Journal of Cell Science 108, 3485-3499 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
JCS3108
3485
The Aspergillus nidulans bimE (blocked-in-mitosis) gene encodes multiple
cell cycle functions involved in mitotic checkpoint control and mitosis
Steven W. James1,2,*, Peter M. Mirabito1,†, Peter C. Scacheri2,‡ and N. Ronald Morris1
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School,
Piscataway, NJ 08854-5635, USA
2Department of Biology, Gettysburg College, Gettysburg, PA 17325, USA
*Author for correspondence at address 2 (e-mail: [email protected])
†Present address: Molecular and Cell Biology Group, T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, KY 40506-0225 USA
‡Present address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
SUMMARY
The bimE (blocked-in-mitosis) gene appears to function as
a negative mitotic regulator because the recessive bimE7
mutation can override certain interphase-arresting treatments and mutations, causing abnormal induction of
mitosis. We have further investigated the role of bimE in
cell cycle checkpoint control by: (1) coordinately
measuring mitotic induction and DNA content of bimE7
mutant cells; and (2) analyzing epistasis relationships
between bimE7 and 16 different nim mutations. A combination of cytological and flow cytometric techniques was
used to show that bimE7 cells at restrictive temperature
(44°C) undergo a normal, although somewhat slower cell
cycle prior to mitotic arrest. Most bimE7 cells were fully
reversible from restrictive temperature arrest, indicating
that they are able to enter mitosis normally, and therefore
require bimE function in order to finish mitosis. Furthermore, epistasis studies between bimE7 and mutations in
cdc2 pathway components revealed that the induction of
INTRODUCTION
The eukaryotic cell cycle consists of a temporally ordered and
strictly regulated sequence of events to ensure the exact duplication and segregation of genetic material during cell division.
The cell cycle begins with a gap phase, G1, which is followed
by nuclear DNA replication at S phase. After S the cell
traverses another gap phase, G2, and then finishes the cycle by
undergoing mitosis, or M phase, where nuclear division occurs.
The essential order and fidelity of the cell cycle is enforced
through a variety of molecular controls referred to as ‘checkpoints’ (Hartwell and Weinert, 1989) or ‘feedback’ controls
(Murray and Kirschner, 1989). These controls act by monitoring completion of earlier processes, thereby establishing a
dependent order of cell cycle events. The mitotic entry checkpoint, for example, couples M phase to S phase by preventing
the onset of M until DNA replication is completed (Dasso and
Newport, 1990; Enoch and Nurse, 1990, 1991; Enoch et al.,
1992; Smythe and Newport, 1992). In addition, this checkpoint
mitosis caused by inactivation of bimE requires functional
p34cdc2 kinase, and that mitotic induction by bimE7
depends upon several other nim genes whose functions are
not yet known. The involvement of bimE in S phase
function and mitotic checkpoint control was suggested by
three lines of evidence. First, at restrictive temperature the
bimE7 mutation slowed the cell cycle by delaying the onset
or execution of S phase. Second, at permissive temperature
(30°C) the bimE7 mutation conferred enhanced sensitivity
to the DNA synthesis inhibitor hydroxyurea. Finally, the
checkpoint linking M phase to the completion of S phase
was abolished when bimE7 was combined with two nim
mutations that cause arrest in G1 or S phase. A model for
bimE function based on these findings is presented.
Key words: bimE, nimQ, p34cdc2, Aspergillus, cell cycle, checkpoint,
mitosis
or a similar one also monitors DNA damage and functions to
delay cells in G2 until damaged DNA has been repaired
(Weinert and Hartwell, 1988; Rowley et al., 1992; Shiestl et
al., 1989; Al-Khodairy and Carr, 1992; Weinert, 1992). Checkpoints have also been discovered which monitor: (a) the status
of pre-START G1 cells and restrain M until G1 is finished
(Moreno and Nurse, 1994); (b) the status of the spindle
apparatus during mitosis (Hoyt et al., 1991; Li and Murray,
1991); and (c) the status of the DNA during S phase (Navas et
al., 1994).
We are investigating the role of the bimE gene of Aspergillus
nidulans in regulation of the mitotic entry checkpoint. bimE
was identified by a recessive, temperature sensitive lethal
mutation, bimE7, that causes nuclei to accumulate and persist
in a pre-anaphase mitotic state with tightly condensed
chromatin and a normal-appearing spindle apparatus at restrictive temperature (44°C). Molecular disruption of the gene phenocopies the bimE7 defect, indicating that loss of bimE
function leads to mitotic arrest (Osmani et al., 1988a). The
3486 S. W. James and others
bimE gene encodes a large protein of predicted Mr 229 kDa
which may possess membrane-spanning domains (Engle et al.,
1990) and which shares homology with a recently discovered
mouse gene (Starborg et al., 1994).
bimE was proposed to act as a negative regulator of mitosis
because bimE7 mutant cells advance into M when previously
blocked in S phase by treatment with hydroxyurea (HU). Upon
germination in HU at restrictive temperature, approximately
30% of bimE7 cells formed a spindle apparatus, as compared
with <1% in identically-treated wild-type (WT) cells. Also,
inactivation of bimE induced M in the presence of two cell
cycle mutations reported to block in S (nimK14) or G2 (nimA5)
(Osmani et al., 1988a). For example, during germination at
restrictive temperature nimK14;bimE7 double mutant cells
accumulated M-arrested nuclei with kinetics identical to single
mutant bimE7 cells, and formed spindle apparatuses, suggesting that the bimE7 mutation overrides prior cell cycle events
and causes premature entry into M. The phenotype of
nimA5;bimE7 cells is more complex. The nimA gene encodes
a protein kinase, p79nimA, the expression and activity of which
peaks at G2/M (Osmani et al., 1987, 1991a). A recessive, tslethal mutation in this gene (nimA5) causes G2 arrest, and
deregulated expression of the wild-type gene causes premature
induction of and terminal arrest in M, leading to the conclusion that nimA functions as a mitotic inducer (Osmani et al.,
1988b). nimA5;bimE7 cells also arrest in M, and exhibit mitotic
specific protein phosphorylation. However, entry into M is
delayed relative to bimE7 single mutants, and M-arrested
double mutant cells display aberrant morphologies, such as
mono- and tri-polar spindles and nuclear membrane abnormalities, enlargement of nuclei and multiple nucleoli, and
endomembrane proliferation. The reason for these mitotic
abnormalities is not known, but suggests the possibility that the
bimE7 mutation may allow bypass of nimA functions which
contribute to the mitotic process (Osmani et al., 1991a). Altogether, mitotic induction by bimE7 cells treated with HU or
when combined with interphase-arresting nim mutations was
interpreted to mean that bimE normally functions to inhibit M,
that it is a component of a checkpoint which prevents M from
occurring until prior cell cycle events have been completed,
and that in the absence of bimE function, cells advance prematurely into M.
Entry into mitosis in eukaryotic cells is controlled by a
universal mechanism involving the activation at G2/M of a
serine-threonine protein kinase, p34cdc2 (for a review, see
Nurse, 1990; Hartwell, 1991). Studies in the fission yeast
Schizosaccharomyces pombe revealed that activation of
p34cdc2 kinase is partially achieved by binding with a regulatory subunit, cyclinB (p56cdc13), the levels of which accumulate during the cell cycle, reach a maximum at G2, and then
disappear rapidly during M (Booher et al., 1989). Activation
also requires the removal of inhibitory phosphorylations
(Gould and Nurse, 1989; Lundgren et al., 1991) and addition
of stimulatory phosphorylations (Solomon et al., 1993; Fesquet
et al., 1993; Poon et al., 1993). The major inhibitory phosphorylation on Tyr-15 of the S. pombe p34cdc2 kinase is modulated
by competing activities of the weel/mik1 kinases (Featherstone
and Russell, 1991; Lundgren et al., 1991) and the p80cdc25
tyrosine phosphatase (for a review, see Millar and Russell,
1992).
In Aspergillus, three members of the universal cdc2 pathway
have been isolated, two of which, nimE and nimT, were originally identified by ts-lethal G2-arresting mutations (Morris,
1976a; Bergen et al., 1984). Cloning and molecular characterization of nimE and nimT revealed them to be close homologs
of the S. pombe p56cyclinB and p80cdc25 tyrosine phosphatase,
respectively (O’Connell et al., 1992). Both genes were subsequently shown to be necessary for activation of the Aspergillus
p34cdc2 kinase and entry into M (O’Connell et al., 1992;
Osmani et al., 1991b). In addition, three ts-lethal alleles of the
Aspergillus homolog of p34cdc2 kinase were recently created
and characterized (Osmani et al.,1994).
The induction of mitosis caused by inactivation of bimE
could be mediated through at least one of several different
mechanisms. Loss of bimE function could perturb either the
timing mechanism (Murray and Kirschner, 1989) or the checkpoint controls (Hartwell and Weinert, 1989) which together
ensure that S is completed before M is begun. For instance, the
bimE7 mutation might accelerate the timing of mitosis without
abolishing the checkpoint for DNA replication similar to the
way that, for example, wee1− mutations or certain dominant
cdc2 mutations in S. pombe cause cells to divide prematurely
by shortening the G2 phase (reviewed by Enoch et al., 1992).
Alternatively, the bimE7 mutation could perturb the checkpoint
which monitors DNA replication and cause precocious mitotic
induction before DNA replication is finished. In fission yeast,
members of the p34cdc2 pathway are involved in the checkpoint
that restrains M phase until completion of S phase. For
example, the dominant cdc2-3w mutation abolishes the checkpoint that monitors S phase completion, causing cells to enter
M with unreplicated DNA (Enoch and Nurse, 1990, 1991).
Loss of checkpoint control also occurs when Tyr-15 of cdc2 is
mutated to Phe, indicating that phosphorylation of this residue
is critical for detecting unreplicated DNA. In an analogous
fashion, overexpression of the p80cdc25 tyrosine phosphatase
leads to the premature onset of M (Enoch and Nurse, 1991),
although it may not normally be essential for coupling S and
M (Enoch et al., 1992). Override of the replication checkpoint
also occurs as a result of mutations in the hamster RCC1 gene
and its homologs, and in several mutations of budding and
fission yeast that fail to initiate DNA replication after traversing G1 START (reviewed by Enoch and Nurse, 1990, 1991;
Enoch et al., 1992; Dasso, 1993; Li and Deshaies, 1993). If
bimE is involved in checkpoint regulation, it could act through
any of the timing or checkpoint control mechanisms described
above, either as an inhibitor of a positive mitotic regulator(s),
or an inducer of a negative mitotic regulator. Or, bimE might
function independently of these identified pathways and
checkpoint control elements.
In this report we examine the hypothesis that bimE functions
as a negative cell cycle regulator through new phenotypic characterization of the bimE7 mutation. To determine if the bimE7
mutation causes precocious mitotic induction, flow cytometric
and cytological approaches were combined to measure cell
cycle length and to correlate changes in DNA content with the
onset of mitosis in bimE7 mutant cells grown at restrictive temperature. In addition, epistasis studies were carried out between
bimE7 and 16 different nim mutations to determine which
interphase-arresting mutations bimE7 can bypass to induce
mitosis. Since bimE7 does appear to override a defect in the
mitotic induction pathway involving nimA, we particularly
wished to determine if bimE7 can also override G2-arresting
bimE and cell cycle checkpoint control 3487
mutations in members of the universal cdc2/cyclinB pathway.
We further demonstrate the efficacy of flow cytometry for
analyzing DNA content in Aspergillus nidulans, and present a
new phenotypic characterization of the Aspergillus nidulans
nim collection.
MATERIALS AND METHODS
Strains, media and genetic analysis
Strains used in this study are listed in Table 1. All of the mutant nim
and bim strains used in this study were obtained after a minimum of
two outcrossings from the original mutants generated in strain FGSC
154 (Morris, 1976a). Defined minimal media (Kafer, 1977) with
appropriate supplementation were used throughout the study. Genetic
analysis was performed as described by Pontecorvo et al. (1953).
Candidate double mutant strains carrying heat-sensitive nim and
bimE7 mutations were recovered from nim × bimE7 crosses by
screening progeny for enhanced heat-sensitivity, reduced growth,
and/or enhanced sensitivity to the DNA synthesis inhibitor hydrox-
Table 1. Aspergillus nidulans strains
Strain
A559
FGSC122
R153
GR5
SWJ002
SWJ008
SWJ035
SWJ010
SWJ011
SWJ162
MMb001
SWJ191
PMM161
PMM171
SO15
SWJ180
SWJ232
SWJ230
SWJ227
SWJ193
SWJ194
SWJ258
SWJ274
SWJ171
SWJ251
SWJ113
KE8
SWJ187
SWJ145
SWJ310
SWJ312
SWJ334
SWJ170
SWJ168
SWJ203
SWJ224
SWJ205
SWJ248
SWJ207
SWJ209
SO61
SWJ394
SO62
SWJ389
SO63
SWJ388
Genotype
cnxG− ; biA1
riboB2; nicA2; yA2
pyroA4; wA2
pyrG89; pyroA4; wA2
pyrG89
argB2; pabaA1
pyrG89; choA1; yA2
bimE7; pabaA1; yA2
bimE7; pabaA1; fwA1
bimE7 methB3; nicA2; wA2
bimE7 methB3; yA2
bimE7 methB3; pyroA4; wA2
nimA5; pabaA1; wA2
nimA5
nimA5; bimE7; pabaA1; riboB2; wA2
nimB2; nicA2; wA2
nimB2; bimE7 methB3; yA2
nimC3; pabaA1; nicA2
nimC3; bimE7; pabaA1; nicA2
nimE6; methB3; pabaA1; wA2 (+ yA2?)
nimE6; bimE7 methB3; yA2
nimH11; choA1; fwA1
nimH11; bimE7; riboA1
nimI12; pabaA1
nimI12; bimE7; pabaA1
nimK14; pyroA4; riboA1; chaA1
nimK14; bimE7; riboA1; chaA1
nimQ20; methB3; choA1; wA2
nimQ20; bimE7; methB3; choA1
nimR21; pabaA1
nimR21 wA2; pabaA1; yA2
nimR21; bimE7 methB3; yA2
nimT23; pabaA1
nimT23; bimE7; pabaA1
nimU24; methB3; pabaA1
nimU24; bimE7 methB3
hfaB3; pyroA4
hfaB3; bimE7; pyroA4
sodVIB1; pabaA1; yA2
sodVIB1; bimE7; pabaA1; yA2
nimX1 (G25S); pyrG89; pyroA4; wA2
nimX1 (G25S); bimE7; pyroA4; wA2
nimX2 (F223L); pyrG89; pyroA4; wA2
nimX2 (F223L); bimE7; pabaA1; wA2
nimX3 (Y306H); pyrG89; pyroA4; wA2
nimX3 (Y306H); bimE7; pabaA1; wA2
Source
FGSC
FGSC
FGSC
FGSC
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yurea (HU). Following this, candidate double mutants were tested for
the presence of both mutations by complementation in heterokaryons
against strains carrying the single nim and bimE7 mutations. Presumptive double mutants were confirmed by outcrossing them to wild
type and assaying nuclear morphology of heat-sensitive progeny by
DAPI staining (described below) for nim and bim phenotypes.
Analysis of epistasis relationships
To study nim-bimE7 epistasis, the chromosome mitotic index (CMI)
and the number of nuclei per cell were measured during germination
of uninucleate, haploid conidia at restrictive temperature (44°C).
Conidia (1×105 per ml) were germinated on a coverslip (22 mm × 22
mm) submerged in 6 ml of medium in a 60 mm × 15 mm Petri dish.
For staining nuclei, coverslips were inverted onto 0.2 ml of a solution
consisting of 5% glutaraldehyde, 0.1 M potassium phosphate, pH 7.0,
0.2% Triton X-100, and 0.25 µg/ml 2,4-diamidino-2-phenylindole
(DAPI). After staining for 15-20 minutes, coverslips were rinsed for
10-15 minutes by immersion in an excess volume of Tris-buffered
saline (TBS; 25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2.7 mM KCl),
air-dried, and then mounted for observation, as described by Bergen
et al. (1984). For each sample, at least 150 individual cells were scored
on a Zeiss Axioplan or Nikon Optiphot photomicroscope equipped
with epifluorescence optics.
The DNA content of some strains was measured flow cytometrically. The vegetative conidia, or asexual spores, of Aspergillus
provide suitable material for flow cytometric studies because they are
haploid and uninucleate, have a uniform spherical shape (prior to germination) and are produced in great abundance. Most importantly,
conidia are arrested in the G1 stage of the cell cycle (Bergen and
Morris, 1983). The spores break dormancy semi-synchronously,
nearly all conidia beginning germination within a one-hour window,
and grow semi-synchronously for the first 8-10 hours.
Preparation of cells for flow cytometry was carried out essentially
as described by Costello et al. (1986). Fresh spores were inoculated
into minimal medium at a concentration of 2×105 per ml, and cultures
were grown with shaking in a water bath. Samples were taken at
appropriate intervals by removing a 1 ml aliquot (2×105 spores),
adding 1 µl of 10% NP-40, pelleting cells by centrifugation for 2-3
minutes, resuspending in 300 µl PBS + 0.01% NP-40, then adding
700 µl 100% ethanol and fixing for ≥12 hours at 4°C. The fixed cells
were washed with 1 ml phosphate-buffered saline (PBS) + 0.01% NP40, and resuspended in 0.5 ml of the same buffer. Preboiled RNAse
(Sigma Chemical Company) was added to 0.2 mg/ml, and the samples
were incubated at 37°C for 2-3 hours. Propidium iodide (PI; Sigma
Chemical Company) was added to 5 µg/ml. Just prior to cytometric
analysis, cell samples were sonicated to break up clumps.
Total fluorescence intensities were measured by quantitative flow
cytometry using an Epics Profile II Cytometer (Coulter Electronics)
equipped with a 25 mW argon-ion laser (Omnichrome 150) operated
at 15 mW at a wavelength of 488 nm. Light to the forward angle
scatter amplifier was filtered using a 1.0 optical density filter (Coulter
Electronics). The red fluorescence emission signal of PI was
processed through a 630 nm long pass filter. The resulting DNA
content distribution of particles was determined on linear scale.
Analyses were based upon the accumulation of 10,000 cells.
As reported in flow cytometric studies of Schizosaccharomyces
pombe using PI fluorescence (Costello et al., 1986), we observed artifactually high fluorescence readings at later time points during the
experiments. This phenomenon appeared to be correlated to the
lengthening hyphal germ tube and mitochondrial DNA proliferation,
and could be avoided by confining measurements of DNA content to
cells germinated for ≤8-10 hours. In addition, dormant Aspergillus
conidia were observed to bind PI to the cell wall, and gave an anomalously broad peak of fluorescence until the spores broke dormancy
and began to germinate after incubation at 44°C for approximately
four hours. At this time, cell walls ceased to bind PI, as judged by
3488 S. W. James and others
direct observation using epifluorescence microscopy, and the cells
developed a tight 1C DNA peak.
A
RESULTS
bimE7 mutant phenotype
At restrictive temperature (44°C) ts-lethal bimE7 cells arrest at
a pre-anaphase stage of mitosis (M) with condensed chromatin
and short polymerized mitotic spindles (Osmani et al., 1988a).
In this study the chromatin of M-blocked bimE7 cells was very
highly condensed (Fig. 1B), appearing hypercondensed
relative to the less compacted chromatin both in metaphase
wild-type cells (Fig. 1C) and in bimA1, the only other
Aspergillus bim which confers a pre-anaphase M block (see
O’Donnell et al., 1991). At restrictive temperature, bimE7
conidia showed very limited growth, often barely initiating a
germ tube, and the nuclei persisted in a highly condensed state
for 4-12 hours before they began to decondense and dissipate,
as reflected by slowly decreasing chromosome mitotic indices
(CMI) at later times during germination of conidia (e.g. see
Fig. 4).
The discovery that bimE7 cells at restrictive temperature
(44°C) form mitotic spindles even when S phase has been previously blocked by hydroxyurea (HU) suggested that bimE
may act as part of a checkpoint mechanism that monitors DNA
replication and prevents spindle formation until replication is
completed (Osmani et al., 1988a; Morris et al., 1992). To
further study its role in checkpoint control, bimE7 was tested
for enhanced sensitivity to hydroxyurea and radiation.
Mutations which disable the mitotic entry checkpoint often
confer enhanced sensitivity to hydroxyurea or radiation, presumably because abolition of the checkpoint allows cells to
advance into M prior to completion of S or repair of DNA
damage (reviewed by Li and Deshaies, 1993).
To test for HU-sensitivity, growth of bimE7 and wild-type
cells was compared on minimal agar media supplemented with
HU over a range of concentrations. At permissive temperature
(30°C), bimE7 was twofold more sensitive than wild type to
HU, failing to grow at [HU] ≥20-25 mM, whereas wild-type
cells were able to grow at [HU] ≤50 mM (data not shown).
Cosegregation of the ts and HU-sensitive phenotypes was
confirmed by analysis of random spore progeny from a cross
of bimE7 (SJ010) × wild type (R153) (n=56).
Radiation sensitivity was tested by comparing the survival
of two bimE7 strains and two wild-type strains at ultraviolet
(UV) dosages designed to kill 50% and 90% of wild-type cells.
Radiation-sensitive checkpoint mutants in S. pombe typically
confer 100- to 1,000-fold greater sensitivity than wild type to
UV dosages sufficient to kill approximately one half of wildtype cells (Enoch et al., 1992; Al-Khodairy and Carr, 1992).
In Aspergillus, bimE7 mutant and wild-type strains exhibited
similar survival rates at both UV exposures, indicating that the
bimE7 mutation does not confer enhanced radiation sensitivity
at permissive temperature (data not shown).
bimE function was further investigated by examining the
reversibility of the bimE7 mitotic arrest. Previous studies have
shown that bimE7-induced mitotic arrest is approximately 80%
reversible in cells incubated for 90 minutes at a restrictive temperature of 41°C, but the viability of cells decreased rapidly
with longer incubation at this temperature (Morris, 1976b). We
B
C
Fig. 1. Nuclear morphology in wild type vs bimE7. Conidia from
wild type (R153) or bimE7 (SWJ010) were incubated at restrictive
temperature (44°C) for 9 hours. Samples were fixed in
glutaraldehyde and stained with DAPI, and the nuclei observed.
(A) Wild-type cell in interphase; (B) bimE7 cells arrested in mitosis;
(C) Wild-type cells in metaphase of mitosis. Bar, 10 µm.
extended the previous study by assaying reversibility of the
bimE7 mitotic arrest in cells incubated for a longer period, and
at the maximum restrictive temperature of 44°C. bimE7
conidia were first grown for 9 hours at permissive temperature
(30°C) and then arrested at the restrictive temperature (44°C)
for 150 minutes, the time equivalent of between one and two
cell cycles (Bergen and Morris, 1983). The cells were then
returned to permissive temperature (30°C) and incubated for
an additional 45 minutes. Under these conditions, most cells
readily reversed the mitotic arrest, finishing mitosis to produce
interphase daughter nuclei (Table 2). The mitotic index
increased from about 3% at permissive temperature to 97.5%
at the restrictive arrest point, and then decreased to about 20%
after shifting down to permissive temperature. Measurement of
the increase in number of nuclei per cell between restrictive
arrest and release at permissive temperature revealed that at
least 70% of cells finished M and produced interphase daughter
nuclei. Thus, most bimE7 cells at restrictive temperature enter
mitosis normally, and can complete mitosis normally upon
relaxation of the restrictive condition. These results show that
the bimE7 mutation is reversible, and suggest that bimE
function is normally required for completion of mitosis.
DNA content analysis of the bimE7 cell cycle using
flow cytometry
To further investigate the role of bimE in cell cycle regulation, flow cytometric methods were developed to measure the
DNA content of Aspergillus cells. DNA content analysis can
be successfully performed by using propidium iodide to stain
the uninucleate, haploid conidia during the first 8-10 hours of
germination and germ tube formation. At later times the
hyphal germ tube becomes too long and causes a variety of
bimE and cell cycle checkpoint control 3489
Table 2. The bimE7 induced mitotic arrest is reversible
Proportion of nuclei/germling*
Condition
Chromosome
mitotic index (%)
1
2
4
8
16
3.50
97.51
22.82
2.26
3.77
0.00
31.22
17.92
4.62
65.61
64.62
27.69
0.90
13.21
65.13
0.00
0.47
2.57
Before arrest (30°C)
Restrictive arrest (44°C)
After release (30°C)
Minimum proportion reversible = 71%
A bimE7-containing strain (SJ010) was grown in minimal medium for 9 hours at 30°C before shifting to 44°C for 150 minutes. Following restrictive arrest,
cultures were shifted down to 30°C for an additional 45 minutes. Samples were removed at 9 hours, at the 150 minute arrest, and 45 minutes after shifting down.
Fixed samples were stained with DAPI and the chromosome mitotic index and number of nuclei per germling were determined. For both measurements, at least
200 cells were counted at each time point.
*The table shows the proportion of germlings with 1, 2, 4, 8, or 16 nuclei at each time point.
complications, including clumping of germlings and artifactually high fluorescence readings, possibly from increased
mitochondrial DNA mass. Fig. 2 shows typical DNA content
profiles for wild-type conidia germinated at 44°C in the
absence and presence of the DNA synthesis inhibitor hydroxyurea (HU). G1-arrested conidia broke dormancy and began
to grow after 4-5 hours, displaying a predominantly 1C DNA
content at 4 hours, and an approximately equal combination
of 1C and 2C cells at 5 hours. Nearly all cells in the population achieved a 2C DNA content by 7 hours, and by 8 hours,
the cells began to accumulate a 4C peak of fluorescence (Fig.
2A). By contrast, germination of wild-type conidia in 50 mM
HU at 44°C caused cells to arrest uniformly with a 1C DNA
content after a delay of about one hour in the breaking of spore
dormancy (Fig. 2B). Fig. 3 shows a comparison of DNA
content, nuclear number, and nuclear morphology for wild
type and bimE7 conidia germinated at restrictive temperature
(44°C). At each 30-minute time point over the 8 hour time
course, two cell samples were taken. One sample was
prepared for flow cytometry to analyze DNA content and the
other was fixed in glutaraldehyde and stained with DAPI to
analyze nuclear number and nuclear morphology. DNA
content profiles of bimE7 germinating conidia were similar to
wild type, except that bimE7 germlings were delayed either in
the onset or execution of S phase, completion of S being
delayed approximately 30 minutes relative to wild type. In
addition, bimE7 germlings arrested with a 2C DNA content.
Although completion of the bimE7 S phase was substantially
delayed, there is no evidence that the mutant cells entered
mitosis prematurely, before the completion of DNA replica-
Fig. 2. Flow cytometric analysis of
DNA content of wild-type cells. Wildtype conidia (R153) were grown at
44°C in the absence (A) or presence
(B) of the DNA synthesis inhibitor
hydroxyurea (HU; 50 mM). Samples
were withdrawn hourly beginning at 4
hours, fixed in ethanol, and stained
with propidium iodide as described in
Materials and Methods. Linear
fluorescence histograms show relative
DNA content in arbitrary units on the
horizontal axis, and the cell number
on the vertical axis. Each histogram is
based on counts of 10,000 cells.
tion. The rate at which bimE7 cells entered mitosis was similar
to the rate at which wild-type cells became binucleate, indicating that bimE7 cells likely complete S and G2 phases before
condensing chromatin. For instance, at 8 hours, when 63.4%
of wild-type germlings had undergone one mitosis to become
binucleate, 59.4% of bimE7 germlings had entered M, as
judged by the CMI. Furthermore, the CMI of the bimE7
culture began to rise above the wild-type level of 2-5% only
after a large majority of bimE7 nuclei had completed replication and achieved a 2C DNA content, at 6.5 and 7 hours. At
these times, when the proportion of bimE7 cells having a 2C
content of DNA was approximately 75% and 90%, respectively, the corresponding mitotic indices of bimE7 germlings
rose to 8.1% and 16.9%. By comparison, the wild-type culture
at 6.5 and 7 hours had achieved twice the frequency of binucleate cells (16.3% and 30.3%, respectively). Although DNA
content and CMI was not directly compared in individual
cells, this experiment demonstrates that mitotic induction is
not accelerated in the bimE7 mutant cell cycle, and that these
cells enter M, as judged by the condensation of chromatin,
after DNA replication has been completed. Combined with the
observation that the bimE7 mitotic arrest is reversible, these
results strongly indicate that bimE function is required to
complete mitosis.
Induction of mitosis by bimE7 requires functional
p34cdc2 kinase
To determine whether p34cdc2 kinase is required for the
induction of mitosis caused by inactivation of bimE, double
mutant strains carrying bimE7 and mutations in three cdc2-
3490 S. W. James and others
B
A
pathway genes were constructed. Epistasis in nim;bimE7
double mutant strains was assessed using four criteria. First, to
determine if bimE7 was able to bypass the nim arrest point and
enter M, the arrest point of double mutants was determined by
DAPI staining to measure the proportion of nuclei with
condensed (mitotic) versus uncondensed (interphase)
chromatin. Second, because the length of the hyphal germ tube
at terminal arrest differed greatly between bimE7 and most
nims, being very short in bimE7 and long in some nims, the
growth of the germ tube was monitored and used as an
indicator of epistasis. Third, in some cases the cell cycle arrest
point was determined with respect to DNA content by using
flow cytometry. And fourth, the number of nuclei per germling
was monitored to estimate the leakiness of some nim mutations
during incubation at restrictive temperature.
Double mutant strains carrying the bimE7 mutation and
either the nimE6 (cyclinB), nimT23 (cdc25 tyrosine phosphatase), or nimX1, -2, or -3 (cdc2) mutations were analyzed
by microscopy after germination at restrictive temperature.
All five of the ts nim mutations were epistatic to bimE7,
because the nim single mutant strains and the nim;bimE7
double mutant strains became blocked predominantly with a
single uncondensed nucleus and a long germ tube (Figs 4, 5).
For nimE6 and nimT23, the DNA content of single and double
mutants was monitored during germination at restrictive temperature. A representative flow cytometry profile is shown
only for nimE6 and nimE6;bimE7 strains (Fig. 6) because the
nimT23 and nimT23;bimE7 DNA profiles were very similar.
As expected, both single mutant nimE6/T23 and double
mutant nimE6/T23;bimE7 strains underwent one normal cell
cycle, and arrested in G2 phase with a 2C content of DNA.
Since the ts nim mutations are recessive, loss-of-function
defects, these observations indicate that p34cdc2 activity is
required for the induction of mitosis caused by inactivation of
bimE.
In the case of nimE6;bimE7 and nimT23;bimE7, a minor
proportion of nuclei blocked in M; this can be attributed to the
leakiness of the two nim mutations. For example, at terminal
arrest (10-11 hours post-germination) the proportion of single
mutant nimE6 and nimT23 nuclei that had divided once (9.3%
and 21.8%, respectively) was similar to the proportion of Mblocked nuclei in the double mutants (12.5% and 28.1%,
respectively). Thus, double mutant cells which leaked past the
nimE6 or nimT23 block points became trapped at the downstream bimE7 block point. Interestingly, the chromatin of
mitotically arrested double mutants typically exhibited a fragmented morphology which did not occur in the bimE7 single
mutant (Fig. 5E).
Fig. 3. Analysis of the DNA content, nuclear number, and
chromosome mitotic index of wild type (A) and bimE7 (B) at
restrictive temperature. Conidia of strains R153 and SWJ010 were
grown at 44°C. At 30 minute intervals, two samples were withdrawn
from each culture. One of the samples was fixed in ethanol, stained
with propidium iodide, and used to determine the relative DNA
content by flow cytometry. The other sample was fixed in
glutaraldehyde, stained with the DNA-specific dye DAPI, and used
to measure the number of nuclei per germling and the chromosome
mitotic index (CMI). Nuclear number was recorded as the percentage
of cells which became binucleate over the time course. All bimE7
cells remained uninucleate.
bimE and cell cycle checkpoint control 3491
Fig. 4. (a-f) Chromosome mitotic index
and nuclear division of single and double
mutants at restrictive temperature. Conidia
were germinated at 44°C for 11 or 12
hours. Beginning at 4 hours, samples were
taken hourly and fixed and stained with
DAPI to determine nuclear number and
chromosome mitotic index. All
measurements are based on observations
of at least 150 cells per time point. s,
wild type (R153); d, bimE7 (SWJ010);
h, nim single mutant (see Table 1); j,
bimE7 + nim double mutant (see Table 1).
3492 S. W. James and others
A
B
C
Fig. 5. Mitotic induction by bimE7
requires functional p34cdc2. Conidia
from nim single mutant and
nim;bimE7 double mutant strains
were germinated at restrictive
temperature (44°C) for 9 or 10 hours.
Samples were fixed in
glutaraldehyde and stained with
DAPI. nimE6 and nimE6;bimE7 are
not shown since they were like
nimT23 and nimT23;bimE7. (A)
nimX1 (SO61); (B) nimX1;bimE7
(SWJ394); (C) nimT23 (SWJ170);
(D and E) nimT23;bimE7 (SWJ168).
Bar, 10 µm.
D
E
bimE7 overrides the interphase arrest caused by
three nim mutations
The idea that bimE plays a role in mitotic checkpoint regulation
stems partly from the discovery that bimE inactivation induced
mitosis in the presence of the G2-arresting nimA5 mutation. The
nimA5 mutant arrests with a moderate-length germ tube and a
single interphase nucleus, and nimA5;bimE7 double mutants at
arrest appear identical to bimE7 with short germ tubes and
tightly condensed chromatin (Osmani et al., 1988a). Additional
electron microscopic and biochemical characterization of
mitotic induction in nimA5;bimE7 revealed a number of abnormalities involving spindle and membrane morphology, but
Fig. 6. Flow cytometric analysis of DNA
content of nimE6 (A) and nimE6;bimE7 (B).
Conidia of strains SWJ193 and SWJ194 were
germinated at 44°C. Samples were withdrawn
at two-hour intervals beginning at 4 hours, and
prepared as described in Materials and
Methods. Linear fluorescence histograms show
relative DNA content in arbitrary units on the
horizontal axis, and cell number on the vertical
axis. Each histogram is based on counts of
10,000 cells.
supported the conclusion that the p79nimA kinase is not required
for mitotic entry resulting from inactivation of bimE (Osmani et
al., 1991a). In this study nimA5 and nimA5;bimE7 behaved as
previously described (Osmani et al., 1988a), exhibiting tight tslethality with no leakage to form binucleate cells (Fig. 4f). In
addition, nimA5 and nimA5;bimE7 arrested with a uniform 2C
DNA content (Fig. 7A,B), but the double mutant cell cycle was
much slower than wild type or nimA5. Compared to bimE7, a
pronounced 3-4 hour delay occurred before nimA5;bimE7 began
to enter mitosis (Fig. 4f). In contrast, Osmani et al. (1988a)
reported a much shorter one hour lag between single and double
mutants. This discrepancy is caused by nutritional differences,
bimE and cell cycle checkpoint control 3493
rich medium (yeast extract-glucose, YAG) having been used in
the original study, and minimal medium in this study. To
confirm this, we repeated the experiments with both minimal and
rich media (YAG). The double mutant experienced a one-hour
lag in rich medium, as previously reported, and a four-hour delay
in minimal medium (not shown).
To determine whether bimE7 can override other interphasearresting nim mutations to cause mitosis, we surveyed the
Aspergillus nim collection by constructing nim;bimE7 double
Fig. 7. Flow cytometric
analysis of DNA content of
nim single and nim;bimE7
double mutants at restrictive
temperature. Conidia were
germinated at 44°C.
Samples were withdrawn at
two-hour intervals (A and
B), or hourly (C-F), and
prepared for flow cytometry.
The chromosome mitotic
indices of nimQ20 and
nimQ20;bimE7 (D and E)
were determined in parallel
by DAPI staining, and are
shown under the
fluorescence histograms.
(A) nimA5 (PMM171);
(B) nimA5;bimE7 (SO15);
(C) wild type (R153);
(D) nimQ20 (SWJ187);
(E) nimQ20;bimE7
(SWJ145); (F) nimR21
(SWJ310).
mutants and characterized them by microscopy and flow
cytometry. A summary of results is shown in Table 3. In a clear
and striking case, bimE7 entirely bypassed the arrest point of
the nimQ20 mutation, causing rapid mitotic induction in cells
with unreplicated DNA. At restrictive temperature, nimQ20
conidia grew moderate to long germ tubes, they blocked tightly
with a single uncondensed nucleus (Fig. 8A) and arrested with
a 1C DNA content (Fig. 7D). nimQ20;bimE7 double mutants
also became blocked with a 1C DNA content (Fig. 7E), but
3494 S. W. James and others
Table 3. Summary of epistasis relationships between
bimE7 and Aspergillus nim (never-in-mitosis) mutations
A
nim gene product
nim mutations epistatic to bimE7*
nimB2
nimC3
nimE6
nimH11
nimK14
nimT23
nimU24
nimX1
nimX2
nimX3
sodVIB1
nim mutations overridden by bimE7†
nimA5
nimQ20
nimR21
nim mutations with uncertain epistasis‡
nimI12
hfaB3
?
?
Cyclin B
?
?
cdc25 tyrosine phosphatase
?
p34cdc2 kinase
p34cdc2 kinase
p34cdc2 kinase
?
p79 NIMA kinase
?
?
?
?
*These bimE7;nim double mutants arrested growth at restrictive
temperature (44°C) with predominantly uncondensed (interphase) chromatin.
†These bimE7;nim double mutants arrested growth at restrictive
temperature (44°C) with condensed (mitotic) chromatin and short germ tubes
characteristic of the bimE7 single mutant.
‡The epistasis relationship between bimE7 and these nim mutations was
difficult to ascertain due to the leakiness of the nim mutations.
arrested with the tightly condensed chromatin and short germ
tubes characteristic of bimE7 (Fig. 8B). In approximately 10%
of cases the condensed chromatin was fragmented (Fig. 8C).
Furthermore, the CMI of bimE7 single mutants and
nimQ20;bimE7 double mutants increased with identical
kinetics (Fig. 4g); the increase in CMI was not delayed in these
double mutants as it was in nimA5;bimE7. Taken together,
these observations demonstrate that while bimE7 and nimQ20
by themselves do not appear to bypass the requirement for
DNA replication, the double mutant completely abolishes the
dependency of M phase on S phase.
nimQ20 also displayed enhanced sensitivity to hydroxyurea
at permissive temperature, failing to grow on HU-supplemented minimal agar media at concentrations ≥35 mM,
whereas wild-type cells (R153) were able to grow at HU concentrations up to 50 mM. nimQ20;bimE7 double mutants
exhibited additively enhanced sensitivity to HU, failing to
grow at HU concentrations above 15-20 mM (data not shown).
bimE7 also appeared to bypass the requirement for nimR21,
but less strongly than in the case of nimQ20. Like nimQ20,
nimR21 blocked with a 1C DNA content (Fig. 7F) and a single
uncondensed nucleus, but unlike nimQ20, mutant nimR21 cells
arrested with a very short germ tube and diffusely staining
DNA (not shown). nimR21;bimE7 double mutant cells also
arrested with 1C DNA and a very short germ tube (not shown),
but a significant proportion of nuclei, 35-40%, blocked with
highly condensed chromatin after a delay of about three hours
by comparison to the bimE7 single mutant (Fig. 4h).
Epistasis analysis between bimE7 and nim
mutations with leaky phenotypes
The study of nim;bimE7 epistasis was extended to include three
additional nim mutations reported to cause G2 arrest, nimB2,
nimU24, and hfaB3 (high-frequency-aneuploidy); three nims
B
C
Fig. 8. bimE7 overrides the nimQ20 mutation. Conidia from nimQ20
and nimQ20;bimE7 were germinated at restrictive temperature
(44°C) for 11 hours. Samples were fixed in glutaraldehyde and
stained with DAPI. (A) nimQ20 (SWJ187); (B and C)
nimQ20;bimE7 (SWJ145). Bar, 10 µm.
reported to block cells at S phase, nimC3, nimK14, and sodVIB1
(stabilizer-of-disomy); and two nims, nimH11 and nimI12,
with undetermined arrest points (Morris, 1976a; Bergen et al.,
1984). These nims were found to have leaky phenotypes. For
instance, hfaB3 single mutants grew very long germ tubes and
failed to arrest until the 4-8 nucleate stage, and nimI12 single
mutants progressed further to at least the 32-nucleate stage
before arrest. Not surprisingly, hfaB3;bimE7 and
nimI12;bimE7 double mutants arrested growth with short germ
tubes and tightly condensed chromatin characteristic of bimE7
(see Fig. 1C). Mitotic induction in these double mutants
therefore likely reflects the trapping of cells at the bimE7 block
point after nim leakthrough, and as a result these nims were
eliminated from further study. The remaining six nims were
also leaky, but the phenotypes were tight enough to assess
nim;bimE7 epistasis. Between 7% and 92% of these nim single
mutants became binucleate by the end of a germination time
course (Figs 9, 10). In nim;bimE7 double mutants with
nimH11, nimU24, nimC3, and sodVIB1, the rise in CMI of
double mutants occurred concomitantly with the leakthrough
of nim single mutants to form binucleate cells (Fig. 9a-d), suggesting that mitotic entry resulted from nim leakthrough.
Leakiness aside, nimH11 and nimU24 were epistatic to bimE7
at least for hyphal growth, as in double mutants the germ tubes
grew to a substantial length before mitotic arrest occurred (Fig.
10B,D). Also, the rise in CMI of the double mutants was
delayed by 2-4 hours relative to the single bimE7 mutant. This
delay likely reflects the time required to leak past the nimH11
or nimU24 block points (Fig. 9a,b). Additionally and surprisingly, in the case of nimU24;bimE7 some of the double mutant
cells (9.0%) became binucleate and thus appeared to progress
beyond the bimE7 block point (Fig. 9b). This observation is
unprecedented, and suggests that nimU24 may permit mitotic
progression in the absence of bimE function.
Assessing epistasis in nimC3;bimE7 and sodVIB1;bimE7
double mutants was problematic, due to the nim leakiness (Figs
9c,d, 10E,F), and in the case of sodVIB1 the cells arrested with
little or no germ tube growth (Fig. 10F). Double mutants
arrested with short germlings and condensed chromatin characteristic of bimE7 (see Fig. 1C). Given the parallel rise in CMI
of double mutants and the accumulation of binucleate cells in
the nim single mutants (Figs 9c,d, 10E,F), mitotic arrest in the
double mutants likely occurred from leakthrough, and the nim
bimE and cell cycle checkpoint control 3495
mutations, if they are truly nims, are therefore probably
epistatic to bimE7.
bimE7 was reported to override the nimK14 mutation and
advance cells into M (Osmani et al., 1988a). As previously
documented, nimK14;bimE7 cells advanced into M with
kinetics identical to bimE7 (Fig. 9e), and arrested with short
Fig. 9. (a-f) Chromosome
mitotic index and nuclear
division of single and double
mutants at restrictive
temperature. Conidia were
germinated at 44°C for 12 or
15 hours. Beginning at 4
hours, samples were taken
hourly and fixed and stained
with DAPI to determine
nuclear number and
chromosome mitotic index.
All measurements were based
on observations of at least 150
cells per time point. s, wild
type (R153); d, bimE7
(SWJ010); h, nim single
mutant (see Table 1);
j, bimE7 + nim double
mutant (see Table 1).
germ tubes and tightly condensed chromatin. However, the
nimK14 mutation was leaky, arresting with long, binucleate
germlings in which the nuclei became elongated at later stages
(Fig. 10G). Furthermore, the DNA replication cycle was unperturbed in nimK14. At restrictive temperature, most cells
underwent two cell cycles and achieved a 4C DNA content by
3496 S. W. James and others
A
B
C
D
E
F
G
Fig. 10. Cytology of nim and nim;bimE7 strains. Conidia from nim single and nim;bimE7 double mutants were germinated at 44°C for 9-11
hours. Samples were fixed in glutaraldehyde and stained with DAPI. (A) nimH11 (SWJ258); (B) nimH11;bimE7 (SWJ274); (C) nimU24
(SWJ203); (D) nimU24;bimE7 (SWJ224); (E) nimC3 (SWJ230); (F) sodVIB1 (SWJ207); (G) nimK14 (SWJ113) showing elongated strip of
chromatin between two nuclei. Bar, 10 µm.
the end of the time course (Fig. 11A), indicating that the
mitotic arrest in nimK14;bimE7 probably resulted from
nimK14 leakthrough.
The nimB2 mutation was originally reported to cause G2
arrest (Bergen et al., 1984), but by examining the DNA content
of cells during germination at restrictive temperature, nimB2
was observed to undergo one or two DNA replication cycles
and arrest with a mixture of 2C and 4C DNA (Fig. 11B).
However, only a small proportion of nimB2 mutant cells
became binucleate (Fig. 9f), indicating that nuclear division is
defective in this mutant. The nimB2 mutation did not prevent
chromatin condensation, because during germination at restrictive temperature, mutant nimB2 cells underwent a substantial
rise in CMI which peaked early in the time course (6 hours)
and gradually diminished over the remainder of the experiment
(Fig. 9f). nimB2 mutants arrested predominantly with one
uncondensed nucleus and a moderate length germ tube (not
shown). nimB2;bimE7 cells arrested with condensed chromatin
and short germ tubes characteristic of bimE7 and blocked with
a 2C DNA content (Fig. 11C) after a delay of one hour relative
to bimE7 (Fig. 9f). This observed delay, combined with the discoveries that nimB2 achieved a 4C DNA content and
underwent transient chromatin condensation, indicates that
mitotic induction in this double mutant results from leakthrough of the nimB2 mutation.
DISCUSSION
The bimE gene was hypothesized to act as a negative regulator
of mitosis based on the observations that at restrictive temperature, bimE7 mutant cells treated with hydroxyurea (HU) form
spindle apparatuses at an elevated frequency (30% in bimE7
versus less than 1% in wild type); and, bimE7 reportedly
bypassed the arrest points of the nimA5 and nimK14 mutations,
causing the nim;bimE7 double mutant cells to arrest in M
(Osmani et al., 1988a). These phenomena suggested that
bimE7 may perturb a checkpoint control mechanism which
normally prevents cells from entering mitosis prematurely. We
tested this hypothesis by examining: (1) sensitivity to agents
such as HU and UV-radiation which have proven useful for
identifying checkpoint-defective mutants in the budding and
fission yeasts (Enoch et al., 1992; Weinert, 1992; Al-Khodairy
and Carr, 1992); (2) reversibility of the bimE7 mitotic arrest;
(3) timing of DNA replication and mitosis; and (4) epistasis
relationships between bimE7 and other nim mutations,
including members of the universal p34cdc2 pathway. The
results support a model for bimE as a multifunctional protein
which is required for the completion of mitosis, plays a role in
the onset or execution of S phase, and which participates in
controlling the mitotic checkpoint.
Several lines of evidence demonstrate roles for bimE in M
and S phases. At restrictive temperature (44°C) bimE7
undergoes a normal cell cycle: bimE7 cells finish DNA replication and accumulate 2C DNA before entering mitosis. Furthermore, most cells can recover from mitotic arrest and
continue cycling, demonstrating that bimE7 does not bypass or
disrupt events prior to M that are necessary for its completion.
Taken together, these observations show that the extended
mitotic arrest of bimE7 results primarily from the inability to
complete mitosis, rather than from premature induction of M.
Although the bimE7 cell cycle appears normal at restrictive
temperature, the cells are delayed in the completion of S phase
by approximately 30 minutes relative to wild type, and the
bimE7 mutation confers enhanced sensitivity to HU at permissive temperature. Together, these observations suggest a
bimE and cell cycle checkpoint control 3497
Fig. 11. Flow cytometric analysis of
DNA content of nim single and
nim;bimE7 double mutant cells.
Conidia were germinated at 44°C.
Samples were withdrawn hourly (A)
or every two hours (B and C), and
prepared for flow cytometry. The
chromosome mitotic index of nimK14
(A) was determined in parallel by
DAPI staining, and is shown under the
fluorescence histograms.
role for bimE in S phase and may well be manifestations of the
mitotic checkpoint defect of bimE7 (discussed below).
Mitotic induction by bimE7 is dependent upon activation of
the universal p34cdc2 kinase. In combination with mutations in
nimXcdc2, nimEcyclinB, and nimTcdc25, double mutants behave
like the nim single mutants, arresting with long germ tubes and
an uncondensed nucleus. These observations support our contention that bimE function is required to finish mitosis, since
inactivation of bimE neither bypasses the requirement for
nimXcdc2 in mitotic induction, nor apparently does it prematurely induce the activation of p34cdc2 kinase prior to completion of S phase (except, presumably, when combined with the
nimQ20 or nimR21 mutations). Instead, these results are consistent with a model whereby one function of bimE, necessary
for completion of M, is executed downstream of p34cdc2.
Induction of M by bimE7 is also dependent upon a number
of genes of unidentified function, including nimB, nimH, nimU,
and probably nimC and sodVIB. The case of nimU24;bimE7 is
particularly interesting, as some cells appear to progress
beyond the bimE7 block point to form two nuclei. This finding
shows that nimU24 may allow mitosis to continue without
bimE function, and suggests a role for nimU in controlling M
phase progression.
Results from nim;bimE7 epistasis analyses demonstrate that
bimE is involved in mitotic checkpoint function. For example,
nimQ20;bimE7 and nimR21;bimE7 double mutants exhibit loss
of checkpoint control, causing the cells to enter M prematurely
with unreplicated DNA. The case of nimQ20;bimE7 is most
convincing: both the nimQ20 single mutant and
nimQ20;bimE7 double mutant cells arrest uniformly with a 1C
DNA content, but in contrast to nimQ20 cells which arrest with
one uncondensed nucleus and a long germ tube, the
nimQ20;bimE7 cells bypass the nimQ20 block point altogether
and undergo mitotic arrest comparable in timing and degree to
bimE7 alone. The same features hold true for nimR21 and
nimR21;bimE7 except that double mutants delay induction of
M relative to bimE7, and only some of the cells (35-40%)
advance into mitosis. Nonetheless, both the nimQ20 and
nimR21 mutations are exceptional in that combination with
bimE7 clearly abolishes the dependency of M on completion
of S. Perhaps the nimQ/R and bimE gene products interact
redundantly with a common component of the checkpoint
machinery, such that impairment of both gene functions is
necessary to abolish the signal generated by their common
target. Function by either would be sufficient to enforce the
checkpoint, but loss of both nimQ and bimE would render the
checkpoint inactive, and thereby permit precocious mitotic
induction. Precedent for such a model occurs in the case of the
wee1 and mik1 genes of the fission yeast Schizosaccharomyces
pombe. These genes encode kinases which inhibit activation of
p34cdc2 kinase. Inactivation of the wee1 gene advances the
timing of mitosis but does not bypass the requirement for DNA
replication, and impairment of mik1 function causes no discernible phenotype. But when combined, wee1− mik1− double
mutant cells display a ‘mitotic catastrophe’ phenotype in which
cells rapidly enter a lethal mitosis without completing DNA
replication or other required cell cycle events (Lundgren et al.,
1991).
Alternatively, nimQ/R and bimE could act in different, but
parallel pathways of checkpoint regulation. By this model,
function of either pathway would be sufficient to enforce
checkpoint control, but loss of both would abolish the need to
complete S before the onset of M. Although no specific
examples of redundant, parallel checkpoints have yet been
3498 S. W. James and others
proven, recent studies have revealed a large number of genes
whose products act in different aspects of mitotic checkpoint
control, and some have been suggested to participate in parallel
checkpoint pathways (for review, see Li and Deshaies, 1993;
Downes and Wilkins, 1994).
Finally, loss of checkpoint control in nimQ20/R21;bimE7
could resemble the premature mitotic induction caused by
mutation of the cut5+, cdc18+, and cdt1+ genes of S. pombe, in
which cells that fail to replicate DNA after traversal of START
enter a lethal mitosis (Saka and Yanagida, 1993; Kelly et al.,
1993; Hofmann and Beach, 1994). The model emerging from
these studies posits that DNA replication serves as the signal
for activating the mitotic checkpoint. Failure to synthesize
DNA after commitment to the cell cycle therefore renders the
cell incapable of monitoring the state of the DNA, with the
result that cells bypass S phase and prematurely enter mitosis
(reviewed by Li and Deshaies, 1993). By this model
nimQ20/R21 would not be part of a checkpoint mechanism.
Rather, loss of checkpoint control in the double mutants would
simply reflect the bimE7 checkpoint defect in cells that are
unable to replicate DNA. It is not yet known if nimQ20 and
nimR21 mutants fail to initiate DNA replication, or if they
initiate but cannot finish S phase. Further understanding of the
checkpoint loss suffered by nimQ20/R21;bimE7 must await
identification of the nimQ/R gene products.
bimE7 appears to bypass the late G2 phase requirement for
p79nimA kinase and cause nimA5;bimE7 cells to enter M after
completion of DNA replication, albeit with aberrant morphologies, including mono- and tri-polar spindles, abnormalities in nuclear and other endomembrane systems, and enlarged
nuclei with multiple nucleoli. Since nimA is required for
induction of M, the mitotic abnormalities of the double mutant
were suggested to mean that bimE7 bypasses nimA functions
which contribute to mitosis (Osmani et al., 1991a). The
meaning of the nimA5;bimE7 mitotic arrest is difficult to
interpret, owing to the fact that nimA encodes a late G2 function
and because mitotic induction in double mutants is substantially delayed relative to bimE7, suggesting the possibility that
this induction could result from leakage of the nimA5 mutation.
This issue could be resolved by deleting nimA from the genome
in a bimE7 background. The significance of the nimA5;bimE7
mitotic induction is additionally complicated by the findings
that two other mitotic arrest mutations, bimA1 and bimG11,
bypass the G2 block point of nimA5 and cause induction of M
(P. M. Mirabito and J. H. Doonan, personal communication).
bimA encodes a member of the tetratricopeptide repeat family
of proteins which is required for completion of M (O’Donnell
et al., 1991), and localizes to the spindle pole body (Mirabito
and Morris, 1993). bimG encodes a homolog of mammalian
phosphoprotein phosphatase 1 and is also required for completion of M (Doonan and Morris, 1989). bimE encodes a large
protein of 229 kDa (Engle et al., 1990) which localizes to the
endomembrane system of the cytoplasm (S. W. James et al.,
unpublished results). It is nominally difficult to see how
mutations in these three genes could induce bypass of the
nimA5 arrest point. However, it is possible that the gene
products function through the same pathway, and for this
reason exert similar effects in a nimA5 background.
We thank Ed Yurkow, director of the UMDNJ Flow Cytometry
facility, for assistance and advice with DNA content analysis, and we
are grateful to Herb Geller for generously allowing us the use of his
photomicroscope. We are also grateful for the advice and critical
thinking of Matthew O’Connell, Steve Osmani, Michelle Mischke,
and Karen Kirk, and for strains provided by John Doonan and Steve
Osmani. We thank Maggie Tobin for assistance with preparation of
photographs, and Kim McNeal for help in preparing the manuscript.
This study was supported by a grant from the National Institutes of
Health to NRM (GM-34711-08), by an Anna Fuller Foundation postdoctoral fellowship to S.W.J., and by a grant from Gettysburg College
to PCS.
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(Received 19 August 1994 - Accepted 7 August 1995)