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Antimicrobial Agents and Chemotherapy, August 1999, p. 1993-1999, Vol. 43, No. 8
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Unusual Antimicrotubule Activity of the Antifungal
Agent Spongistatin 1
Yulia Y.
Ovechkina,1
Robin K.
Pettit,2
Zbigniew A.
Cichacz,2
George R.
Pettit,2 and
Berl R.
Oakley1,*
Department of Molecular Genetics, The Ohio
State University, Columbus, Ohio 43210,1 and
Departments of Microbiology and Chemistry, Cancer Research
Institute, Arizona State University, Tempe Arizona
852872
Received 22 March 1999/Returned for modification 28 April
1999/Accepted 7 June 1999
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ABSTRACT |
Spongistatin 1, a macrocyclic lactone from the marine sponge
Hyrtios erecta, has broad-spectrum antifungal activity.
Since this compound is a potent antimicrotubule agent in mammalian
cells, we examined its effects on the filamentous fungus
Aspergillus nidulans to determine if its antifungal effects
are due to antimicrotubule activity. At 25 µg/ml (twice the MIC),
spongistatin 1 caused a greater-than-twofold elevation of the
chromosome and spindle mitotic indices. Immunofluorescence microscopy
revealed that mitotic spindles were smaller and shorter than in control
germlings. However, late-anaphase and telophase nuclei were seen
occasionally, and this suggests that the spindles are capable of
segregating chromosomes. Spongistatin 1 had more dramatic effects on
cytoplasmic microtubules. At 30 min after initiation of treatment, 83%
of germlings contained fragmented microtubules and after 2 h of
treatment, microtubules had disappeared completely from 82% of
germlings. In contrast, microtubules disappeared rapidly and completely
from germlings treated with benomyl. We conclude that spongistatin 1 has antimicrotubule activity in A. nidulans and that its
mechanism of action may involve a novel microtubule-severing activity.
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INTRODUCTION |
Spongistatin 1 is an experimental
antineoplastic agent that has recently been found to have
broad-spectrum antifungal activity (11). The relative
scarcity of effective antifungal drugs and the increasing incidence of
serious fungal infections, particularly in immune-compromised patients,
makes the existence of a new class of antifungals particularly
exciting. In mammalian cells, spongistatin 1 is a potent antimitotic,
antimicrotubule agent (1), and we wished to determine if its
antifungal activity is due to antimicrotubule effects as well. We chose
Aspergillus nidulans as our experimental organism because
good immunofluorescence procedures are available for this organism and
because other, related, species of Aspergillus cause severe
problems in immune-compromised patients. For comparison, we have also
examined the effects of the important antifungal, antimicrotubule agent
benomyl on A. nidulans mitosis and microtubules.
We found that benomyl causes a complete loss of mitotic spindles and a
nearly complete loss of cytoplasmic microtubules in A. nidulans. Spongistatin 1, at twice the MIC, also has substantial effects on microtubules. Rather than causing the rapid loss of microtubules, however, spongistatin 1 causes a fragmentation of cytoplasmic microtubules. These microtubule fragments disappear over
time. Mitotic spindles form in spongistatin 1-treated germlings, but
they do not achieve the maximum size seen in untreated material. These
findings suggest that spongistatin 1 is an antimicrotubule agent in
A. nidulans and that it acts by a mechanism different from
that of benomyl and other antimicrotubule agents.
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MATERIALS AND METHODS |
Media.
YG (5 g of yeast extract per liter, 20 g of
dextrose per liter) was used as a complete liquid medium. YAG (YG with
15 g of agar per liter) and FYG (YG with 25 g of Pretested
Burtonite 44c [TIC Gums, Inc., Belcamp, Md.] per liter) were used as
solid media. Both YAG and FYG were supplemented with trace elements
(3).
Broth macrodilution susceptibility testing.
Broth
macrodilution susceptibility testing of A. nidulans was
performed in accordance with a proposed standardized procedure (4) with some modification. To induce conidiospore
formation, A. nidulans FGSC4 (Glasgow wild type) was grown
on a potato dextrose agar (PDA) slant at 35°C for 3 days. The slant
was covered with 1 ml of sterile 0.05% Tween 80, and a suspension was
made by gently probing the colonies with the tip of a sterile Pasteur
pipette. The resulting mixture was transferred to a sterile, clear
microcentrifuge tube, and heavy particles were allowed to settle for 5 min. The upper homogeneous suspension was transferred to a sterile
microcentrifuge tube, vortexed for 15 s, adjusted
spectrophotometrically, and diluted in sterile 0.165 M
morpholinepropanesulfonic acid (MOPS)-buffered RPMI 1640 medium, pH
7.0, to yield a final inoculum of 0.5 × 103 to
2.5 × 103 CFU/ml. The inoculum was vortexed
repeatedly during the broth macrodilution assay to ensure the
suspension of small spores. The assay was performed in sterile plastic
tubes (12 by 75 mm) containing twofold dilutions of spongistatin 1. One
tube was left drug free for a turbidity control. Tubes were incubated
without agitation at 35°C. The MIC was read after 24 h, when
heavy growth was seen in the control. The MIC was defined as the lowest
concentration of spongistatin 1 that inhibited all visible growth of
A. nidulans FGSC4. The minimum fungicidal concentration
(MFC) was determined by subculturing 0.1 ml from each tube with no
visible growth in the broth macrodilution series onto drug-free PDA
plates. The plates were incubated at 35°C for 48 h, and the MFC
was defined as the lowest concentration of spongistatin 1 that
completely inhibited growth on PDA plates.
Treatment with antifungal agents.
Benomyl (Sigma Chemical
Co., St. Louis, Mo.) was dissolved in 95% ethanol at a concentration
of 200 µg/ml. Spongistatin 1 was isolated from the marine sponge
Hyrtios erecta as described elsewhere (10).
Spongistatin 1 was dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 2.5 mg/ml immediately before use. Conidia from
A. nidulans FGSC4 (Glasgow wild type) were inoculated at a
final concentration of 2.5 × 106/ml into 4-dram (1 fluidram = 3.697 ml) vials containing 1.1 ml of YG medium with
0.1% agar (agar was added to reduce clumping). Conidial suspensions
were incubated for 6 h at 37°C with shaking (115 rpm), after
which time most conidia had germinated. Samples (0.1 ml) were taken
immediately before the addition of spongistatin 1 or benomyl and at
30-min intervals for 2 h afterwards. Spongistatin 1 was used at a
final concentration of 25 µg/ml (twice the MIC). Since benomyl has
been used commercially and experimentally for many years, we did not
carry out macrodilution assays on multiple strains to define the MIC
for benomyl. Rather, we tested FGSC4 and determined that the minimum
benomyl concentration required to inhibit colonial growth on solid
medium (YAG) is 1.2 µg/ml (9). This result is consistent
with values previously obtained with wild-type A. nidulans
strains (8, 13). Based on these data, we used benomyl at a
final concentration of 2.4 µg/ml for our experiments. This
concentration is twice the concentration required to inhibit colonial
growth on YAG medium and should be equivalent to twice the MIC in the
liquid medium we used, which differs from YAG only in that it contains
0.1% agar instead of 1.5% agar. For solvent-alone controls, DMSO or
ethanol was added to give appropriate final concentrations.
Fluorescence microscopy.
Samples were fixed with 0.9 ml of
freshly prepared fixative solution [8% formaldehyde in 50 mM
piperazine-N,N'-bis(2-ethanesulfonic acid)
(PIPES), pH 6.7; 25 mM EGTA, pH 7.0; 5 mM MgSO4; and 5% DMSO] for 30 or 40 min. The samples were then washed twice for 10 min
in 50 mM PIPES (pH 6.7). Cells from each sample were passed through a
26-gauge needle 25 times to break clumps apart. Fifty microliters of
the cell suspension was spread on a coverslip, which had previously
been coated with a poly-L-lysine solution (0.1% [wt/vol]
in deionized water) (Sigma Chemical Co.), and allowed to dry.
To visualize DNA alone, the fixed samples were stained with
4',6-diamidino-2-phenylindole (DAPI) at 0.015 µg/ml for 2 h,
washed twice (10 min per wash) in deionized distilled water and mounted in Citifluor AF1 (Marivac, Ltd., Halifax, Nova Scotia, Canada). To
visualize both DNA and microtubules, coverslips were washed in 50 mM
PIPES (pH 6.7) and transferred to a solution containing Driselase
(InterSpex Products, Inc., Foster City, Calif.) at 10 mg/ml,
-D-glucanase (InterSpex Products, Inc.) at 16 mg/ml, and lyticase (catalog no. 36-094; ICN Biomedicals, Inc., Costa Mesa, Calif.) at 81.4 U/ml in 50 mM sodium citrate, pH 5.8, with 50% (vol/vol) egg white. (All washes in this and subsequent steps were for
5 to 10 min.) In some experiments, -D-Glucanase was treated to reduce protease activity by the following method (based on
the method of Roncal et al. [12]).
-D-Glucanase (200 mg/ml) was dissolved in 100 mM sodium
citrate, pH 4.5, and then incubated at 55°C for 5 min, kept on ice
for 30 min, transferred to 
70°C for storage, and used at 16 mg/ml
in the digestion solution. Coverslips were incubated with digestion
solution at 25°C for 1 h. They were then washed three times in
PE buffer (50 mM PIPES [pH 6.7], 25 mM EGTA) and incubated in
20°C methanol for 10 min. They were washed two times in PE before
incubation for 1 to 3 h in primary antibody diluted in PE with
0.5% (vol/vol) Nonidet P-40. The primary antibody was a mouse
monoclonal anti--tubulin antibody, TU27B, from a cell line
generously provided by Lester Binder (Northwestern University Medical
School, Chicago, Ill.) via G. Lozano (M. D. Anderson Cancer
Center, Houston, Tex.). The coverslips were washed three times in PE
buffer before incubation for 1 h or overnight with a secondary
antibody (CY3-conjugated goat anti-mouse immunoglobulin G [Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pa.]) diluted in PE
containing 0.5% (vol/vol) Nonidet P-40. The diluted secondary antibody
was preadsorbed against an A. nidulans acetone powder
prepared by the method of Harlow and Lane (5). After three
washes in 50 mM PIPES (pH 6.7), coverslips were postfixed in 2 mM
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma
Chemical Co.) in 50 mM PIPES (pH 6.7) for 20 min. They were then rinsed
briefly in 50 mM PIPES (pH 6.7), stained with DAPI at 0.075 µg/ml in
50 mM PIPES (pH 6.7) for 15 to 45 min, rinsed in 50 mM PIPES (pH 6.7),
and mounted in Citifluor AF1.
Photographs were taken with T-MAX 400 film rated at 400 or 800 ASA
using a Zeiss standard microscope. The film was developed
in T-MAX
developer, and negatives were scanned into Adobe Photoshop
with a
Polaroid SprintScan 35 Plus scanner. Images were also captured
by using
a Nikon Eclipse E800 microscope equipped with a Princeton
Instruments
MicroMax charge-coupled device camera and IPLab Spectrum
software.
Images were processed by using NIH Image and Adobe Photoshop.
Composite
images were prepared by using
ClarisWorks.
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RESULTS |
Determination of the MIC and MFC of spongistatin 1 for A. nidulans.
Similar to those for other filamentous fungi
(11), the MIC of spongistatin 1 for A. nidulans
FGSC4 was 12.5 µg/ml. The MIC was unchanged after 72 h. The
MFC/MIC ratio equaled 1.
Determination of CMIs.
Since microtubules are the major
component of the mitotic apparatus, antimicrotubule agents cause the
mitotic apparatus to fail to function normally or, in many cases, to
assemble at all. The absence of a functional mitotic apparatus causes
cells to become blocked in mitosis, and the percentage of cells in
mitosis (the mitotic index) increases relative to that of untreated
cells. We consequently determined the effects of spongistatin 1 on the chromosome mitotic index (CMI), the percentage of germlings in which
nuclei have condensed chromosomes. Here it should be noted that
A. nidulans is coenocytic and that the nuclei within a
single cell enter mitosis together. We examined germlings at 6 to
8 h after inoculation. At this time, the great majority of
germlings were a single cell.
Conidia (asexual spores) were incubated for 6 h at 37°C.
Spongistatin 1 was added to a final concentration of 25 µg/ml (twice
the MIC). Samples were taken immediately before the addition of
spongistatin 1 and at 30-min intervals thereafter. Samples were
collected over 2 h during which untreated cells would pass through
approximately 1.5 cell cycles (
2). As controls, samples were
treated with the antifungal, antimicrotubule agent benomyl at
2.4 µg/ml or with the solvents in which spongistatin 1 and benomyl
were
dissolved. Results are shown in Fig.
1.
In spongistatin 1-treated
material, the CMI rose gradually and reached
a value of 14% at
the 2-h time point. This value was significantly
higher than that
of the solvent-alone control, which maintained a CMI
of under
5% for the duration of the experiment.
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FIG. 1.
CMIs (percentages of germlings in which nuclei have
condensed chromatin) of A. nidulans germlings treated with
spongistatin 1 (25 µg/ml) and benomyl (2.4 µg/ml). Samples were
taken immediately before the addition of spongistatin 1 or benomyl
(zero time point) and at 30-min intervals afterward. Appropriate
concentrations of the solvents used to dissolve these compounds were
used as controls. Spongistatin 1 caused a gradual elevation of the CMI,
whereas benomyl caused a much more rapid elevation of the CMI, which
then dropped to 3.1%. Each point represents the average of three
separate experiments with 400 germlings scored at each time point in
each experiment. Error bars indicate standard deviations. When error
bars are not shown, the standard deviations fell within the boxes.
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The results obtained with benomyl were more dramatic (Fig.
1). The CMI
reached 32% at 30 min after the addition of benomyl
and 37% at 1 h. By 2 h, the CMI had dropped to approximately 3%.
Interestingly, in benomyl-treated samples, an unusual class of
nuclei
was seen at 30 min and later. These nuclei did not have
a typical
interphase or mitotic appearance (Fig.
2A, D, and
G),
but rather, the nuclei had chromatin
that appeared partially condensed
(Fig.
3A). (Nuclei with partially condensed
chromatin were not
counted as mitotic for Fig.
1.) After 2 h the
percentage of nuclei
with partially condensed chromatin reached 37%
(±20%). Such nuclei
were not seen in the spongistatin-treated
material and only one
such nucleus was seen in over 6,000 germlings
scored in the material
treated with solvent only. As will be discussed
below, such nuclei
have been seen previously in germlings carrying a
-tubulin disruption
(
7).
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FIG. 2.
Mitotic and interphase microtubules and nuclei in
control A. nidulans germlings. (A, D, and G) Chromatin as
revealed by DAPI staining. (B, E, and H) Microtubules as revealed with
an anti--tubulin antibody. (C, F, and I) Phase-contrast micrographs.
(A to C) An interphase germling shows nuclei with uncondensed chromatin
(A). The nucleoli (arrows) do not stain and are visible as dark
regions. A normal array of cytoplasmic microtubules is present (B). (D
to F) A portion of a germling in medial nuclear division shows two
mitotic nuclei (arrows) with condensed chromatin and no visible
nucleoli (D) and normal mitotic spindles (arrows) with astral
microtubules (arrowheads) (E). (G to I) A portion of a mitotic germling
with two nuclei (arrows), one in early anaphase (upper) and one in
telophase (lower), is shown (G). Two spindles (arrows) with astral
microtubules (arrowheads) are present (H). The lower spindle is
partially out of the plane of focus. All micrographs are at the same
magnification. The scale in panel C is 5 µm.
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FIG. 3.
Microtubules and nuclei in A. nidulans
germlings treated with benomyl (A to C) or spongistatin 1 (D to J). (A
and H) Chromatin as revealed by DAPI staining. (B, D, F, and I)
Microtubules as revealed with an anti--tubulin antibody. (C, E, G,
and J) Phase-contrast micrographs. (A to C) A germling after 2 h
of treatment with benomyl. (A) Nuclei (e.g., arrows) with partially
condensed chromatin and no visible nucleoli. (B) Complete absence of
microtubules in the germling. (D) An interphase germling after 30 min
of treatment with spongistatin 1 showing fragmented microtubules (e.g.,
arrows). Additional fragments of microtubules are present out of the
plane of focus. (F) An interphase germling after 1 h of treatment
with spongistatin 1 showing complete absence of microtubules. (H to J)
A mitotic germling after 1 h of treatment with spongistatin 1. (H)
An anaphase nucleus (arrow) in which the chromatin has moved toward the
poles. (I) A spindle (arrow) is present. Three more spindles are
present but out of the plane of focus. Note the absence of astral
microtubules. All micrographs are at the same magnification. The scale
in panel E is 5 µm.
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Spongistatin 1 effects on microtubules.
The elevation of
the CMI in spongistatin 1-treated material suggested that
spongistatin 1 might be inhibiting mitotic spindle function (although
less completely than benomyl). To look more directly at the effects of
spongistatin 1 on microtubules, we prepared germlings for
immunofluorescence microscopy by using an anti--tubulin antibody to
visualize microtubules.
As expected, in germlings prior to treatment and in solvent-only
controls, microtubules were present in virtually all germlings.
Cytoplasmic microtubules were present in virtually all interphase
germlings, and normal spindles were present in mitotic germlings
(Fig.
2B, E, and H). Benomyl caused a rapid disassembly of nearly
all
microtubules (Fig.
3B and
4). Thirty
minutes following the
addition of benomyl, microtubules were absent
from more than 99%
of germlings and the number of germlings with
detectable microtubules
remained very low throughout the experiment
(Fig.
4). Mitotic
spindles were completely absent from benomyl-treated
material
at all time points.
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FIG. 4.
Benomyl effects on microtubules. Samples were taken
immediately before the addition of benomyl at 2.4 µg/ml (zero time
point) and at 30-min intervals afterward. Shown are the percentages of
benomyl-treated germlings with fragmented microtubules (abbreviated
mts), normal cytoplasmic microtubules, spindles, or no microtubules at
each time point. Benomyl caused disassembly of all microtubules in more
than 99% of germlings by the first time point after addition. Spindle
and normal cytoplasmic microtubules were completely absent from
benomyl-treated material. There was no significant loss or
fragmentation of microtubules in a solvent-only control (data not
shown). Each point represents the average of three separate experiments
with 400 germlings scored at each time point in each experiment. Error
bars indicate standard deviations. When error bars are not shown, the
standard deviations fell within the boxes.
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The results obtained with spongistatin 1 were quite different from
those obtained with benomyl. At 30 min after the addition
of
spongistatin 1, the great majority of germlings contained microtubules
(Fig.
5A). The microtubules were in
numerous short fragments and
were arranged randomly with respect to the
growth axis of the
germling (Fig.
3D). In control germlings, the
cytoplasmic microtubules
were much longer and were arranged more or
less parallel to the
growth axis (Fig.
2B). The fragmented microtubules
gradually disappeared
with time (Fig.
3F and
5A). Mitotic spindles were
present at all
time points but were generally small. The spindle
mitotic index
(SMI), the percentage of germlings with spindles, of
spongistatin
1-treated material reached 7% by 30 min following the
addition
of spongistatin 1 and remained at similar levels over the
course
of the experiment (Fig.
5B). These values were about twice the
SMI in solvent-only controls. Apparent late-anaphase or telophase
spindles were observed occasionally (Fig.
3H to J) but were always
smaller and shorter than those seen in anaphase and telophase
control
germlings (Fig.
2G to I). In addition, astral microtubules
(microtubules extending from spindle pole bodies into the cytoplasm,
which are usually seen in later stages of mitosis in control germlings
[Fig.
2E and H] [
6]) were completely absent in
spongistatin
1-treated material (Fig.
3I).
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FIG. 5.
Spongistatin 1 effects on microtubules. Samples were
taken immediately before the addition of spongistatin 1 at 25 µg/ml
(zero time point) and at 30-min intervals afterward. An appropriate
concentration of the solvent used to dissolve this compound was used as
a control. (A) Shown are the percentages of germlings with fragmented
microtubules (abbreviated mts), normal cytoplasmic microtubules, or no
microtubules in control or spongistatin 1-treated material.
Spongistatin 1 caused the fragmentation and eventual loss of
cytoplasmic microtubules, whereas they remained intact in the control.
(B) SMIs (percentages of germlings with spindles) of A. nidulans germlings treated with spongistatin 1. Spongistatin 1 caused a twofold elevation of the SMI by 30 min, and the SMI remained
at that level for the rest of the experiment. The SMIs in the control
remained similar to those of untreated (t = 0)
germlings. Each point represents the average of three separate
experiments with 400 germlings scored at each time point in each
experiment. Error bars indicate standard deviations. When error bars
are not shown, the standard deviations fell within the boxes.
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DISCUSSION |
Our results demonstrate that spongistatin 1 is an antimicrotubule
agent in A. nidulans, and it is reasonable to infer that it
has antimicrotubule activity in other fungi as well. Spongistatin 1 appears to act differently from benomyl. Benomyl causes a relatively rapid loss of all microtubules, as shown by the fact that microtubules are completely absent from 99% of germlings by 30 min after the addition of benomyl. Spongistatin 1 does not completely block mitotic-spindle formation at twice the MIC. Spindles are present and at
least partially functional because we have seen apparent telophase
configurations suggesting that the spindles are capable of moving
chromosomes to the poles. The spindles in spongistatin-treated material
do not reach the maximum length reached by spindles in untreated
material, however, and astral microtubules, cytoplasmic microtubules
extending from the spindle pole body, were universally absent in
spongistatin 1-treated material (Fig. 3H to J).
The most dramatic effect of spongistatin 1 is the rapid fragmentation
of cytoplasmic microtubules, with 83% of germlings having fragmented
cytoplasmic microtubules at 30 min after the initiation of treatment
(Fig. 3D and 5A). The simplest explanation for the fragmentation of
microtubules is that long interphase microtubules are simply broken
into smaller fragments. It is important to note, however, that light
microscopy does not afford the resolution necessary to distinguish
between individual microtubules and small bundles of microtubules. The
fragmentation we have seen could, in theory, result from the shortening
of individual microtubules within extended bundles so that they no
longer overlap. The possibility that spongistatin 1 causes breakage of
microtubules is potentially important. All of the antimicrotubule
agents studied to date are thought to alter the dynamics of microtubule
assembly and/or disassembly by altering tubulin dimer exchange at the
ends of microtubules (14). If spongistatin 1 does cause
microtubule breakage, its mechanism of action would be different from
that of all known antimicrotubule agents. It will be important to
observe the effects of spongistatin 1 on microtubules in vitro by
real-time video microscopy to determine if it really does cause
microtubule severing.
The fragmented cytoplasmic microtubules disappear with time, and
eventually most germlings lack any detectable microtubules (Fig. 3F and
5A). We have considered two explanations for the gradual disappearance
of the microtubules. The first, and most obvious, possibility is that,
after fragmentation, spongistatin 1 simply causes a gradual disassembly
of cytoplasmic microtubules. The second possibility is that disassembly
might be related to the entry of germlings into mitosis. In untreated
germlings, cytoplasmic microtubules disassemble as cells enter mitosis
and reassemble as cells complete mitosis (reviewed in reference
6). Perhaps spongistatin 1 causes cytoplasmic
microtubules to fragment, but complete disassembly is a result of the
normal cytoplasmic microtubule disassembly process that occurs as cells
enter mitosis. If so, spongistatin 1 must prevent the reassembly of
cytoplasmic microtubules after mitosis; otherwise, cytoplasmic
microtubules would be present in a greater percentage of germlings at
later time points. The absence of astral microtubules, cytoplasmic
microtubules extending from the spindle pole bodies of mitotic nuclei,
in spongistatin 1-treated material is consistent with inhibition of the
reassembly of cytoplasmic microtubules. It should also be noted that
the absence of astral microtubules could also be due to a direct effect of spongistatin 1 on the spindle pole body. Such an effect would have
to be directed mainly at the cytoplasmic face of the spindle pole body,
since spindle formation is not blocked. If spongistatin 1 severed
microtubules from the cytoplasmic face of the spindle pole bodies, it
might lead to some of the misorientation and fragmentation of
microtubules that we have noted.
Our benomyl data also make an important point about checkpoint
regulation in A. nidulans. The simplest interpretation of
our data is that in the presence of benomyl, the cell cycle proceeds until germlings transit from G2 to M. At this point,
chromosomes condense but benomyl prevents mitotic-spindle formation and
germlings are blocked in M. The blockage is presumably due to one or
more checkpoints that monitor spindle formation or other essential mitotic events and inhibit transition to G1 without
successful completion of mitosis. We deduced that this checkpoint is
eventually overcome because we saw a decrease in the CMI with time
(Fig. 1). The decrease in the CMI does not appear to be due to the
successful completion of mitosis. We have seen no evidence that mitosis
is completed successfully in benomyl-treated samples (e.g., no anaphase or telophase nuclei), and it would be very surprising, indeed, if
mitosis were completed successfully in the absence of mitotic spindles.
Interestingly, the decondensation of the chromatin in benomyl-blocked
nuclei appears to be a gradual process. The entire mitotic process
normally takes less than 5 min at 37°C (2) and chromosomal
decondensation only takes a fraction of this period. If decondensation
occurred at a normal rate in benomyl-treated material, decondensing
nuclei would be very rare, and we have seen a mean of 37% of germlings
with apparently decondensing nuclei at the 2-h time point. The slow
rate of decondensation does not appear to be caused directly by
benomyl. Similar high percentages of nuclei with decondensing chromatin
have been seen in germlings that carry a disruption of the -tubulin
gene that prevents mitotic-spindle formation (7). It thus
appears that nuclei blocked in mitosis without a mitotic spindle
gradually reenter interphase without completing mitosis (i.e., the
mitotic checkpoint is eventually overridden).
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ACKNOWLEDGMENTS |
This work was supported by grant GM31837 from the NIH and
by the Arizona Disease Control Research Commission.
We thank M. Katherine Jung and C. Elizabeth Oakley for helpful
discussions and critical reading of the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics, The Ohio State University, 484 W. 12th Ave.,
Columbus, OH 43210. Phone: (614) 292-3472. Fax: (614) 292-4466. E-mail: Oakley.2{at}OSU.EDU.
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Abstract
Introduction
