Department of Microbiology, University of Connecticut Health Center, Farmington, CT 06030-3205, USA1
Tel: +1 860 679 2774. Fax: +1 860 679 1239. e-mail: sharris{at}nso2.uchc.edu
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ABSTRACT |
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Keywords: morphogenesis, polarity, cell cycle, spore germination
Abbreviations: CMI, chromosome mitotic index; HU, hydroxyurea; ts, temperature-sensitive
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INTRODUCTION |
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Cellular homeostasis depends upon the existence of regulatory mechanisms that ensure that morphogenesis is integrated with growth and nuclear division. In budding yeast, these processes are coordinated with each other during a step in the cell cycle termed START (Pringle & Hartwell, 1981 ). The nutritional status of the cell is thought to be assessed by the protein kinase A (PKA) pathway, which in turn modulates the temporal expression of the G1 cyclins (Baroni et al., 1994
; Tokiwa et al., 1994
). Cyclin-dependent kinase (cdk) activity associated with the G1 cyclins subsequently directs a number of events, including entry into S phase (Dirick et al., 1995
), polarization of the actin cytoskeleton (Lew & Reed, 1993
) and activation of a mitogen-activated protein (MAP) kinase pathway dedicated to promoting cell wall biosynthesis (Madden et al., 1997
). The switch from apical to isotropic growth that occurs upon entry into G2 is triggered by cdk activity associated with the mitotic cyclins (Lew & Reed, 1993
). In contrast to budding yeast, very little is known about the mechanisms which coordinate morphogenesis with growth and nuclear division in filamentous fungi. One pathway that could potentially serve this function is the PKA pathway. In particular, several studies have shown that PKA-mediated signalling plays a pivotal role in controlling polarized growth in many filamentous fungi (Gold et al., 1994
; Bruno et al., 1996
; Kronstad, 1998
). However, it is not yet known if this pathway acts to regulate growth in hyphal cells as it does in yeast.
The filamentous fungus Aspergillus nidulans forms asexual spores (conidia) which contain a single nucleus arrested in G1 (Bergen & Morris, 1983 ). During spore germination, conidia undergo an initial period of isotropic expansion before switching to polarized growth and forming an elongating germ tube. Concomitant with these morphogenetic events, spores resume general metabolic activity and re-enter the nuclear division cycle (dEnfert, 1997
). The mechanisms regulating the process of spore germination in A. nidulans are just beginning to be understood. It has been shown that the constitutively activated form of Ras prevents the switch from isotropic to polarized growth, which led to the proposal that down-regulation of Ras activity is required for spore polarization (Som & Kolaparthi, 1994
). Although this result suggests that Ras signalling plays a direct role in the control of morphogenesis, it remains possible that Ras may be involved in growth control and the establishment of a cell size threshold for polarization. In addition, the relationship between spore polarization and nuclear division has not been carefully examined in A. nidulans. Although conidia have been observed to undergo nuclear division prior to spore polarization, it is not known if the switch to polarized growth is dependent upon the completion of a specific cell cycle event.
Nuclear division in A. nidulans is controlled by the activity of two distinct protein kinases (Osmani & Ye, 1996 ). The cdk encoded by nimX, p34nimX, is required for passage through START and for entry into mitosis (Osmani et al., 1994
). Activation of p34nimX requires association with the cyclin p56nimE (OConnell et al., 1992
). Furthermore, during G2, p34nimX is converted into an active form by the tyrosine phosphatase p80nimT (OConnell et al., 1992
). In addition to p34nimX, mitotic entry in A. nidulans requires the activation of the NIMA protein kinase. NIMA activity gradually accumulates as hyphal cells progress through G2 and reaches a maximum in early mitosis (Osmani et al., 1988
). Attainment of maximal activity requires hyperphosphorylation of NIMA by p34nimX at the G2/M boundary (Ye et al., 1995
). Mitotic progression is dependent upon the subsequent destruction of NIMA and attenuation of its kinase activity (Pu & Osmani, 1995
). It has recently been shown that NIMA is required for nuclear localization of the p34nimXp56nimE cdk complex (Wu et al., 1998
).
The experiments described in this paper were designed to determine if spore polarization is coordinated with nuclear division, and if so, to provide initial insight into the nature of the underlying regulatory mechanism(s). By examining the kinetics of spore polarization in a battery of strains possessing defined defects in nuclear division, the following conclusions were reached: (i) spore polarization is coupled to cell cycle progression, and (ii) the mitosis-promoting kinase NIMA may play a role in inhibiting spore polarization. In addition, evidence is presented that suggests that the link between spore polarization and nuclear division can be uncoupled by growth signals. The data supporting these conclusions and a tentative model for the temporal regulation of spore polarization are described below.
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METHODS |
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Induction of the alcA promoter was accomplished by providing the relevant strains with 100 mM threonine as sole carbon source (Waring et al., 1989 ). For these experiments, strains were inoculated into YGV and incubated at 37 °C for 3 h to hasten the breaking of dormancy (conidia inoculated directly into MNV+thr take >6 h to germinate). During this period of incubation, conidia underwent isotropic swelling but did not polarize. Coverslips were then washed twice with prewarmed MNV+thr, and were subsequently transferred to a Petri dish containing fresh prewarmed MNV+thr at 37 °C. Control (alcA uninduced) coverslips were kept in YGV at 37 °C. Samples were taken at 2 h intervals following the media shift.
For the nutritional shift, wild-type (A28) conidia were incubated at 37 °C in YGV (rich media) for 2·5 h. One set of coverslips were transferred to a Petri dish containing pre-warmed YGV (rich media) and the incubation continued at 37 °C. The remaining coverslips were washed twice in pre-warmed MNV (poor media), and then transferred into a Petri dish containing fresh pre-warmed MNV at 37 °C. Samples were taken at 30 min intervals following the nutritional shift. For some experiments, nocodazole (Sigma) was added to rich or poor media at a final concentration of 5 µg ml-1 (from a stock solution of 5 mg ml-1 in DMSO).
Staining and microscopy.
Preparation of samples, staining with Calcofluor and Hoechst 33258, and mounting of stained coverslips on glass slides were performed as previously described (Harris et al., 1994 ). Slides were observed using an Olympus BMAX fluorescent microscope and DPLANAPO x40 and x100 (oil immersion) objectives. Spore size was measured using a calibrated eyepiece micrometer.
Measurements.
For each time point, 200 spores were counted to determine the fraction that had polarized. Currently, the most convenient marker for spore polarization is the presence of a germ tube. Although recent observations suggest that polarization of the actin cytoskeleton occurs prior to germ tube emergence (Harris et al., 1999 ), this event cannot be reliably detected under the growth conditions used for the experiments described here (unpublished results). Thus, a spore was counted as polarized if it possessed a germ tube. Since conidia are spherical in shape, recently emerged germ tubes were readily detectable as small protruberances on the spore surface.
Levels of NIMA activity in different strains were indirectly assessed by determining the chromosome mitotic index (CMI). This is a fairly reliable indicator since activated NIMA kinase is known to trigger chromosome condensation in A. nidulans and other organisms (Osmani et al., 1991 ; OConnell et al., 1994
). Thus, for some of the experiments described here, the fraction of spores (n=200) possessing highly condensed nuclei was determined at each time point. As an example, the level of chromosome condensation (22% vs 65%) observed in strains PL11-4 (four integrated copies of alcA::nimA) and PL11-7 (seven integrated copies of alcA::nimA) 10 h after alcA(p) induction is directly proportional to nimA copy number.
All experiments reported here were repeated at least twice with essentially indentical results. In all cases, representative results from one experiment are shown. Although the overall kinetics of spore polarization displayed some variability, there was always a consistent relationship between the different mutants and wild-type (e.g. nimA5 mutants always polarized 1530 min earlier than wild-type).
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RESULTS |
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The possibility that completion of the first mitotic division is a prerequisite for spore polarization was assessed by performing block and release experiments with inhibitors of cell cycle progression. Spores from the wild-type strain A28 were incubated in the presence of either HU or benomyl for 5 h, followed by release into inhibitor-free media. Both drugs were found to significantly impair spore polarization (Fig. 1). Following 5 h in the presence of either HU or benomyl, no spore had undergone nuclear division and only 2·5% were polarized. In contrast, at the same time point, 87% of untreated spores were binucleate and a total of 79% had polarized. Furthermore, the degree of inhibition of spore polarization caused by HU was found to be dose-dependent, as lower doses caused only a brief delay in polarization (unpublished results). These results suggest that the inability to complete the first mitotic division prevents spore polarization.
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Spore polarization in ts cell cycle mutants
In A. nidulans, a large collection of ts mutants defective in cell cycle progression have been identified and characterized in detail (Morris, 1976 ; Doonan, 1992
; Morris & Enos, 1992
; James et al., 1995
). Although these mutants fail to complete the first mitotic division when conidia are incubated directly at restrictive temperature, most of them do undergo spore polarization and form an elongating hypha. However, it is not known if these mutations cause delays in spore polarization similar to those induced by treatment of wild-type spores with HU or benomyl. To address this question, the timing of spore polarization was examined in individual mutants incubated at restrictive temperature and compared to that of a wild-type strain. As described below, these mutations fell into three distinct categories based upon their effects on spore polarization (Table 2
).
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The second group of mutations essentially prevented spore polarization (Table 2). Even after 24 h incubation at restrictive temperature, fewer than 30% of the spores possessing these mutations were polarized. In each mutant, the spores that did polarize were uninucleate (Table 2
) and polarization occurred at a time corresponding to the onset of aneuploidy and lethality (unpublished results). One of the mutations in this group (bimE7) affects the regulation of mitosis and causes spores to arrest with an elevated CMI (~75%; Osmani et al., 1988
). The other two mutations (nimL15 and nimN17) appear to cause arrest in interphase by preventing the completion of replication (Doonan, 1992
).
The third group of mutations had no apparent effect on the timing of spore polarization (Table 2). Three of the four mutations in this group (nimA5, hfaB3, nimT23) affect the regulation of mitosis (Doonan, 1992
; Morris & Enos, 1992
), whereas the fourth (nimX3) causes arrest at START in late G1 (Osmani et al., 1994
). Despite being unable to complete the first mitotic division (Table 2
), nimA5 and nimX3 mutants exhibited polarization kinetics similar to those of a wild-type strain. In principle, the failure of the nimT23 and hfaB3 mutations to delay spore polarization could be attributed to their inherent leakiness (e.g. by 12 h, 96% of nimT23 spores and 73% of hfaB3 spores were binucleate; Table 2
). However, although 90% of the spores from a nimT23 strain had polarized following 6 h incubation at restrictive temperature, only 34% had undergone nuclear division. Thus, these mutations appear to relieve the dependency of spore polarization on the completion of the first mitotic division.
The NIMA kinase may link spore polarization to the completion of mitosis
A possible mechanism by which spore polarization could be coupled to the completion of the first mitotic division was revealed by the analysis of the ts cell cycle mutants. Specifically, a mutation which prevents the accumulation of active NIMA kinase (nimA5; Ye et al., 1995 ) permits spore polarization to occur without delay in the absence of mitosis. In contrast, a mutation which leads to abnormally high levels of NIMA kinase activity (bimE7; Ye et al., 1996
) prevents polarization. Furthermore, polarization of wild-type spores is prevented by treatment with benomyl, which leads to a mitotic arrest with high levels of NIMA kinase activity (Osmani et al., 1991
). The inhibition of spore polarization does not appear to be due to defective mitosis per se since bimA1 and bimC4 mutants are able to polarize despite undergoing aberrant mitosis. These results suggest that the inhibition of spore polarization may in some manner be due to the effects of NIMA kinase activity. The following two experiments were performed to further test this idea.
If the increase in NIMA kinase activity which accompanies progression through G2 prevents spores from polarizing, then it might be expected that the loss of kinase activity in nimA mutants would lead to premature polarization. Consistent with this notion, spores from a nimA5 strain were observed to consistently undergo polarization 1530 min earlier than those from an isogenic wild-type strain (Fig. 2). Moreover, the volume of nimA5 spores at the time of polarization was less than that of wild-type spores (43·8±15·7 vs 62·6±20·3 µm3). This result suggests that NIMA kinase activity may play a role in delaying spore polarization until mitosis is complete.
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The link between spore polarization and mitosis can be uncoupled by growth signals
The experiments described above demonstrate that spore polarization is coupled to the completion of the first mitosis when spores are incubated in rich media (i.e. YGV). To determine if the timing of spore polarization relative to nuclear division could be modulated by growth signals, the kinetics of spore polarization and nuclear division were monitored in germinating spores subjected to a nutritional shift. Transfer of germinating spores from rich to poor media (YGV to MNV; Fig. 4) caused an apparent slowing of cell cycle progression. For example, the percentage of binucleate spores at 2 h post-shift was dramatically lower in the population shifted to MNV compared to a control population shifted to YGV (i.e. 23 vs 77%; Fig. 4a
). Despite the delay in nuclear division, spore polarization was accelerated by the shift to a poorer media source. Whereas only 3·5% of the spores in the control population had polarized by 2 h post-shift, 61·5% of the spores shifted to MNV had polarized by this time (Fig. 4a
). Furthermore, since accelerated polarization occurred even when spores were shifted to poor media containing nocodazole (Fig. 4b
), the shift-induced stimulation of spore polarization did not require nuclear division. These observations support the notion that growth signals can overrule the effect of cell cycle signals on spore polarization.
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DISCUSSION |
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Spore polarization is coupled to completion of the first mitosis
Conidiospores germinating on rich media (i.e. YGV) undergo a round of nuclear division prior to the emergence of a germ tube. Two independent lines of evidence demonstrate that completion of this intraconidial mitosis is a prerequisite for spore polarization. First, polarization does not occur if cell cycle progression is blocked by the inhibitors HU or benomyl. In addition, as would be expected if polarization depends upon the completion of mitosis, spores recovering from a mitotic block (benomyl; Orr & Rosenberger, 1976 ) polarized much more rapidly than spores recovering from an S-phase block (HU; Bergen & Morris, 1983
). Second, spore polarization was delayed by a number of different ts mutations that block cell cycle progression during interphase (nimE6, nimO18, nimP22, nimQ20 and nimR21) or mitosis (bimA1 and bimC4). The extent of the polarization delay depended upon the particular mutation, though it typically ranged from 1 to 6 h. The delay is presumably not caused by an indirect effect on growth since, for example, HU has no effect on RNA or protein synthesis in A. nidulans (Bergen & Morris, 1983
). Instead, the most likely explanation for the delay is failure to reach a specific point in the nuclear division cycle (i.e. an event associated with the completion of mitosis) at which the spore commits to polarization. The observation that the mutant spores ultimately did polarize despite the failure to complete mitosis is critical since it demonstrates that these mutations do not prevent morphogenesis per se. It also suggests that the normal coupling of polarization to completion of mitosis can be overidden by other regulatory responses (i.e. stress responses).
The NIMA kinase may play a role in coupling spore polarization to mitosis
Analysis of the kinetics of spore polarization in the ts cell cycle mutants provided insight into a mechanism that could conceivably couple polarized morphogenesis to nuclear division. Specifically, polarization was essentially inhibited in a mutant that accumulates excess amounts of fully active, hyperphosphorylated NIMA kinase (i.e bimE7; Ye et al., 1996 ). Similarly, polarization was severely delayed in another mutant that displays higher than normal NIMA kinase activity (bimA1; Lies et al., 1998
). Since BIMA and BIME are components of the anaphase-promoting complex (Peters et al., 1996
; Lies et al., 1998
), which is required for the targeted ubiquitination of cell cycle regulators (Townsley & Ruderman, 1998
), activated NIMA may accumulate in these mutants because it cannot be properly degraded. In contrast, polarization proceeded without delay in mutants that fail to accumulate hyperphosphorylated NIMA (i.e. nimA5, nimT23; Ye et al., 1995
). Indeed, polarization was slightly advanced relative to wild-type when nimA5 mutants were examined in detail. These results suggest that the inhibition of polarized morphogenesis is associated with the presence of the fully active, hyperphosphorylated form of NIMA. Two additional observations provide support for this idea. First, increased expression of the nimA gene leads to a dose-dependent inhibition of spore polarization, even under conditions in which nimA mutations do not normally affect polarization (i.e. minimal media). Second, expression of a truncated form of NIMA that can be hyperphosphorylated, but not degraded (Pu & Osmani, 1995
), prevents polarization (unpublished results). The simplest interpretation of these results is that activated NIMA kinase may inhibit spore polarization.
The potential role of NIMA in coupling spore polarization to the completion of mitosis can be explained by the following model. As spores pass through the G2/M boundary of the first mitosis, hyperphosphorylation of NIMA by p34nimX leads to attainment of full kinase activity (Ye et al., 1995 ). The activated kinase promotes events required for mitosis, and in addition inhibits spore polarization. Proteolytic degradation of NIMA triggers exit from the ensuing mitosis (Pu & Osmani, 1995
), and also permits spore polarization by removing the inhibitory signal. The ability of activated NIMA kinase to prevent spore polarization could be either a direct or indirect effect. For example, NIMA could directly phosphorylate, and hence inactivate, a substrate that is required for polarization. Alternatively, activated NIMA could indirectly inhibit polarization by promoting a mitotic event such as chromosome condensation (Osmani et al., 1991
; OConnell et al., 1994
). In this scenario, expression of a gene required for morphogenesis is suppressed when chromosomes are in a condensed state. Distinguishing between these possibilities will not be straightforward, since a mutation or condition that uncouples NIMA activation from chromosome condensation does not yet exist.
Additional mechanisms prevent spore polarization prior to mitosis
Results presented here demonstrate that drugs (i.e. HU; Bergen & Morris, 1983 ) or ts mutations (i.e. nimL15, nimN17; Doonan, 1992
) that block progression through S phase also inhibit spore polarization. Since NIMA levels are very low in S-phase cells (Osmani et al., 1991
), this effect is presumably not attributable to NIMA kinase activity. In support of this notion, spore polarization is blocked in nimA5 mutants treated with HU, or in nimL15 nimA5 double mutants (unpublished results). How then is polarization prevented when spores are progressing through S phase? Activation of the checkpoint pathway that responds to incompletely replicated DNA could conceivably inhibit polarization. However, the observation that polarization is still blocked by HU when the replication checkpoint is abrogated rules out this possibility (unpublished results). Alternatively, expression of a gene required for morphogenesis may be suppressed when chromosomes are in a replicative state. This idea could be tested by examining the expression pattern of genes required for polarized morphogenesis.
Growth signals can uncouple spore polarization from nuclear division
The coupling of polarization to the completion of mitosis in germinating conidiospores could reflect the activity of a size control mechanism that establishes a ratio of nuclei to cytoplasm that is optimal for subsequent hyphal growth (Trinci, 1978 ). If so, poorer growth conditions should then alter the size setting and thus influence the timing of spore polarization relative to nuclear division. Consistent with this notion, conidiospores germinating on MNV media polarize prior to the first mitosis and, under these conditions, polarization is not blocked when spores are trapped in mitosis with condensed chromosomes (unpublished results). Moreover, shifting germinating conidiospores from rich to poor media induces polarization in the absence of nuclear division. These observations suggest that conidiospores can rapidly sense growth conditions and adjust the size control accordingly. In addition, they show that the effects of the cell cycle on spore polarization can be overridden by growth signals.
Why is spore polarization not delayed in nimX3 mutants?
Under the conditions used in the experiments described here, the nimX3 mutation arrests cell cycle progression in G1 (Osmani et al., 1994 ). However, despite failing to proceed through the cell cycle to the completion of mitosis, spore polarization is not delayed by the nimX3 mutation. Furthermore, HU has no effect on the kinetics of spore polarization in nimX3 mutants (unpublished results). Why then, do these mutants undergo spore polarization despite not completing mitosis? One possible explanation is that growth signals can promote polarization in the absence of inhibitory cell cycle signals (i.e. generated when conidiospores are in S phase or mitosis). In this model, although spores germinating in rich media are competent to polarize at START, cell cycle signals delay polarization until the completion of mitosis. This delay may ensure the optimal ratio of nuclei to cytoplasm for hyphal growth. In nimX3 mutants, or in wild-type spores germinating on poor media, the cell cycle signal is not generated and spores can thus polarize prior to the completion of mitosis.
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ACKNOWLEDGEMENTS |
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This work was supported by a New Investigator Award from The Patrick and Catherine Weldon Donaghue Medical Research Foundation.
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Received 16 March 1999;
revised 17 June 1999;
accepted 5 July 1999.