Microbiology (2000), 146, 3279–3284
Printed in Great Britain
Landmarks in the early duplication cycles of
Aspergillus fumigatus and Aspergillus
nidulans : polarity, germ tube emergence and
septation
Michelle Momany and Ian Taylor†
Author for correspondence : Michelle Momany. Tel : j1 706 542 2014. Fax : j1 706 542 1805.
e-mail : momany!dogwood.botany.uga.edu
Department of Botany,
University of Georgia,
Athens, GA 30602 7271,
USA
When the spores of filamentous fungi break dormancy, nuclear division is
accompanied by a series of ordered morphological events including the switch
from isotropic to polar growth, the emergence of a second germ tube from the
conidium and septation. Correlation of these morphological events with
nuclear number allows them to serve as duplication cycle landmarks. Early
duplication cycle landmarks have been characterized in Aspergillus nidulans,
but not in other filamentous fungi. To learn more about duplication cycle
control in filamentous fungi, a study was undertaken to compare the timing of
landmarks in Aspergillus fumigatus and A. nidulans. Nuclear duplication took
approximately 45 min in A. fumigatus, with mitosis occupying roughly 5 % of
this period. Under the same conditions, nuclear duplication in A. nidulans took
approximately 60 min, with mitosis occupying roughly 4 % of this period. In A.
fumigatus the isotropic to polar switch preceded the first mitosis in 22 % of
cells, while in A. nidulans the isotropic to polar switch did not occur until after
the first mitosis. In both A. fumigatus and A. nidulans the earliest emergence
of a second germ tube from the conidium occurred after the third mitotic
division. However, by the fifth mitosis only 19 % of A. fumigatus conidia had a
second germ tube, compared to 98 % of A. nidulans conidia. In both A.
fumigatus and A. nidulans, formation of the first septum occurred after the
fourth mitotic division. In all experiments a few cells lagged behind the others
in nuclear number. In this delayed group, it was common to see landmark
events at an earlier mitotic division. Differences in nuclear number when
identical landmarks occur in A. fumigatus versus A. nidulans, and uncoupling
of mitotic division and landmarks in delayed cells suggest that nuclear division
and morphogenesis lie in parallel pathways, perhaps coordinated by
checkpoints.
Keywords : cell cycle, mitosis, morphogenesis
INTRODUCTION
The realization that ordered morphological landmarks
correlate with mitotic state in Saccharomyces cerevisiae
provided the framework for understanding the cell cycle
of this yeast (reviewed by Pringle & Hartwell, 1981 ; Lew
et al., 1997). During the G1 phase the unbudded mother
cell grows isotropically, adding new material uniformly
in all directions. Late in G1 the bud emerges and grows
in a polar manner, adding new material at the tip of the
bud. This polar growth continues during S phase until
sometime in G2, when the bud switches to isotropic
growth. As M phase ends, septation occurs, separating
the mother and daughter cells. Many biochemical,
genetic and ultrastructural landmarks have been added
to this simple morphological framework, giving a view
of relationships among the pathways required to replicate and divide the components of the yeast cell.
.................................................................................................................................................
† Present address : Cereon Genomics, LLC, Discovery, 45 Sidney Street,
Cambridge, MA 02139, USA.
Morphological landmarks correlate with mitotic state in
filamentous fungi as well. Because the cells of filamen-
0002-4143 # 2000 SGM
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M. MOMANY and I. TAYLOR
METHODS
Strains and growth conditions. A. fumigatus strain 237 was a
clinical isolate provided by Dr David Holden, Department of
Infectious Disease and Bacteriology, Royal Postgraduate
Medical School, Hammersmith Hospital, London W12 ONN,
UK. Aspergillus nidulans strain A28 (pabaA6 ; biA1) was
received from the Fungal Genetics Stock Center, Dept of
Microbiology, University of Kansas Medical Center, Kansas
City, KS, USA. All incubations were at 37 mC in complete
medium (1 % glucose, 0n2 % peptone, 0n1 % yeast extract,
0n1 % Casamino acids, nitrate salts, trace elements and 0n01 %
vitamins, pH 6n5). Trace elements, vitamins, nitrate salts and
amino acid supplements are described in the appendix to
Kafer (1977).
Staining and microscopy. The protocol of Harris et al. (1994)
was used for growth and staining of A. fumigatus and A.
nidulans as follows. Ten millilitres of complete liquid medium
were inoculated with 1–5i10% conidia ml−", poured into a
Petri dish containing a glass coverslip and incubated at 37 mC
for the time indicated in each experiment. Coverslips with
adhering germlings were fixed in 3n7 % formaldehyde, 50 mM
phosphate buffer (pH 7n0) and 0n2 % Triton X-100 for
30–60 min. Coverslips were then washed with water, incubated for 5 min with 10 µg calcofluor white ml−" (Bayer) and
100 ng Hoechst 33 258 ml−" (Sigma), washed again and
mounted on a microscope slide for viewing. Germlings were
photographed using a Zeiss Axioplan microscope and Zeiss
MC100 microscope camera system with Kodak Tmax 100
film.
Because germinating conidia are not perfectly synchronous,
mitotic division number in Fig. 1 is based on the earliest time
point when 50 % of the population shows the appropriate
nuclear number. Morphological landmarks shown in Fig. 4
were scored at the time points represented in Fig. 1, i.e. the
earliest time point when 50 % of the population had
completed the indicated mitotic division. Only those germlings
that had completed the appropriate division were used to
determine the percentage showing a landmark. For example,
11 % of A. nidulans cells that contained two nuclei (had
completed the first mitotic division) were polar. A. fumigatus
experiments were repeated at least three times and A. nidulans
experiments were repeated twice, with essentially identical
results. Typical data sets are shown.
RESULTS AND DISCUSSION
Length of the nuclear cycle
When the uninucleate, asexual spores (conidia) of A.
fumigatus or A. nidulans are inoculated into rich
medium they break dormancy and remain roughly
synchronized through the first several mitotic divisions.
We took advantage of this synchrony to monitor the
length of time between nuclear doublings (Fig. 1).
4
3
Mitotic division
tous fungi remain attached after septation, these landmarks also correlate with mitotic history. A cell containing two nuclei has undergone one mitotic division. A
cell containing four nuclei has undergone two divisions,
and so on. The mitotic cycle in filamentous fungi is often
called the duplication cycle to distinguish it from cell
cycles where daughters completely separate (Fiddy &
Trinci, 1976).
The Aspergillus nidulans duplication cycle is the best
characterized among filamentous fungi (reviewed by
Harris, 1997). After breaking dormancy, the uninucleate
asexual spores of A. nidulans grow isotropically until
the first mitosis. After this first nuclear division, an axis
of polarity is established and the germ tube begins to
emerge (Momany et al., 1999 ; Harris et al., 1999 ;
Harris, 1999). The axis of polarity is maintained as the
germling elongates by tip growth. The first nuclear
division after the germling reaches a predetermined size
triggers synthesis of the first septum at the base of the
germ tube (Harris et al., 1994 ; Wolkow et al., 1996).
This can occur as early as the third mitosis. Subsequent
septa are laid down at regular distances along the hypha
after each round of mitosis. The nuclei in the subapical
compartments are arrested in interphase, while the
nuclei in the apical compartment undergo synchronous
mitosis. When a second germ tube emerges from the
spore, it is most often at 180m to the first, in a bipolar
arrangement (Harris et al., 1999 ; Momany et al., 1999).
Aspergillus fumigatus, an important pathogen of
humans, is an increasing problem in immunocompromised patients (Kwon-Chung & Bennett, 1992). Because it lacks a sexual cycle, A. fumigatus is often
studied in conjunction with the related saprobe A.
nidulans (Tang et al., 1994 ; Borgia et al., 1994 ; Guest &
Momany, 2000). To better understand the biology of
A. fumigatus we have undertaken studies comparing its
duplication cycle with that of A. nidulans. We have
uncovered differences in the isotropic to polar switch,
emergence of the second germ tube and septation. Our
results suggest that nuclear duplication and morphogenesis lie in parallel pathways in filamentous fungi.
2
1
0
3·5
4
4·5
5
5·5
6
6·5
7
Time (h)
.................................................................................................................................................
Fig. 1. Nuclear division in A. fumigatus and A. nidulans. Conidia
were inoculated into rich media and samples were taken every
15 min. Germlings were fixed and stained with Hoechst 33258
and calcofluor white to visualize nuclei and chitin, respectively.
The number of nuclei per germling was counted. The earliest
time point when at least 50 % of the population had completed
each mitotic division is shown for A. fumigatus (black triangles
with solid line) and A. nidulans (white squares with broken
line).
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Aspergillus landmarks
(a)
(d)
(b)
(c)
(e)
(f)
(g)
.................................................................................................................................................................................................................................................................................................................
Fig. 2. Morphology of the early A. fumigatus duplication cycle. Germlings were grown and stained as described in Fig. 1.
Representatives of each mitotic division are shown. (a) A conidium that has not completed mitosis contains one nucleus
(left) and a conidium that has completed one mitotic division contains two nuclei (right). Nuclei are in interphase. (b, c)
Germlings that have completed two mitotic divisions. (b) The nuclei are in mitosis. (c) Nuclei are in interphase. (d) A
germling that has completed three mitotic divisions. The nuclei are in interphase. (e) Germling that has completed four
mitotic divisions. Nuclei are in interphase. Not all nuclei are in the plane of focus. (f, g) Germlings that have undergone
five mitotic divisions. Not all nuclei are in the plane of focus. (f) Nuclei are in mitosis. (g) Nuclei are in interphase. Arrows
mark septa. Bar, 5 µm.
Conidia were inoculated into rich medium, incubated,
fixed at 15 min time points and stained with Hoechst
33258 and calcofluor white to visualize nuclei and chitin,
respectively. Cells treated in this way are easily scored
for nuclear number and morphological events (Fig. 2). In
A. fumigatus the first nuclear division occurred after a
lag of 4 h 15 min with subsequent nuclear divisions
every 45 min. In A. nidulans the initial lag was 3 h
45 min with subsequent divisions every 60 min. This is
somewhat faster than previously reported nuclear cycle
lengths of 90–120 min for A. nidulans (Robinow &
Canten, 1969 ; Bergen & Morris, 1983), but agrees with
doubling times observed under similar conditions
(Harris et al., 1994).
As illustrated in Fig. 2, after staining, mitotic nuclei are
small and bright, while interphase nuclei are ellipsoidal
with dark spots (compare Fig. 2f and g). The distinctive
appearance of mitotic versus interphase nuclei allowed
us to determine that approximately 5 % of A. fumigatus
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M. MOMANY and I. TAYLOR
Table 1. Nuclear state in germlings
100
.................................................................................................................................................
4h
A. fumigatus
Mitotic
Total
CMI*
A. nidulans
Mitotic
Total
CMI*
13
219
5n9
5h
9
207
4n4
6h
8
221
3n6
7h
Mean
11
222
5n0
80
Germlings (%)
Spores were incubated at 37 mC for the indicated time, fixed
and stained with Hoechst 33258 to stain nuclei. Germlings
containing small, bright nuclei were counted as mitotic.
Germlings containing larger nuclei with dark spots were
counted as interphase.
60
40
20
4n7
0
7
200
3n5
9
200
4n5
8
200
4n0
9
200
4n5
0
1
2
3
4
5
Mitotic division no.
4n1
* Cell mitotic index l percentage of germlings in mitosis.
.................................................................................................................................................
Fig. 3. Polar growth can begin before mitosis in A. fumigatus.
Germlings were grown and stained as described for Fig. 1. Both
cells contain one nucleus. The cell on the left is polar ; the cell
on the right is nonpolar. Right panel, fluorescent image. Left
panel, phase-contrast image. Bar, 5 µm.
cells are in mitosis at any time (Table 1). Thus mitosis
occupies about 5 % of the 45 min duplication cycle, or
2n25 min, in A. fumigatus. Under the same conditions
approximately 4 % of A. nidulans cells are in mitosis so
that mitosis occupies about 2n4 min. This agrees with
the previously reported value of 3–4 % mitotic cells in A.
nidulans (Robinow & Canten, 1969 ; Bergen & Morris,
1983).
Polarity establishment
The conidia of both A. fumigatus and A. nidulans are
initially uninucleate and spherical. After a growth
period, but before the first nuclear division, 22 % of A.
fumigatus spores took on a pear shape (Figs 3, 4),
indicating that an axis of polarity had been established.
After the first mitosis, greater than 90 % of A. fumigatus
cells were polar. In A. nidulans, however, no cells were
polar before the first mitosis, 12 % were polar after the
first mitosis, and greater than 90 % were polar after
the second mitosis.
Our results are consistent with inhibitor studies that
suggest a mitotic division is normally needed for polarity
.................................................................................................................................................
Fig. 4. Timing of morphological landmarks. Germlings were
grown and stained as described for Fig. 1. A. fumigatus (black
symbols, solid line) and A. nidulans (white symbols, broken line)
cells were monitored for polarization (circles), emergence
of the second germ tube (squares) and septum formation
(triangles).
establishment in A. nidulans in rich medium (Harris,
1999). Interestingly, A. nidulans spores grown in poor
medium do not require completion of mitosis for
polarization (Harris, 1999). A size-control mechanism
that monitors nuclear to cytoplasmic ratio for optimal
hyphal growth has been previously suggested (Trinci,
1978). Harris (1999) postulates that the ability of A.
nidulans to polarize without mitosis in poor medium
may reflect a lowering of this size threshold when
nutrients are scarce. When we grew A. fumigatus in
minimal medium rather than complete medium, we
observed no difference in polarization (data not shown).
If a nuclear to cytoplasmic ratio must be achieved for
polarity establishment, it appears that the size threshold
is not lowered by nutrient scarcity in A. fumigatus.
Emergence of the second germ tube
We observed the earliest conidia with second germ tubes
after the third mitosis in both A. fumigatus and A.
nidulans (Fig. 4). However, by the fifth mitosis just 19 %
of A. fumigatus cells possessed a second germ tube,
while 98 % of A. nidulans cells possessed a second germ
tube. Three patterns of germ tube emergence were
observed. The second germ tube was either 180m from
the first (bipolar), 90m from the first (quarterpolar) or at
some other angle (random). For both A. fumigatus and
A. nidulans, the bipolar pattern was seen in just over
half of germlings, the quarterpolar pattern was seen in
just over a third of germlings and the random pattern
was seen in the remaining tenth. Though our observed
percentages are different, the relative frequency of each
pattern is consistent with previously published reports
in A. nidulans which showed bipolar 84 % of the time,
quarterpolar 16 % of the time and random 1n5 % of the
time (Harris et al., 1999).
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Aspergillus landmarks
(a)
(b)
(c)
.................................................................................................................................................
Fig. 5. The first A. fumigatus septum is at the basal end of the
germ tube. Germlings were grown and stained as described for
Fig. 1. (a) Septum at the base of the germ tube. (b) Septum
about 7 µm from the base of the germ tube. (c) Septum about
12 µm from the base of the germ tube. Bar, 5 µm.
Emergence of the second germ tube is analogous to the
emergence of a second bud from the mother cell in S.
cerevisiae. In yeast, the site of bud emergence is
controlled by actin, septins and Bud proteins (Pringle et
al., 1995). It seems likely that a similar marker system
acts to establish polarity in both A. fumigatus and A.
nidulans. Our data suggest however, that polarity
markers may be regulated differently in these two fungi,
with emergence of the second germ tube either suppressed in A. fumigatus or enhanced in A. nidulans.
Perhaps by directing growth primarily in one direction,
the lower percentage of second germ tubes allows A.
fumigatus to scavenge nutrients more efficiently in the
host.
Septation
We observed the first septum in 21 % of A. fumigatus
germlings that had undergone four rounds of mitosis
(Fig. 4). The first septum was found anywhere within
about 20 µm of the base of the germ tube (Fig. 5). In A.
nidulans we observed the first septum in 55 % of
germlings that had undergone four rounds of mitosis.
This is in contrast to previous reports that the first
septum in A. nidulans is usually laid down after the third
nuclear division (Harris et al., 1994). This difference in
reported nuclear number at first septation probably
arose from differences in incubation temperature and
the methods used to define nuclear state of the population. Harris et al. (1994) grew cells at 28 mC, took
hourly time points and separately counted nuclear
number in the population and the presence of the first
septum. They then correlated the two events, i.e. the
majority of the population had 8 nuclei when the first
septa appeared. We, on the other hand, grew cells at
37 mC, took time points every 15 min and counted septa
in germlings with 8 nuclei separately from those with 16
nuclei. The data diagrammed in Fig. 4 reflect the earliest
time points when 50 % of the population had
completed the appropriate division and only that part of
the population with the indicated nuclear number. A
few A. nidulans cells did lag behind the others in mitotic
divisions, having only eight nuclei when the majority of
the population had 16 or even 32 nuclei. In these delayed
cells it was common to see septation after the third
mitosis (results not shown). The lower incubation
temperature, less frequent time points, separate scoring
of mitotic number and septation, and the presence of
lagging cells probably account for the difference in
timing observed by Harris et al. (1994). We never
observed septa forming in A. nidulans germlings with
fewer than eight nuclei or in A. fumigatus germlings
with fewer than 16 nuclei.
Using A. nidulans conditional mutants that do not go
through mitosis, but still grow, Wolkow et al. (1996)
established that the formation of the first septum is
triggered by a mitotic division after the germling reaches
a critical size. They also showed that the position of the
first septum is determined by the position of mitotic
nuclei. Previous work (Fiddy & Trinci, 1976) had
established that mitosis proceeds in a parasynchronous
wave starting at the tip of the hypha and moving back
toward the conidium. Wolkow et al. (1996) postulated
that an inhibitor of septation with a tip-high gradient is
diluted out by cell growth before mitosis triggers
septation. During apical extension, the inhibitor would
be diluted below the inhibition threshold first at the base
of the germ tube. When the mitotic wave intersects this
inhibition-free zone at the base of the germ tube,
septation would be triggered nearby. They further
postulated that formation of a septum suppresses
formation of septa at adjacent nuclei. Their model is
supported by the observation that delays in mitosis
cause A. nidulans to misplace septa away from the base
of the germ tube. By giving the hypha more time to
extend before mitosis, the zone of cytoplasm below the
critical inhibitor threshold is enlarged. The slightly later
septation of A. fumigatus may result from some difference in size control. Alternately, because A. fumigatus
hyphae are about half the diameter of A. nidulans
hyphae, perhaps they must extend further to dilute the
inhibitor enough to allow septum formation.
Uncoupling of mitosis and morphogenesis
In our experiments a few cells ( 1 %) always lagged
behind the others in mitotic divisions. For example,
some germlings had only a single nucleus when the
majority of the population had 16 nuclei. In delayed A.
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M. MOMANY and I. TAYLOR
nidulans cells it was common to see polarization with a
single nucleus, emergence of the second germ tube after
only two mitotic divisions and septation at the third
mitotic division (data not shown). This uncoupling of
nuclear division from morphological landmarks suggests that these events are not inter-dependent. Rather,
nuclear division and morphogenesis appear to lie in
parallel pathways in filamentous fungi. The delay in
mitosis seen along with the delay in polarization further
suggests that these pathways may be coordinated by
checkpoints in much the same way that the nuclear
division and bud emergence pathways are coordinated
by the morphogenesis checkpoint in S. cerevisiae (Pringle
& Hartwell, 1981 ; Lew et al., 1997).
ACKNOWLEDGEMENTS
A Burroughs Wellcome Young Investigator Award in Pathogenic Molecular Mycology to M. M. supported this work. We
thank Dr Steve Harris and members of the Momany lab for
critical reading of the manuscript.
REFERENCES
Bergen, L. G. & Morris, N. R. (1983). Kinetics of the nuclear
division cycle of Aspergillus nidulans. J Bacteriol 156, 155–160.
Borgia, P. T., Dodge, C. L., Eagleton, L. E. & Adams, T. H. (1994).
Bidirectional gene transfer between Aspergillus fumigatus and
Aspergillus nidulans. FEMS Microbiol Lett 122, 227–232.
Fiddy, C. & Trinci, A. P. J. (1976). Mitosis, septation, branching
and the duplication cycle in Aspergillus nidulans. J Gen Microbiol
97, 169–184.
Guest, G. M. & Momany, M. (2000). Analysis of cell wall sugars in
the pathogen Aspergillus fumigatus and the saprophyte Aspergillus nidulans. Mycologia 92, 1047–1050.
Harris, S. D. (1997). The duplication cycle in Aspergillus nidulans.
Fungal Genet Biol 22, 1–12.
Harris, S. D. (1999). Morphogenesis is coordinated with nuclear
division in germinating Aspergillus nidulans conidiospores.
Microbiology 145, 2747–2756.
Harris, S. D., Morrell, J. L. & Hamer, J. E. (1994). Identification and
characterization of Aspergillus nidulans mutants defective in
cytokinesis. Genetics 136, 517–532.
Harris, S. D., Hofmann, A. F., Tedford, H. W. & Lee, M. P. (1999).
Identification and characterization of genes required for hyphal
morphogenesis in the filamentous fungus Aspergillus nidulans.
Genetics 151, 1015–1025.
Kafer, E. (1977). Meiotic and mitotic recombination in Aspergillus
and its chromosomal aberrations. Adv Genet 19, 33–131.
Kwon-Chung, K. & Bennett, J. E. (1992). Aspergillosis. In Medical
Mycology, pp. 201–247. Philadelphia : Lea and Febiger.
Lew, D. J., Weinert, T. & Pringle, J. R. (1997). Cell cycle control in
Saccharomyces cerevisiae. In The Molecular and Cellular Biology
of the Yeast Saccharomyces : Cell Cycle and Cell Biology, pp.
607–695. Edited by J. R. Pringle, J. R. Broach & E. W. Jones.
Plainview, NY : Cold Spring Harbor Laboratory.
Momany, M., Westfall, P. J. & Abramowsky, G. (1999). Aspergillus
nidulans swo mutants show defects in polarity establishment,
maintenance and hyphal morphogenesis. Genetics 151, 557–567.
Pringle, J. R. & Hartwell, L. H. (1981). The Sacccharomyces
cerevisiae cell cycle. In The Molecular Biology of the Yeast
Saccharomyces : Life Cycle and Inheritance. Edited by J. N.
Strathern, E. W. Jones & J. R. Broach. Cold Spring Harbor, NY :
Cold Spring Harbor Press.
Pringle, J. R., Bi, E., Harkins, H. A., Zahner, J. E., DeVirglio, C.,
Chant, J., Corrado, K. & Fares, H. (1995). Establishment of cell
polarity in yeast. Cold Spring Harbor Symp Quant Biol LX,
729–744.
Robinow, C. F. & Canten, C. E. (1969). Mitosis in Aspergillus
nidulans. J Cell Sci 5, 403–431.
Tang, C., Smith, J. M., Arst, H. N. & Holden, D. W. (1994).
Virulence studies of Aspergillus nidulans mutants requiring lysine
or p-aminobenzoic acid in invasive pulmonary aspergillosis.
Infect Immun 62, 5255–5260.
Trinci, A. P. J. (1978). The duplication cycle and vegetative
development in moulds. In The Filamentous Fungi : Developmental Mycology, pp. 132–163. Edited by J. E. Smith & D. R.
Berry. New York : Wiley.
Wolkow, T. D., Harris, S. D. & Hamer, J. E. (1996). Cytokinesis in
Aspergillus nidulans is controlled by cell size, nuclear positioning,
and mitosis. J Cell Sci 109, 2179–2188.
.................................................................................................................................................
Received 27 March 2000 ; revised 21 August 2000 ; accepted 30 August
2000.
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