking these proteins in combination with loss of
G1 or mitotic cyclins) and biochemical data (Gin4p binds to
Nap1p affinity columns, and both Nap1p and Cla4p are required for
maximal phosphorylation of Gin4p in G2/M) (2, 8, 16,
62). We therefore examined whether cla4 and
nap1 mutants also displayed a Swe1p-dependent
elongated-bud phenotype. Indeed, both mutants displayed mild
elongated-bud phenotypes (Fig. 2G and J) that were eliminated upon
deletion of SWE1 or introduction of
CDC28Y19F (Table 3) (unpublished results). The
elongated-bud phenotype was dramatically exacerbated in cla4
gin4 and nap1 gin4 double mutants (Fig. 2H and
K), but even these extreme phenotypes were suppressed by deletion of
SWE1 (Fig. 2I and L; Table 3) or introduction of
CDC28Y19F (unpublished results).
Roles of Cla4p and Nap1p in septin organization and function.
Given the phenotypic similarities and genetic and biochemical
interactions among Gin4p, Cla4p, and Nap1p, it seemed possible that
Cla4p and Nap1p might also play roles in septin organization. Indeed,
both cla4 and nap1 mutants exhibited
aberrant septin organization similar to that seen in gin4
mutants (Fig. 3A to D). As in
gin4 mutants, the abnormal septin organization in
cla4 and nap1 mutants was typically
reflected in a loss of the normal asymmetry of chitin deposition at the
mother-bud neck (Table 3).
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FIG. 3.
Effects of gin4, cla4, and
nap1 mutations, but not of hsl1 or
kcc4 mutations, on septin organization. Cells of the
indicated strains were grown to exponential phase in YM-P medium at
30°C and examined by immunofluorescence microscopy to localize
Cdc11p. (A) YEF473; (B) M-272; (C) M-515; (D) M-546; (E) M-522; (F)
M-544; (G) M-601; (H) M-286. The arrow and arrowhead in panel D
indicate cells displaying septin misorganization, classified as
"fuzzy" and "bars," respectively, as discussed in Table 3.
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As both Cla4p and Nap1p are required for maximal phosphorylation of
Gin4p (2, 62), it seemed possible that the roles of Cla4p
and Nap1p in septin organization are simply to localize and/or activate
Gin4p. Consistent with this hypothesis, deletion of CLA4 or
NAP1 resulted in the loss of detectable localization of
Gin4p to the neck in a large fraction of the cells (Table
4). However, an alternative possibility
is that Cla4p and Nap1p promote normal septin organization through a
pathway(s) separate from that of Gin4p and that the septin
misorganization in cla4 and nap1 cells
precludes the efficient recruitment of Gin4p to the neck. Consistent
with this hypothesis, both cla4 gin4 and nap1 gin4 double mutants displayed more severe defects in septin
organization (and in the associated localization of chitin deposition)
than did the single mutants (Fig. 3E and F; Table 3).
Although deletion of SWE1 restored normal bud morphology to
the gin4, cla4, nap1,
cla4 gin4, and nap1 gin4 strains (see
above), it did not restore normal septin organization or localization
of chitin deposition (Table 3). Thus, taken together, the data suggest
that Cla4p and Nap1p, like Gin4p, have roles in proper septin
organization and that one consequence of the defective septin
organization observed in their absence is the Swe1p-dependent
inhibition of Cdc28p.
Distinct roles of Nim1p family kinases in septin organization and
cell cycle control.
In addition to Gin4p, S. cerevisiae
contains two other kinases, Hsl1p (or Nik1p) and Kcc4p (or Ycl024Wp),
in the family defined by S. pombe Nim1p (6, 27, 40,
48). These proteins have closely related kinase domains at their
N termini and long, generally dissimilar C-terminal regions. We asked
whether Hsl1p and Kcc4p, like Gin4p, play roles in septin organization.
However, hsl1 and kcc4 single mutants and
hsl1 kcc4 double mutants all displayed essentially
normal septin organization and localization of chitin deposition (Fig.
3G and H; Table 3). Moreover, the gin4 hsl1 and
gin4 kcc4 double-mutant strains and even the
triple-mutant gin4 hsl1 kcc4 strain did not display
a significant exacerbation of the defects seen in the
gin4 single mutant (Table 3) (unpublished results). Thus,
of the three Nim1p family kinases in S. cerevisiae, only
Gin4p appears to play a major role in septin organization or function.
In S. pombe, Nim1p directly phosphorylates the Swe1p-related
kinase Wee1p, inhibiting its catalytic activity (15, 49, 64). Given the similarities between the kinase domains of Nim1p, Gin4p, Hsl1p, and Kcc4p, it was recently suggested that the three S. cerevisiae kinases play redundant roles in the
down-regulation of Swe1p (6). Consistent with other evidence
for a direct role of Hsl1p in the down-regulation of Swe1p (43,
44), we observed a mild elongated-bud phenotype in
hsl1 mutants grown to high cell density (Fig.
4B), although not in exponential-phase
cells (Table 3). Moreover, the hsl1 mutation was lethal
when combined with deletion of MIH1 (to enhance any effect
of a partial loss of Swe1p regulation [44]). In
contrast, no such effects were seen in the kcc4 mutant,
which grew well and formed buds of normal shape even when
MIH1 was also deleted (Fig. 4C; Table 3). In addition, the
role of Gin4p in Swe1p regulation seems likely to be an indirect
consequence of its role in septin organization, as similar degrees of
bud elongation were observed upon deletion of GIN4,
CLA4, or NAP1, all of which affect septin
organization (see above). Moreover, bud elongation was neither more
frequent nor more pronounced in the gin4 hsl1 double
mutant or the gin4 hsl1 kcc4 triple mutant than in
the gin4 single mutant (Fig. 4D to F; Table 3),
suggesting that these proteins do not independently inhibit Swe1p.
Thus, of the three Nim1p family kinases in S. cerevisiae, only Hsl1p appears to play a direct role in Swe1p regulation.
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FIG. 4.
Effects of gin4, hsl1, and
kcc4 mutations on bud morphology. The indicated strains were
grown to exponential phase or (for the hsl1 strain) to a
high cell density in YM-P medium at 30°C and examined by DIC
microscopy. (A) YEF473; (B) M-601; (C) M-1031; (D) M-272; (E) M-603;
(F) M-604.
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Interestingly, the mutations that we have examined that affect septin
organization caused a considerably greater Swe1p-dependent elongated-bud phenotype than did deletion of HSL1 in the
same genetic background (see above). Thus, perturbing septin
organization appears to promote both the loss of Hsl1p-mediated Swe1p
down-regulation (so that once septins have been perturbed, deletion of
HSL1 has little or no further effect) and an additional
Hsl1p-independent pathway(s) that impinges on Swe1p-mediated Cdc28p phosphorylation.
Septin-dependent, hierarchical localization of Swe1p, Hsl1p, and
Hsl7p to the neck.
To investigate the basis for the
Swe1p-dependent cell cycle delay observed upon perturbation of septin
organization, we examined the localization of Swe1p, Hsl1p, and Hsl7p
(another negative regulator of Swe1p; see Introduction) using
epitope-tagged versions of these proteins that appeared to be fully
functional (44, 59) (see Materials and Methods). In unbudded
wild-type cells, Swe1p either was not detected or was detected only in
the nucleus (32% of the cells examined) (Fig.
5A). In budded wild-type cells, Swe1p was
detected only in the nucleus (12% of the cells examined), only at the
neck (23% of the cells), or at both locations (39% of the cells)
(Fig. 5A). The Swe1p nuclear staining varied greatly in intensity,
being most intense in unbudded and small-budded cells and essentially
undetectable in postanaphase budded cells. The Swe1p neck staining was
observed predominantly in cells with medium-size or large buds, where
Swe1p appeared to form a ring on the daughter side of the neck (Fig.
5A). As recently reported by others (6, 58), Hsl1p and Hsl7p
were also localized primarily to a ring on the daughter side of the
neck (unpublished results). Double-label immunofluorescence experiments
showed that Hsl1p and Hsl7p precisely colocalized at all stages and
that both Hsl1p and Hsl7p were always localized entirely within the
septin-containing region of the neck (unpublished results).
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FIG. 5.
Localization of Swe1p in wild-type, hsl1
mutant, and hsl7 mutant cells. The indicated strains were
grown to exponential phase in YPD medium at 30°C and stained for
Swe1p (left-hand panels) or DNA (right-hand panels) as described in
Materials and Methods. (A) JMY1441; (B) JMY1477; (C) JMY1475; (D)
JMY1479; (E) DLY1. Strains JMY1441, JMY1477, JMY1475, and JMY1479 all
contain two integrated copies of SWE1myc, whereas strain
DLY1 lacks SWE1myc as a negative control for the antibody
sandwich protocol.
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To determine if the neck localization of Swe1p, Hsl1p, and Hsl7p
depends on the septins, we examined the localization of these proteins
in a temperature-sensitive septin mutant. After a 30-min shift to
restrictive temperature, Hsl1p and Hsl7p were both delocalized from the
neck in the mutant cells (Fig. 6B and C)
but not in control wild-type cells (Fig. 6A). Swe1p was delocalized
from the neck even in wild-type cells under these conditions (Fig. 6A).
However, by 60 min after the temperature shift, Swe1p was again
localized to the necks of wild-type cells but not of septin mutant
cells (strain JMY1498) (unpublished results). Thus, Swe1p, Hsl1p, and Hsl7p are all localized to the neck in a septin-dependent manner.
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FIG. 6.
Septin dependence of Hsl1p and Hsl7p localization. Cells
of the indicated strains were grown to exponential phase in YM-P medium
at 23°C and fixed either before (left-hand panels) or 30 min after
(right-hand panels) a shift to 37°C. (A) Cells of wild-type strains
JMY1441 (SWE1-myc), M-1427 (HSL1-13myc), and
M-1423 (HSL7-3HA) were stained with antibodies to the tagged
proteins. (B and C) Cells of cdc12-6 strains M-1552
(HSL1-13myc) (B) and M-1554 (HSL7-3HA) (C) were
double stained with antibodies to Cdc11p and to the tagged protein.
|
|
In addition to colocalizing at the neck, Hsl1p, Hsl7p, and Swe1p appear
to interact physically with one another (44, 58). Thus, we
determined if the neck localization of each of these proteins depends
on the others. Deletion of either HSL1 or HSL7 eliminated any detectable neck localization of Swe1p (Fig. 5B, C),
whereas deletion of SWE1 (or of MIH1) did not
affect neck localization of either Hsl1p or Hsl7p (Fig. 7A and
B). In addition, deletion of
HSL1 eliminated the neck localization of Hsl7p (Fig. 7B)
(58), whereas deletion of HSL7 did not affect the
neck localization of Hsl1p (Fig. 7A) (6). These data suggest
the existence of a localization hierarchy in which the septins link
Hsl1p to the neck, Hsl1p links Hsl7p to the neck, and Hsl7p links Swe1p
to the neck.
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|
FIG. 7.
Localization of Hsl1p and Hsl7p in hsl7,
hsl1, swe1, and mih1 mutant cells.
Cells of the indicated strains were grown to exponential phase in YM-P
medium at 30°C and examined by fluorescence microscopy to localize
Hsl1p-GFP (A) or fixed and examined by immunofluorescence microscopy to
detect Hsl7p-HA (B). (A) HSL1-GFP strains M-1272
(hsl7), M-1426 (swe1), and M-1158
(mih1). (B) HSL7-3HA strains M-1445
(hsl1), M-1440 (swe1), and M-1421
(mih1).
|
|
In the hsl1 and hsl7 single mutants, as
well as in an hsl1 hsl7 double mutant, Swe1p was still
localized to the nucleus (Fig. 5B to D). However, in these strains,
Swe1p remained detectable in the nuclei of the majority (~90%) of
anaphase and postanaphase cells, where it was rarely (<3%) detected
in wild-type cells (Fig. 5A).
Effects of abnormal septin organization and of actin
depolymerization on the localization of Hsl1p and Hsl7p.
The
abnormally organized septins present in gin4,
cla4, and nap1 mutants appear to retain much of
their function, as judged by the ability of the mutant cells to
localize bud site-selection markers and components of the chitin
synthase III complex (40) (Table 3). Nonetheless, it seemed
possible that changes in the localization of Hsl1p and/or Hsl7p might
contribute to the apparent activation of Swe1p in these mutant strains.
Indeed, both Hsl1p and Hsl7p were undetectable at the neck in 
73% of
gin4, cla4, or nap1 cells
(Fig. 8; Table
5). Double labeling showed that Hsl1p and
Hsl7p were delocalized from the neck even in a majority of the cells
with relatively normal septin-staining patterns (unpublished results).
In contrast to the effect of GIN4 deletion on Hsl1p localization, deletion of HSL1 (or SWE1 or
MIH1) did not affect Gin4p localization to the neck (Table
4). Thus, not only a loss of septin localization (such as seen in
septin mutants; see above) but also more subtle perturbations of septin
organization are associated with delocalization of Hsl1p and Hsl7p (and
thus presumably of Swe1p) from the neck. In contrast, deletion of
HSL1 (Fig. 3G), HSL7 (Table 3), or
SWE1 (unpublished results) produced no obvious perturbation
of septin organization.
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|
FIG. 8.
Loss of Hsl1p and Hsl7p localization in gin4
and cla4 mutants. Strains M-1443 (gin4
HSL1-13myc) (A), M-1451 (cla4 HSL1-13myc) (B),
M-1442 (gin4 HSL7-3HA) (C), and M-1454 (cla4
HSL7-3HA) (D) were grown to exponential phase in YM-P medium at
30°C and examined by immunofluorescence microscopy to localize
Hsl1p-myc or Hsl7p-HA. Arrows indicate the minority of cells that show
localization of the tagged proteins to the neck.
|
|
In contrast to the dramatic effects of septin perturbations, complete
depolymerization of F actin using latrunculin-A (4) had
little effect on the localization of Hsl1p or Hsl7p to the neck (Fig.
9A, 30 min), although prolonged exposure
to the drug caused an eventual reduction in the staining intensity
(Fig. 9A, 3 h). In contrast, Swe1p disappeared progressively and
relatively rapidly from the necks of cells exposed to latrunculin-A,
with a concomitant increase in the proportion of cells displaying
nuclear staining (Fig. 9B). Thus, although perturbations of both actin and the septins promote Swe1p-dependent delays of the cell cycle, such
perturbations have very different effects on the localization of Hsl1p,
Hsl7p, and Swe1p. In this regard, it is also of interest that
temperature shock, which produces a transient depolarization of the
actin cytoskeleton (36), also affects the localization of
Swe1p while having no detectable effect on the localizations of Hsl1p
and Hsl7p (Fig. 6A).
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|
FIG. 9.
Localization of Hsl1p, Hsl7p, and Swe1p following actin
depolymerization. (A) HSL1-13myc (M-1427) and
HSL7-3HA (M-1423) cells growing exponentially in YM-P medium
at 30°C were treated with 100 µM latrunculin-A for 30 min or 3 h, as indicated, and Hsl1p-myc and Hsl7p-HA were localized by
immunofluorescence. Equal exposure times were used for all four panels.
(B) SWE1myc cells (JMY1441) were grown to exponential phase
in YPD medium at 30°C and treated with 100 µM latrunculin-A. The
proportions of preanaphase budded cells displaying Swe1p staining at
the neck ( ) or in the nucleus ( ) were determined at the indicated
times. (Cells with Swe1p at both locations were counted in both
categories.) More than 200 cells were counted per sample.
|
|
| |
DISCUSSION |
Roles for Gin4p, Cla4p, and Nap1p in septin organization.
Previous observations had suggested that both Gin4p and Cla4p are
important for normal organization of the mother-bud neck and that
Gin4p, Cla4p, and Nap1p all function in related processes (2, 8,
16, 40, 62). Consistent with these observations, we found that
both cla4 and nap1 single mutants exhibited
striking defects in septin organization similar to those observed
previously in gin4 mutants. Moreover, the cla4
gin4 and nap1 gin4 double-deletion mutants displayed
significantly more severe defects in septin organization than did any
of the single mutants. It was reported previously that cla4
nap1 double-deletion mutants display a synthetic growth defect
compared to cla4 or nap1 single mutants
(62). Taken together, these synthetic effects appear to rule
out a linear pathway such as that proposed by Tjandra et al.
(62). Instead, the results suggest that Gin4p, Cla4p, and
Nap1p function at least partially in parallel to promote normal septin
organization (Fig. 10).
Gin4p becomes hyperphosphorylated as cells progress through the cell
cycle, with maximal phosphorylation in G2/M; this
hyperphosphorylation requires (and appears to stimulate) Gin4p kinase
activity and depends also on the septins, Cla4p, and Nap1p (2, 13,
62). Because Gin4p localization occurs early in the cell cycle
and does not depend on Gin4p kinase activity (40), the
hyperphosphorylation presumably does not affect Gin4p localization. In
contrast, as efficient localization of Gin4p to the neck requires Cla4p
and Nap1p (Table 4) as well as the septins (40), it seems
possible that Gin4p localization to the neck might be important for its subsequent hyperphosphorylation. In this case, the role of the septins,
Gin4p, Cla4p, and Nap1p in activating Gin4p may be indirect by means of
their promotion of a septin organization that allows Gin4p localization
(Fig. 10).
Swe1p-dependent cell cycle delay upon perturbation of septin
organization.
Septin mutants display a modest G2 delay
accompanied by the formation of highly elongated buds. Similarly, the
disorganization of the septins in gin4, cla4,
nap1, cla4 gin4, and nap1 gin4 mutants is also accompanied by the formation of elongated buds. In all cases,
the elongated-bud phenotypes, but not the defects in septin organization and function, were suppressed by deleting SWE1
or by introducing the nonphosphorylatable
CDC28Y19F allele. The simplest interpretation of
these results is that defects in septin organization promote the
accumulation and/or activation of Swe1p, resulting in lowered Cdc28p
activity and thus in a cell cycle delay associated with continued
apical growth of the bud (Fig. 10).
Barral and coworkers recently reported similar observations on septin
mutants and reached similar conclusions (6). In contrast, Kellogg and coworkers have suggested that the cell cycle delays in
septin and gin4, cla4, and nap1
mutants occur after Cdc28p activation, and therefore that the septins,
Gin4p, Cla4p, and Nap1p must function downstream of Cdc28p in a pathway
for executing the switch from apical to isotropic bud growth (2,
13, 30, 62). However, this conclusion was based largely on
measurements of Clb2p-associated histone H1 kinase activity in vitro,
which might not faithfully reflect the levels of relevant activity in vivo; a further caveat is that the specific activities of Cdc28p were
not measured in the various mutants. Moreover, without additional assumptions, the model of Kellogg and coworkers seems very difficult to
reconcile with the observations that either deletion of SWE1 or introduction of CDC28Y19F suppresses the
elongated-bud phenotypes of the various mutants. However, it should
also be noted that none of the models proposed to date explains why the
apical bud growth of septin mutants apparently continues even after the
cells have undergone nuclear division (presumably marking the end of
the Swe1p-dependent G2 delay) (1, 26).
Distinct roles for Nim1p family kinases in septin organization and
cell cycle control.
Gin4p, Hsl1p, and Kcc4p all contain closely
related kinase domains and are localized to the neck, and at least for
Gin4p and Hsl1p, neck localization appears to be important for
function. Based on these similarities, it has been suggested that these three kinases play redundant roles in both septin organization and cell
cycle control (6). However, we found no evidence for a role
of either Hsl1p or Kcc4p in septin organization, even in the absence of
Gin4p, and other tests for redundancy in function between Gin4p and
Kcc4p (which contain large related nonkinase domains that have little
sequence similarity to the corresponding domain in Hsl1p) were also
negative (40). In this regard, it is also worth noting that
the localization patterns of the three kinases at the neck are
distinct: Gin4p typically colocalizes with the septins on both mother
and bud sides of the neck (40), whereas Hsl1p and Kcc4p
localize specifically to the bud side of the neck (reference
6 and this study). In addition, we found no evidence
for a direct role of either Gin4p or Kcc4p in cell cycle control. Swe1p
is fully stabilized (i.e., is as stable in G2/M as it is in
G1) in cells deleted only for HSL1, suggesting that neither Gin4p nor Kcc4p targets Swe1p for degradation
(44), and kcc4 deletion cells showed no evidence
of a G2 delay even when MIH1 was also deleted
(Fig. 4C). Moreover, although gin4 mutant cells did display
a G2 delay that was actually more pronounced than that seen
in hsl1 mutant cells, this delay seems likely to be an
indirect effect of the septin perturbation in the gin4
mutant cells (Fig. 10). Thus, it appears that the three Nim1p-related kinases of S. cerevisiae play distinct roles in the cell:
Gin4p promotes normal septin organization (40), Hsl1p
down-regulates Swe1p (6, 43, 44), and Kcc4p plays a role
that remains to be discovered.
In this context, recent observations on a second S. pombe
Nim1p family kinase, Cdr2p, are also of interest. Deletion of
cdr2 caused a pronounced Wee1p-dependent G2
delay, but simultaneous deletion of nim1 and cdr2
did not cause a greater G2 delay in proliferating cells
than deletion of cdr2 alone (12, 29). This result
seems inconsistent with the hypothesis that Nim1p and Cdr2p play
redundant roles in cell cycle control. Instead, it suggests that
cdr2 mutations (like gin4 mutations) may cause a
cell cycle delay indirectly, consistent with the hypothesis that Nim1p
family kinases may generally play distinct roles as specified by their
large nonkinase domains.
Septin-dependent recruitment of Hsl1p, Hsl7p, and Swe1p to the
neck.
A clue as to how septin perturbations might affect Swe1p
came from the observations that Hsl1p (6; this
study), Hsl7p (58; this study), and a fraction of
cellular Swe1p (Fig. 5) are all colocalized to the neck. The neck
localization of Hsl1p requires the septins but not Hsl7p or Swe1p; the
neck localization of Hsl7p requires the septins and Hsl1p but not
Swe1p; and the neck localization of Swe1p requires both Hsl1p and
Hsl7p, as well as the septins. Consistent with these findings, recent
biochemical evidence indicates that the septin Cdc3p interacts
physically with Hsl1p (6), that Hsl1p interacts physically
with Hsl7p (58), and that Hsl7p interacts physically with
Swe1p (44, 58). Thus, Hsl1p and Hsl7p appear to function as
part of a septin-dependent hierarchy for localization of Swe1p to the
neck, providing another illustration of the general role of the septins
in organizing other proteins at the cell surface (40, 42).
The colocalization of Swe1p and its negative regulators at the neck
raised the possibility that mislocalization of these proteins might
contribute to the apparent increase in Swe1p activity in mutants with
septin defects. Indeed, we found that even the relatively mild
perturbations of septin organization observed in gin4,
cla4, and nap1 mutant cells were associated with
greatly reduced localization of Hsl1p and Hsl7p (and hence, presumably,
of Swe1p) to the neck. In contrast, two functionally unrelated
proteins, Bni4p and Bud4p, were still localized efficiently to the neck
in gin4 mutant cells (40), suggesting that Hsl1p
and Hsl7p localization may be particularly sensitive to perturbations
of septin organization. The involvement of both Hsl1p and Hsl7p in
targeting Swe1p for degradation during G2/M (44,
58) might simply reflect a role for Hsl1p and Hsl7p in delivering
Swe1p to a location at which the ubiquitination complex
SCFMet30 (28) can target it for degradation.
Alternatively (or in addition), Hsl1p and Hsl7p may play a direct role
in targeting Swe1p for degradation (e.g., through phosphorylation of
Swe1p by Hsl1p [44, 58]). In this case, the
localization of the proteins to the neck might be important for the
activity of Hsl1p and/or Hsl7p. Consistent with this possibility, the
autophosphorylation activity of Hsl1p was reduced in extracts from a
septin mutant strain relative to that in extracts from a wild-type
strain (6). However, it should also be noted that cells with
delocalized Hsl1p appear to retain at least partial Hsl1p activity,
because gin4, cla4, and nap1 mutations
are not lethal in combination with deletion of MIH1 (Fig. 2)
(unpublished results), whereas hsl1 mih1 double mutants
undergo a lethal G2 arrest (44).
The stabilized Swe1p in hsl1 and hsl7 mutants
accumulated in the nucleus, which presumably facilitates its inhibition
of nuclear Clb-Cdc28p complexes. It seems likely that septin
perturbations, by delocalizing Hsl1p and Hsl7p, also cause Swe1p
stabilization and nuclear accumulation, contributing to the observed
G2 delay. It is also possible that changes in Swe1p
specific activity contribute to the G2 delay. Indeed, the
Swe1p-dependent cell cycle delays observed in mutants with perturbed
septin organization were more severe than those observed upon deletion
of HSL1 or HSL7, suggesting that septin
organization affects Swe1p (and/or Mih1p) function through at
least one additional Hsl1p/Hsl7p-independent pathway (Fig. 10).
Why are Hsl1p, Hsl7p, and Swe1p localized to the daughter side of
the neck?
There appear to be at least four possible models to
explain the surprising localization of this regulatory module to the
septin ring at the mother-bud neck. These models are not mutually exclusive.
First, as suggested previously (6, 58), Hsl1p and Hsl7p
might serve as sensors that monitor septin organization as part of a
checkpoint pathway that delays nuclear division if the neck is not well
organized for subsequent cytokinesis. However, it is not known whether
yeast cells in their natural environment ever experience perturbations
of septin organization (for instance, neither temperature shock nor
osmotic shock produces obvious alterations of septin organization
[unpublished results]), and it is unclear how introduction of a short
G2 delay might help cells to cope with such perturbations.
Nevertheless, the finding that deletion of SWE1 exacerbates
the growth defects of certain septin mutants (6;
this study) does suggest that under some circumstances the delay is
beneficial, supporting the hypothesis of a septin-monitoring checkpoint.
Second, as also suggested previously (6), septin
misorganization might serve as a sensitive indicator for more general morphogenetic problems, so that the septin sensor postulated above could in fact cause a checkpoint delay in response to perturbations of
actin organization or of bud formation. However, there is no evidence
suggesting that actin perturbations affect septin organization; for
example, septin rings form normally in cells lacking F actin because of
treatment with latrunculin-A (4), and we found that the
actin perturbations induced by heat shock or by latrunculin-A did not
significantly alter the neck localizations of Hsl1p and Hsl7p (Fig. 6A
and 9). These observations suggest that the mechanisms underlying Swe1p
regulation in response to actin perturbations and to septin
perturbations are distinct. Consistent with this hypothesis, actin
perturbations can promote much longer Swe1p-dependent cell cycle delays
(>15 h [46]) than those observed upon septin perturbation (30 to 45 min).
Third, consistent with other evidence for the septins serving as a
scaffold for the assembly of functional complexes (40, 42),
the neck may simply be a convenient place to concentrate Swe1p together
with its regulators. In this regard, it is interesting that another
cell cycle regulator, Cdc14p, is localized to the nucleolus, where it
is prevented from acting for much of the cell cycle (57,
63). It may be that the neck and the nucleolus provide convenient
sites to store (or inactivate or degrade) cell cycle regulators at
times when they are not needed.
A final model is particularly attractive because it also rationalizes
the strikingly asymmetric localization of the regulatory module to the
daughter side of the neck. In particular, this model proposes that the
ability of the module to assemble in this location serves as an
indicator that a bud has been formed. Whereas unbudded and small-budded
cells undergo a Swe1p-dependent G2 arrest upon depolymerization of F actin, larger-budded cells do not
(46). However, constitutive but modest Swe1p overexpression,
which is insufficient to delay the normal cell cycle, allows
larger-budded cells to arrest in G2 in response to actin
depolymerization (46). This observation suggests that
large-budded cells retain the ability to sense actin perturbations but
do not normally contain sufficient Swe1p to enforce a G2
arrest. Perhaps, once a bud has been formed, the cells no longer
require the morphogenesis checkpoint, and Swe1p is then degraded so
that subsequent actin perturbations do not affect the cell cycle.
Assembly of the module on the daughter side of the neck presumably
relies on a particular septin organization that is unique to budded
cells. Activation of Hsl1p and Hsl7p upon such assembly could therefore
couple Swe1p degradation to bud formation.
We thank the other members of the Pringle and Lew laboratories
for providing great working environments and many valuable discussions.
We also thank Yves Barral, Mike Snyder, Doug Kellogg, Mark Shulewitz,
and Jeremy Thorner for valuable discussions and communication of
unpublished results. We also thank Susan Whitfield for her usual
outstanding assistance with the illustrations.
This work was supported by NIH grant GM31006 to J.R.P. and by NIH grant
GM53050 and funds from the Searle Scholars Program/The Chicago
Community Trust to D.J.L.
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Kanoh, J., and P. Russell.
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The protein kinase Cdr2, related to Nim1/Cdr1 mitotic inducer, regulates the onset of mitosis in fission yeast.
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Abstract
Introduction
Present address: Department of Biochemistry and Molecular Biology,
Oklahoma State University, Stillwater, OK 74078-3035.
