Department of Botany, University of Georgia, Athens, GA 30602 7271, USA1
Author for correspondence: Michelle Momany. Tel: +1 706 542 2014. Fax: +1 706 542 1805. e-mail: momany{at}dogwood.botany.uga.edu
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ABSTRACT |
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Keywords: cell cycle, mitosis, morphogenesis
a Present address: Cereon Genomics, LLC, Discovery, 45 Sidney Street, Cambridge, MA 02139, USA.
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INTRODUCTION |
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Morphological landmarks correlate with mitotic state in filamentous fungi as well. Because the cells of filamentous 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 180° 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.
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METHODS |
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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 15x104 conidia ml-1, poured into a Petri dish containing a glass coverslip and incubated at 37 °C for the time indicated in each experiment. Coverslips with adhering germlings were fixed in 3·7% formaldehyde, 50 mM phosphate buffer (pH 7·0) and 0·2% Triton X-100 for 3060 min. Coverslips were then washed with water, incubated for 5 min with 10 µg calcofluor white ml-1 (Bayer) and 100 ng Hoechst 33258 ml-1 (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.
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RESULTS AND DISCUSSION |
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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 180° from the first (bipolar), 90° 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 1·5% of the time (Harris et al., 1999
).
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 °C, 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 °C, 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.
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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. 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
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 March 2000;
revised 21 August 2000;
accepted 30 August 2000.