Download In yeast, the pseudohyphal phenotype induced by isoamyl alcohol

Research Article
3423
In yeast, the pseudohyphal phenotype induced by
isoamyl alcohol results from the operation of the
morphogenesis checkpoint
Claudia Martínez-Anaya1, J. Richard Dickinson2 and Peter E. Sudbery1,*
1Department of Molecular Biology and
2Cardiff School of Biosciences, Cardiff
Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
University, PO Box 915, Cardiff CF10 3TL, UK
*Author for correspondence (e-mail: [email protected])
Accepted 25 April 2003
Journal of Cell Science 116, 3423-3431 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00634
Summary
Isoamyl alcohol (IAA) induces a phenotype that resembles
pseudohyphae in the budding yeast Saccharomyces
cerevisiae. We show here that IAA causes the rapid
formation of linear chains of anucleate buds, each of which
is accompanied by the formation of a septin ring at its neck.
This process requires the activity of Swe1 and Slt2 (Mpk1).
Cdc28 is phosphorylated on tyrosine 19 in a Swe1dependent manner, while Slt2 becomes activated by
dual tyrosine/threonine phosphorylation. Tyrosine 19
phosphorylation of Cdc28 is not dependent on Slt2.
However, the defective response in the slt2∆ mutant
is rescued by an mih1∆ mutation. The IAA response
Introduction
The yeast Saccharomyces cerevisiae can grow either in a
unicellular budding form or in branching chains of elongated
cells known as pseudohyphae (Gimeno et al., 1992).
Pseudohyphae form in diploids in response to nitrogen-limited
growth, and in other stress and starvation conditions. It is
thought that pseudohyphae formation is a strategy by which a
normally sessile organism can forage for microenvironments
that are more favourable. The pathways by which
pseudohyphae form in response to nitrogen-limitation have
been extensively studied (for a review, see Rua et al., 2001).
Two signal transduction pathways are required involving the
mating pheromone MAP kinase module and cAMP/PKA,
respectively. These pathways converge on elements in the
complex promoters of genes such as FLO11. Filamentous
growth requires an extended G2 and a delay in the switch from
polarised to isotropic growth of the bud (Kron et al., 1994).
This involves inhibition of the activity of the Clb2-Cdc28
kinase, but the way in which this comes about is currently
unclear. However, it is known that it does not depend upon
inhibitory tyrosine phosphorylation by Swe1, because
pseudohypha formation occurs normally during nitrogenlimited growth in a swe1∆ mutant (Ahn et al., 1999).
Pseudohyphae can also form upon exposure to branched-chain
or ‘fusel’ alcohols such as isoamyl alcohol (IAA) and 1-butanol
(Dickinson, 1996; La Valle and Wittenberg, 2001; Lorenz et al.,
2000). Fusel alcohols are formed by the catabolism of branchedchain amino acids such as leucine. Reliance on such a poor
still occurs in a cell containing a dominant nonphosphorylatable form of Cdc28, but no longer occurs in
an mih1∆ slt2∆ mutant containing this form of Cdc28.
These observations show that IAA induces the Swe1dependent morphogenesis checkpoint and so the resulting
pseudohyphal phenotype arises in an entirely different way
from the formation of pseudohyphae induced by nitrogenlimited growth.
Key words: Morphogenesis checkpoint, Isoamyl alcohol,
Pseudohyphae, SLT2, SWE1, Yeast
nutrient source may also act to trigger pseudohyphae formation
as a foraging mechanism to search for better conditions. The
mechanism by which fusel alcohols trigger pseudohyphae
formation is poorly understood, but it is clearly different from
that operating during nitrogen-limited growth because it has a
distinct set of genetic requirements. For example, butanolinduced pseudohyphal formation is dependent on SWE1 (La
Valle and Wittenberg, 2001) but is independent of genes such as
FLO8 and FLO11 that are required for pseudohyphal induction
during nitrogen-limited growth (Lorenz et al., 2000). Recently,
it has been shown that 1-butanol causes a rapid cessation of
translation by targeting the eIF2B translation initiation factor
(Ashe et al., 2001). This effect is strain specific, affecting only
certain samples of the W303-1A strain. The difference between
the strains was tracked down to a P180S variation in Gcd1. As
both strain subtypes reacted to 1-butanol by forming filaments,
the relevance of butanol-induced inhibition of translation to
filament formation is currently unclear.
We show that shortly after exposure to IAA, a series of small
buds form accompanied by the appearance of multiple ectopic
septin rings in the absence of nuclear division. These events
are wholly dependent on Swe1, which inhibits Clb2-Cdc28 by
phosphorylation of tyrosine 19. We further show that this
process is dependent on Slt2 (Mpk1), the cell integrity MAP
kinase, and that Slt2 is activated upon exposure to IAA.
However, we show that tyrosine phosphorylation of Cdc28 is
not dependent on Slt2, instead Slt2 acts as a negative regulator
of Mih1, the tyrosine phosphatase that reverses the inhibitory
3424
Journal of Cell Science 116 (16)
90
A
0 buds
1 bud
2 buds
3 buds
4 buds
5 buds
B
80
70
% cells
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
8
5
6
7
8
300
360
420
480
time (h)
25
C
nuclei at the ends
branched buds
nucleus in the filament
>2 nuclei
% cells
20
15
10
5
0
0
1
2
3
4
time (h)
1000
cells ml-1 (106)
D
100
10
1
0
60
120
180
240
time (min)
Fig. 1. Bud formation is uncoupled from nuclear division in diploid cells treated with IAA. An exponentially growing heterozygous
CDC3/CDC3-GFP strain was incubated in YEPD (1% Difco yeast extract, 2% Difco-Bacto Peptone and 2% glucose) plus 0.5% IAA. Samples
were removed at 1-hour intervals and stained with DAPI. The samples were examined with DIC microscopy to record overall appearance, and
fluorescence microscopy to visualise CDC3-GFP (green) or nuclei (blue). (A) Filaments representative of the changes observed. The time of
sampling is indicated in each panel. Arrows in the 1- and 2-hour samples indicate examples of multiple septin patches; the solid arrow in the 8hour sample (right) indicates a branch forming and the open arrow (left) a double septin ring. Scale bar is 5 µm. The figure does not show
examples from all the time points analysed in B and C. (B) The number of buds produced per filament over the 8 hours examined. Over 100
filaments were analysed for each time point. (C) The number of nuclei per filament. Filaments were categorised as indicated, according to
whether they had nuclei in a large cell at either end of the filament or a nucleus within the chain of small buds. (D) At the indicated time points,
samples were withdrawn and fixed with 2.5% w/v formaldehyde and briefly sonicated. Cell number was determined, counting each clump or
filament as a single cell. Filled circles, no IAA; open circles, plus IAA.
phosphorylation applied by Swe1. Taken together these
observations show that IAA acts by inducing the Swe1dependent morphogenesis checkpoint.
Materials and Methods
Strains
Table 1 shows strains used in this study, all were congenic to Σ1278b.
All experiments were carried out at 26°C.
Gene manipulations
Gene deletions and the Cdc3-GFP fusion, were constructed as
described (Longtine et al., 1998), using the plasmids pFA6a-kanMX6
and pFA6a-GFP-(S65T)-kanMX6, respectively. The integrity of all
constructs was confirmed by PCR. A full list of oligonucleotides used
is presented in Table 2.
Protein extractions and western blotting
Cells were harvested by centrifugation, washed in PBS and snap-
Isoamyl alcohol causes uncontrolled budding in yeast
3425
Table 1. Strains
Name
Genotype
Σ1278h
Σ1278b
CMS2
CMS18
CMS19
CMS50
CMS55
SKY903
CMS77
Source
MATa ura3-52
MATa/α ura3-52/ura3-52
MAT a/α CDC3/CDC3-GFP::kanMX6 ura3-52/ura3-52
MATα slt2∆::kanMX6 ura3-52
MATα slt2∆::kanMX6 ura3-52 trp1∆::URA3
MATa swe1∆::kanMX6 ura3-52
MATa/α swe1∆::kanMX6 /swe1∆::CaURA ura3-53
MATα mih1∆::LEU2
MATα mih1∆::LEU2 slt2∆::kanMX6
G. Fink, Cambridge, MA, USA
G. Fink
This study
This study
This study
This study
This study
S. Kron, Chicago, IL, USA
This study
Table 2. Oligonucleotides
Name
SWE1-1
SWE1-2
SWE1-3
SWE1-4
KanMX
CDC3-1
CDC3-2
CDC3-3
CDC3-4
SLT2-1
SLT2-2
SLT2-5
SLT2-4
Sequence
GAT TAC TAC TGA ACA GGT CTT ACT ATT TTT GAT
TGC GTA GCG GAT CCC CGG GTT AAT TAA
ATT GGA TTA TTT ATA CAA TGA GGA CCA TAA GCA
CGT GTG GGA ATT CGA GCT CGT TTA AAC
CGT CTC TAG TAC TGG TAA GC
CGT CTT GTT GGA GTG GAG AT
GAT GGT CGG AAG AGG CAT AA
CCA CTC CCC CGT CCC TAC AAA GAA GAA GGG ATT
TTT ACG TCG GAT CCC CGG GTT AAT TAA
TAA TAG TGT ATG TTT GAA ATT TTT ATA TGT CTT TAT
TTC GGA ATT CGA GCT CGT TTA AAC
AAC AGC TAG AAC TTT CAA TA
GTT AAT TCT GAG CTA ATC AT
TCG GGT ATT TCC AGT GGC AGG TCT CAT CTC CAT CAT
ACT CCG GAT CCC CGG GTT AAT TAA
AAA GAA ATA GGG CAT GGA GCA TAC GGC ATA GTG TGT
GCA GGA ATT CGA GCT CGT TTA AAC
TTC AAG GTC TAG AAG CGT GC
CGA AGA TAC CAC AGT TGC CA
frozen in liquid nitrogen. After thawing, cells were washed once in
STOP solution (1× PBS + 10 mM NaN3 + 50 mM NaF) and once in
20% trichloroacetic acid (TCA). Pellets were resuspended in 200 µl
of 20% TCA with glass beads. Cells were disrupted by three 10second cycles of agitation in a Ribolyser (Hybaid) set at a speed of
6.5. Extracts were separated from glass beads by piercing a hole at
the bottom of the Eppendorf tube and centrifuging at 2000 g for
5 minutes. TCA precipitated proteins were then obtained by
centrifuging 5 minutes at 28,000 g and by discarding supernatant. The
pellet was resuspended in 200 µl of 2× electrophoresis sample buffer
Orientation
Function
Forward
Deletion
Reverse
Deletion
Forward
Reverse
Reverse
Forward
Confirmation
Confirmation
Confirmation
GFP-tagging
Reverse
GFP-tagging
Forward
Reverse
Forward
Confirmation
Confirmation
Deletion
Reverse
Deletion
Forward
Reverse
Confirmation
Confirmation
containing 250 mM Tris pH 8 and boiled for 5 minutes.
Electrophoresis gels were loaded with 45 µl of this TCA extract.
Antibodies used for detection of proteins were as follows:
anti-phospho-tyro-Cdc28 (Y19) (Cdc2-Tyr15; Cell Signaling
Technology); anti-Cdk1/Cdc2 (PSTAIR) (Upstate Biotechnology);
anti-phospho p44/42 (Slt2) (New England Biolabs); anti-rabbit IgG
(H+L) (Jackson ImmunoResearch Labs); anti-mouse IgG-HRP and
anti-rabbit IgG-HRP (BabCO). Detection of phospho-Cdc2 (Tyr15)
and of diphospho-Slt2 (using a three antibody protocol to enhance
sensitivity), were done as described previously (Harrison et al., 2001).
Bands were visualised with ECL solution (Pharmacia)
using the western blot reader GeneGnome (Syngene
Bio Imaging). Images were acquired with GeneSnap
4.00.00 (Synoptics Ltd), and analysed using GeneTools
Fig. 2. IAA uncouples bud formation from nuclear
division in haploid cells. Exponentially growing
haploid Σ1287h cells were added to YEPD containing
0.5% IAA and incubated for 24 hours. (A) Cells
stained with DAPI. (B) DIC images of the cells shown
in A. Open arrow, wide filaments; solid arrow, narrow
filaments; solid arrows with barbed tails, filaments
with a mixture of wide and narrow compartments.
(C) Cells were treated as above for 8 hours and Cdc11
visualised by immunocytofluorescence using an antiCdc11 antibody as described previously (Sudbery,
2001). (D) The same cells as in C, stained with DAPI.
Septin rings form in the absence of nuclear division.
(E) Cells were grown for 16 hours in YEPD plus 0.5%
IAA, treated with zymolyase and stained with DAPI.
Cells that contain nuclei separate after the zymolyase
treatment. Scale bars: 10 µm (A,B,E); 5 µm (C,D).
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Journal of Cell Science 116 (16)
resuspended in 1× PBS buffer and 1 µl of DAPI (0.05
mg/ml) was added to 50 µl of cell suspension and incubated
at room temperature for 1 hour. Actin was visualised using
TRITC-conjugated phalloidin (Sigma) following the
published protocol (Lee et al., 1998).
Zymolyase treatment
Cells were washed twice with 500 µl of 0.1 M K2HPO4, 0.1
M KH2PO4 and 1.2 M sorbitol, resuspended in 500 µl of the
same solution plus 1.2 µl β-mercaptoethanol and 20 µl of 5
mg/ml zymolyase 20-T, and incubated for 40 minutes at
37°C
Flow cytometry analysis
One ml samples of cell culture were centrifuged at 4000 g
for 3 minutes and washed in 200 µl of distilled water; they
were then fixed with 1 ml of ice-cold 70% ethanol and
stored at 4°C. The fixed cells were collected by
centrifugation at 4000 g for 3 minutes, and then resuspended
in 1 ml of 50 mM sodium citrate (pH 7.0). RNA was
digested by the addition of 25 µl of 20 mg/ml RNase A,
followed by incubation for 3 hours with gentle agitation at
37°C. Then, 500 µl of 5 µg m–1 propidium iodine was added
and the samples were kept at 4°C overnight. Before flow
cytometry, cells were briefly sonicated to disrupt clumps.
DNA content was analysed in a Beckton-Dickinson cell
sorter (Franklin Lakes, NJ, USA). After fixation, haploid
cells were treated with zymolyase, as described above, to
separate cells in filaments.
Microscopy
Cells were examined with a Leica DMLB fluorescence
microscope. Digital images were acquired with a cooled
CCD camera (Princeton instruments, model RTE) linked to
an Apple Macintosh G4 computer running Open Lab
software version 2.2.5 (Improvison, Warwick, UK). Images
were exported as TIF files and edited in Adobe Photoshop
version 5.5.
Fig. 3. The response to IAA is dependent on SWE1. (A) A haploid Σ1278h
MATα strain (wild type; wt) and a swe1∆ derivative were incubated for 16
hours in YEPD alone or YEPD plus 0.5% IAA, and photographed using DIC
optics. No filaments were produced by the SWE1 mutant upon IAA treatment.
Scale bar: 10 µm. (B) Flow cytometry traces showing relative DNA content in
untreated Σ1278h cells, and cells exposed to 0.5% IAA for the indicated
times. The proportion of cells in G1 and G2 was determined for each time
point and the proportion of cells in G2 in the treated (open symbols) and
untreated (closed symbols) cultures is plotted against time.
3.00.22 (Synoptics Ltd). Loading controls were either Cdc11, detected
using rabbit anti-Cdc11 polyclonal antisera (Santa Cruz), or Cdc28
detected by rabbit anti-PSTAIR polyclonal antisera (Upstate
Biotechnology). Each experimental value was normalised with respect
to the signal from the appropriate loading control.
Fixation, DAPI and phalloidin staining
Cell suspensions were sonicated for three seconds to separate cells
that remained associated despite having completed cytokinesis. When
cells were to be examined for GFP and DAPI fluorescence, they were
mixed with an equal volume of mounting medium containing 0.1
mg/ml DAPI. Cells that were only to be examined for DAPI staining
were fixed in 70% ethanol for 1 hour and then washed and
Results
Isoamyl alcohol induces the rapid formation of
chains of small buds
To date, investigations on the action of isoamyl
alcohol (IAA) have reported the effects of prolonged
exposure in liquid culture or the appearance of microcolonies on agar plates. We investigated the early
events upon exposure to IAA. Strain CMS2 was
constructed containing a heterozygous CDC3/CDC3GFP fusion in the Σ1278b background, which shows the
strongest response to IAA. Preliminary experiments
established that 0.5% IAA was the optimum concentration to
give a clear response without affecting viability.
CMS2 was exposed to 0.5% IAA in liquid culture, and
samples were withdrawn at 1-hour intervals for 8 hours. The
samples were examined by DIC microscopy to record their
overall appearance, fluorescence microscopy to visualise
Cdc3-GFP and DAPI-stained to visualise the nuclei. Fig. 1A
shows representative cells at various times during the course
of the experiment Within 1 hour of IAA treatment, small
ectopic patches of septin appeared at the tips of pre-existing
buds (arrows, 1- and 2-hour time points). This was followed
Isoamyl alcohol causes uncontrolled budding in yeast
3427
Fig. 4. Cells treated with IAA show polarisation of
the actin cytoskeleton. Wild-type haploid cells
were treated with 0.5% IAA. After 4 hours cells
were fixed and stained with TRITC-conjugated
phalloidin. The figure shows a collage of typical
cells. Scale bar: 5 µm.
by the appearance of further unipolar buds forming chains of
up to five small buds projecting from the mother cell by 8
hours. The formation of each bud was accompanied by the
appearance of a bright septin ring at its neck, which often coexisted with the fainter ring that remained after the previous
round of budding (e.g. 2-hour time point). Quantification
showed that the interval between the formation of each bud was
approximately 60 minutes (Fig. 1B), which is faster than the
110-minute doubling-time of the untreated control culture (Fig.
1D). By 8 hours, 50% of the cells had two or more buds (Fig.
1B). In about 20% of the filaments, large round cells were
observed at either end of the chain (Fig. 1A,C). Some filaments
produced side branches (arrow, Fig. 1A).
Initially, the nucleus in the mother cell remained undivided.
However, after 5 hours mitosis began in some cells, and
filaments with more than one nucleus appeared (Fig. 1A, 6 and
8-hour time points). By 8 hours approximately
28% of the filaments contained two nuclei
(Fig. 1C). The majority (22.5% of the total
population) of these nuclei were in a large cell
at each end of a chain of small buds. One of
these round cells often displayed a double septin
ring at the junction with the filament (open
arrow, Fig. 1A, 8-hour time point), suggesting
that events leading to septum formation were
resuming. In a smaller fraction of filaments at 8
hours, one of the nuclei was located in the chain
of small buds (5% of the total). Filaments with
more than two nuclei appeared after 6 hours, but
such filaments had disappeared from the
population by 8 hours. The proportion of cells
with two nuclei did not increase after 24 hours
incubation. Consistent with the delay of nuclear
division, cell number increase was immediately
halted for 4 hours after IAA addition before
showing a slow increase, possibly due to the
resumption of nuclear division and cytokinesis
(Fig. 1D). Overall, these data show that the
filaments produced upon IAA treatment arise
through repeated rounds of budding, in the
absence of nuclear division.
Haploid cells also responded to the IAA
exposure
by producing chains of anucleate buds,
Fig. 5. Filament formation depends on SLT2. (A) Appearance of slt2∆ cells
although the process took longer than in diploid
incubated for 16 hours in YEPD or YEPD plus 0.5% IAA. Scale bar: 10 µm.
cells. After 8 hours exposure to IAA, only one
(B) Samples of wild-type cultures in the log-phase of growth were taken at the
or two anucleate buds had formed (data not
indicated times after addition of 0.5% IAA. Total protein extracts were probed by
shown). However, by 24 hours, chains of up to
western blotting with anti-active (p44/42) Slt2 antisera. Values on the ordinate are
the ratio of each experimental signal to its loading control.
four buds had formed in 100% of the cells (Fig.
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Journal of Cell Science 116 (16)
fusion protein was mis-localised into large cytoplasmic bars,
even in cells not treated with IAA. We therefore used
immunocytofluorescence with polyclonal antisera raised
against Cdc11 to investigate whether multiple septin rings
formed in haploids (Fig. 2C,D). As with diploids, septin rings
were also observed in the absence of nuclear division.
Immunocytofluorescence requires the cell wall to be removed
with zymolyase, however, the compartments remain associated
showing that they are coenocytic (Fig. 2C,D). After 16 hours
culture, the compartments that contained nuclei separated upon
zymolyase treatment, showing that they were physiologically
autonomous (Fig. 2E).
Fig. 6. Tyrosine phosphorylation of Cdc28 is Swe1 but not Slt2dependent. Cultures of the indicated genotype were grown to mid-log
phase and treated with 0.5% IAA. At the indicated times, total
protein extracts were obtained by TCA precipitation and probed by
western blotting with anti-phosphotyro-Cdc28 (anti-pY19) and antiactive Slt2 antisera. The membrane was then stripped and probed
with anti-PSTAIR antisera. Levels of activated Slt2 and phosphotyroCdc28 were determined using the signal from the lower band
(Cdc28) in the anti-PSTAIR panel as a loading control. Values on the
ordinate are the ratio of each experimental signal to its loading
control.
2). These cells showed considerable heterogeneity in the width
of the filaments: some were the same width as the mother cell
(barbed arrows Fig. 2B), others were much narrower (open
arrows Fig. 2B), while some contained a mixture of wide and
narrow compartments (filled arrows with barbed tails Fig. 2B).
After 24 hours exposure, the filaments become populated with
nuclei: of 173 filaments examined, 56 (32%) remained
anucleate, 59 (34%) had one nucleus in the filament, 29 (17%)
had nuclei at both ends, and 29 (17%) had more than one
nucleus within the filament. There was no obvious pattern in
the way that the compartments of the filaments acquired nuclei
as mitosis was observed at both the neck of the filament and
the mother cell or wholly within the filament. As was the case
in diploids, where there was a nucleus at both ends, the
compartments containing the nuclei were large and round.
We found that in a haploid ∑1278h strain, the Cdc3-GFP
The response to IAA requires Swe1
In S. cerevisiae, the morphogenesis checkpoint has been shown
to delay nuclear division when bud formation has been
disrupted. It is triggered in two different situations. First, when
the actin cytoskeleton has become depolarised because of
genetic lesions such as cdc24 or cdc42 mutations or by
treatment with latrunculin A (Lew and Reed, 1995a; Lew and
Reed, 1995b; McMillan et al., 1998). Second, when septin ring
formation has been disrupted by mutations affecting the
component septins or proteins such as Elm1, Cla4 and Gin4
that are required for the proper organisation of the septins
(Barral et al., 1999). The delay in nuclear division observed
above led us to ask whether Swe1 was involved in the effects
seen with IAA. We found that a swe1∆ mutation abrogated the
response to IAA in haploid (Fig. 3A) and diploid strains (data
not shown). This observation has recently been independently
reported elsewhere (La Valle and Wittenberg, 2001). Swe1
phosphorylates Cdc28 at tyrosine 19 (Sia et al., 1996).
Consistent with this, IAA treatment resulted in tyrosine
phosphorylation of Cdc28 in a Swe1-dependent manner (Fig.
6). Such inhibitory phosphorylation of Cdc28 would be
expected to delay mitosis. Indeed, FACS analysis showed an
increase in the number of cells in G2 (Fig. 3B), indicating that
mitosis was delayed, although there was not a complete block.
The peak of phosphotyro-Cdc28 levels at 3 hours (Fig. 6)
corresponds to start of the increase in the number of cells in
G2 (Fig. 3), whereas the fall after 5 hours may be responsible
for the resumption of nuclear division (Fig. 1C).
Actin is polarised after IAA treatment
The morphogenesis checkpoint is induced by defects in actin
polarisation or in the septin cytoskeleton (see above). Since
septin rings appear normal during IAA treatment, we
investigated whether there were any defects in actin
polarisation. As shown in Fig. 4, actin was highly polarised
towards the tip of the growing bud chains during IAA
treatment. Therefore, a failure to polarise actin is unlikely to
be responsible for the induction of the morphogenesis
checkpoint.
The effect of IAA is dependent on Slt2, the cell integrity
MAP kinase
The cell integrity Pkc1/Slt2 MAP kinase pathway has been
shown to be required for the operation of the morphogenesis
checkpoint, although tyrosine phosphorylation of Cdc28
Isoamyl alcohol causes uncontrolled budding in yeast
3429
some differences compared with wild type,
phosphorylation of Slt2 still occurred in a
swe1∆ mutant, indicating that Slt2 activation
is not downstream of Cdc28 tyrosine
phosphorylation. Interestingly, activation of
Slt2 peaks after 1 hour while the peak of Cdc28
phosphorylation occurs after 3 hours (Fig. 6).
Thus, Slt2 activation occurs much more rapidly
than Cdc28 tyrosine phosphorylation.
During the operation of the morphogenesis
checkpoint, Cdc28 tyrosine phosphorylation is
controlled both by the rate of phosphorylation by
Swe1 and dephosphorylation by Mih1 (Sia et al.,
1996). Harrison et al. (Harrison et al., 2001)
showed that during the operation of the
checkpoint induced by actin depolarisation, an
mih1∆ mutation restored the checkpoint
function that was defective in an slt2∆ mutant,
suggesting that Slt2 acts as a negative regulator
of Mih1, not an upstream activator of Swe1. This
hypothesis predicts that an mih1∆ mutation
should rescue the filamentation defect of an
slt2∆ mutant. We determined whether this was
the case in the response to IAA. Fig. 7 shows
that an mih1∆slt2∆ double mutant does filament
in response to IAA in a manner identical to that
seen in a wild-type strain.
Discussion
IAA uncouples the nuclear and budding
cycles
We have shown here that the effect of exposure
of S. cerevisiae cells to IAA is the appearance
of a series of small buds that form at a rate
faster than the doubling-time of the parent
strain. At the same time, nuclear division is
delayed. As a result, linear chains of small
anucleate buds extend from the mother cell
after 3 hours. In many cells, the first bud
Fig. 7. The slt2∆ filamentation defect is rescued by mih1∆. Cells of the indicated
appears on the surface of a very small bud that
genotype were incubated in YEPD or YEPD plus 0.5% IAA for 16 hours. Scale bar:
has formed just prior to the IAA exposure, and
20 µm.
before the occurrence of mitosis and
cytokinesis that would normally be required to
persists in an slt2∆ mutant (Harrison et al., 2001). We
permit the daughter cell to bud. This loss of budding control
determined whether Slt2 is required for the IAA-induced
is accompanied by loss of both temporal and spatial control
response and found that an slt2∆ mutant failed to respond to
of septin localisation. Patches of septin appear before each
IAA by producing filaments (Fig. 5A). Slt2 is activated by
successive bud, and in some cells, multiple septin patches can
dual threonine and tyrosine phosphorylation by Mkk1 and
be seen. After 5 hours nuclear division resumes and
Mkk2. Upon IAA treatment of a wild-type cell, such
approximately 28% of the filaments eventually become
phosphorylation is detected with polyclonal antisera specific
binucleate. The majority of binucleate filaments were found
to the active diphospho form of Slt2 within 30 minutes
in cases where large round cells were located at either end of
(Harrison et al., 2001; Martin et al., 2000) (Fig. 5B). Tyrosine
chains of between three and five anucleate buds. A similar
phosphorylation of Cdc28 persisted in an slt2∆ mutant (Fig.
process occurs in haploid cells, although the process is a
6), indicating that although Slt2 is required for the response
little slower so that chains of buds were only fully formed
to IAA, it does not act upstream of Cdc28 tyrosine
after 24 hours. These filaments contain a higher proportion
phosphorylation. We also investigated whether tyrosine
of nucleated cells than was observed in diploids and there
phosphorylation of Cdc28 acted upstream of Slt2 activation
was also considerable heterogeneity in the width of the
by examining the effect of a swe1∆ mutation. The membrane
filaments. It is not clear why the process should take longer
that was probed with anti-phosphotyro-Cdc28 was also
in haploids; however, it is clear that a similar mechanism is
probed with anti-active Slt2 antisera. Although there were
operating.
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Journal of Cell Science 116 (16)
IAA induces the morphogenesis checkpoint
The delay in cell division and continued budding observed upon
IAA treatment suggested to us that the morphogenesis
checkpoint has been induced. This checkpoint ensures that
mitosis only takes place when bud formation is proceeding
normally. It has been extensively studied in two situations.
Firstly, when the actin cytoskeleton has been depolarised by
latrunculin A or by cdc24 or cdc42 mutations (Lew and Reed,
1995a; Lew and Reed, 1995b; Lew and Reed, 1993; McMillan
et al., 1998). Secondly, where mutations have disrupted the
organisation of the septin ring (Barral et al., 1999; Bouquin et
al., 2000; Longtine et al., 2000; Sreenivasan and Kellogg, 1999).
Both of these situations cause a delay of mitosis. In addition,
disruption of the septin ring prolongs polarised growth of the bud,
resulting in cells that are similar in appearance to those treated
with IAA. The checkpoint is mediated by the inhibitory tyrosine
19 phosphorylation of Cdc28 by Swe1 and thus cannot occur in
swe1∆ mutant. The checkpoint also requires the action of the
Pkc1/Slt2 MAP kinase pathway (Harrison et al., 2001). However,
checkpoint function is restored in a slt2∆ mih1∆ mutant.
We have shown that filament formation following IAA
treatment in both haploids and diploids requires Swe1 and that
Cdc28 becomes tyrosine-phosphorylated in a Swe1-dependent
fashion. Furthermore, the IAA response is also dependent on
Slt2 and we have shown that Slt2 is rapidly activated by
phosphorylation upon IAA treatment. Moreover, as is the case
when the checkpoint is induced by actin depolarisation, Cdc28
phosphorylation is not dependent on Slt2, but the loss of
checkpoint function in an slt2∆ mutant is rescued by an mih1∆
mutation. Taken together these observations strongly suggest
that the same Swe1-dependent, morphogenesis checkpoint
operating upon latrunculin A treatment also operates to delay
mitosis in response to IAA treatment.
Harrison et al. interpreted the rescue of checkpoint function
in a slt2∆ mih1∆ mutant by placing Slt2 as a negative regulator
of Mih1. This model satisfactorily explains the genetic data.
However, we reproducibly failed to see any decrease of
phophotyro-Cdc28 in an slt2∆ mutant. Moreover, significant
levels of phosphotyro-Cdc28 clearly persisted in an slt2∆
mutant upon latrunculin A treatment (Harrison et al., 2001).
One explanation for this inconsistency is that the western blots
using the anti-phosphotyro Cdc28 antibody do not detect the
residual levels of active Cdc28 and that in the slt2∆ mutant,
sufficient active Cdc28 remains to abrogate the checkpoint. An
alternative explanation is that the Western blots report the total
cellular level of phosphotyro Cdc28, while the Slt2/Mih1
pathway may act to dephosphorylate Cdc28 at a specific
location where its action is critical for cell cycle progression.
These explanations are not mutually exclusive.
La Valle and Wittenberg (La Valle and Wittenberg, 2001)
also showed that Swe1 was required for the production of
pseudohyphae in response to 1-butanol and we have found that
pseudohyphae do not develop in response to 1-butanol in
swe1∆ and slt2∆ mutants. Moreover, the response was restored
in an slt2∆ mih1∆ double mutant (data not shown). Thus the
induction of filamentous growth in response to 1-butanol is also
likely to be due to the morphogenesis checkpoint. In the light
of these results, we suggest that previous reports of alcoholinduced filamentous growth (Lavalle and Wittenberg, 2001;
Lorencz et al., 2000) should be re-interpreted as acting through
the morphogenesis checkpoint.
It is not clear why IAA induces the morphogenesis
checkpoint. Polarisation of the actin cytoskeleton, bud
formation and septin ring formation are not affected by IAA
treatment. It is possible that the initial formation of ectopic
septin patches triggers the checkpoint. However, the septin
rings that subsequently form appear normal. It is more likely
that IAA is acting to trigger the morphogenesis checkpoint for
some other reason. If this is the case, then IAA forms a third
trigger of the morphogenesis checkpoint in addition to defects
in the actin and septin cytoskeletons. The induction of the
checkpoint when both the actin and septin cytoskeletons are
functioning normally, shows that operation of the checkpoint
does not simply result in prolonged polarised growth, but
apparently drives new rounds of bud formation including the
formation of new septin rings.
The mechanism of pseudohyphal development in
response to IAA is entirely different from the one that
occurs in response to nitrogen-limited growth
Both IAA and nitrogen-limited growth induce the formation of
pseudohyphae that look superficially similar and share some
common requirements, such as components of the mating
pheromone-response MAP-kinase pathway. However, our
results presented here show that the production of
pseudohyphae occur differently in each case. In the case of
pseudohyphae induced by nitrogen-limited growth, G2 is
extended producing elongated cells, but no anucleate buds
form. Moreover, although Swe1 has been reported to contribute
to the formation of nitrogen-limited pseudohyphae in certain
conditions (La Valle and Wittenberg, 2001), it is not essential
for the formation of pseudohyphae during nitrogen-limited
growth because normal pseudohyphae are generated in a
homozygous swe1∆/swe1∆ mutant (Ahn et al., 1999) (our
unpublished observations).
At this stage it is not clear whether the induction of the
morphogenesis checkpoint by IAA is a response to a pathology
or whether the morphogenesis checkpoint plays a role in the
normal physiological response to an agent that is commonly
encountered by yeast cells in their natural environment. If
the response is physiological, then the elongated cell chains
resulting from IAA treatment, may still be regarded as
pseudohyphae. The Swe1-dependent pathway inducing this
phenotype would thus be an alternative route to the production
of pseudohyphae in addition to the pathways operating in
response to nitrogen-limited growth. In this connection it is
interesting to note that La Valle and Wittenberg (La Valle and
Wittenberg, 2001) found that Swe1 was required for the
residual pseudohyphal formation observed in the tec1∆ mutant
in response to nitrogen-limited growth. Thus the Swe1 pathway
may contribute to pseudohyphal formation in response to
nitrogen-limiting growth, but does not play an essential role
under normal circumstances.
C.M.A. was supported by a fellowship from CONACYT (Mexico).
We thank Ray Wightman for critical reading of the manuscript.
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