Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, 1428 Buenos Aires, Argentina1
Author for correspondence: Silvia Moreno. Tel: +54 11 4576 3342. Fax: +54 11 4576 3342. e-mail: smoreno{at}qb.fcen.uba.ar
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ABSTRACT |
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Keywords: cAMP, morphogenesis, Mucor rouxii, protein kinase A, polarized growth
Abbreviations: CI-930, 3(2H)-pyridazinone,4,5-dihydro-6-[4-(1H-imidazol-1-yl) phenyl-5-methylmonohydrochloride; dbut-cAMP, dibutyryl-cAMP; IBMX, isobutyl-methylxanthine; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; SQ 65,442, 1-ethyl-4-(ethylthio)-1H-pyrazolo[3,4-b]-pyridine-5-carboxylic acid ethyl ester.
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INTRODUCTION |
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The fungus Mucor rouxii exhibits dimorphic growth; under aerobic conditions it grows as a typical mycelium, and under anaerobiosis the growth is as yeast-like cells (Bartnicki-García & Nickerson, 1962 ). The possible role of cAMP in the morphogenetic process of Mucor has previously attracted the attention of a number of researchers (Orlowski, 1991
, 1995
). We have reported that in the presence of dibutyryl-cAMP (dbut-cAMP) the sporangiospores from M. rouxii grow isodiametrically and that polarized growth is prevented without alteration of growth parameters (Pereyra et al., 1992
).
To study in more detail the transduction pathway involved in the effect of dbut-cAMP upon M. rouxii morphology, we decided to investigate the involvement of cAMP-dependent protein kinase (PKA) in this process. M. rouxii PKA has been thoroughly characterized (Moreno & Passeron, 1980 ; Moreno et al., 1983
; Paveto et al., 1989
; Guthmann et al., 1990
) and the levels of its subunits have been measured during the time course of spore germination in rich medium (Rossi & Moreno, 1994
).
In this report we clarify three points that will help to establish a model system in which to study the involvement of cAMP-mediated phosphorylation-dependent regulation of a cellular process such as hyphal morphogenesis: 1, PKA is involved in the morphogenetic process; 2, the impairment of polarized growth by cAMP analogues is a general effect which can be observed under diverse environmental and nutritional conditions; 3, the concentration of PKA seems to be critical for the differentiation of the germinated sporangiospore.
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METHODS |
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Samples of cultures were withdrawn and observed under the light microscope. Morphologies were classified as follows: M, when cells germinated normally yielding a typical mycelium (Fig. 1a); P, when the effect on morphology was partial and the observed forms comprised round cells, cells with reduced hyphal branching, thicker hyphae with swollen tips and forms consisting of rounded cells in a chain (Fig. 1b
) and T, when the effect was total and the complete population consisted of rounded cells without buds (Fig. 1c
). About 100 cells were scored for each experiment.
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Soluble protein was determined by the Bradford assay (Bradford, 1976 ), using BSA as the standard.
Chemicals.
[-32P]ATP was from New England Nuclear; kemptide, cAMP analogues, papaverine, isobutyl-methylxanthine (IBMX), theophylline, and dipyridamole were from Sigma; CI-930 (3(2H)-pyridazinone,4,5-dihydro-6-[4-(1H-imidazol-1-yl) phenyl-5-methylmonohydrochloride) was a gift from Warner Lambert, Ann Arbor, MI; and SQ 65,442 (1-ethyl - 4 - (ethylthio) - 1H - pyrazolo[3,4 - b] - pyridine - 5 - carboxylic acid ethyl ester) was a gift from E. R. Squibb and Sons, Princeton, NJ, USA. Stock solutions (10 mM) of each compound were prepared as follows: cAMP analogues, papaverine, IBMX and CI-930 in water; dipyridamole and SQ 65,442 in ethanol. Other reagents were of analytical grade.
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RESULTS AND DISCUSSION |
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The results are summarized in Table 1 and representative images are shown in Fig. 1
. N6-benzoyl-cAMP at 0·6 mM and N6-monobutyryl-cAMP at 1·5 mM promoted isodiametric growth of M. rouxii sporangiospores (Fig. 1b, c
). At 0·15 mM dbut-cAMP, total effect was observed as already reported (Pereyra et al., 1992)
. Two controls were performed with O2'-monobutyryl-cAMP and butyrate, assayed at the maximal concentrations that could be derived from the complete hydrolysis of 0·15 mM dbut-cAMP. None of these compounds had an effect on morphology, indicating that the effects of dbut-cAMP are not due to these two hydrolytic products. The results observed in vivo are in accordance with those previously reported in vitro (Paveto et al., 1989
), which indicated that these two cAMP analogues are potent activators of PKA. dbut-cAMP is a very poor activator of PKA, since the concentration required to produce half-maximal activation of M. rouxii PKA is around 300-fold lower than for the two N6-cAMP analogues. This low potency in the activation of PKA has already been reported for higher eukaryotic holoenzymes (Øgreid et al., 1985
). However, its potency can be adequately explained by a sixfold increase in the endogenous cAMP levels (unpublished results). Although the concentration of PDE inhibitor required to produce 50% inhibition of hydrolysis of 1 µM cAMP by the high-affinity cAMP PDE from the fungus is quite low (in the range of 1 mM; Tomes & Moreno, 1990
and unpublished results), it has been reported to be a compound hydrolysed by the PDEs with a very small turnover (Van Haastert et al., 1983
, Braunmann et al., 1986
).
The only PDE inhibitor that promoted an effect on morphology at the concentrations assayed was SQ 65,442. The total effect was attained at 0·1 mM (Fig. 1d), with a morphology similar to the one observed with N6-cAMP analogues and with dbut-cAMP (Fig. 1c
), except for a slight impairment of growth. Two observations guarantee that the lack of polarized growth observed in the presence of SQ 65,442 (see Table 1
and Fig. 1d
) was not the result of growth impairment: 1, cells had a constant and sustained increase in diameter throughout the experiment and 2, they attained a diameter far larger than the one displayed by control cells at the time of germ-tube or bud emergence. The result observed with SQ 65,442 is in accordance with the fact that this compound is the best M. rouxii PDE inhibitor. The IC50 for the hydrolysis of 1 µM cAMP was around 0·01 mM, at least an order of magnitude smaller than the IC50 for the rest of the compounds assayed (Tomes & Moreno, 1990
). The inhibitor has the advantage of being a lipophilic compound. It is not surprising that M. rouxii is insensitive to IBMX and to theophylline since it has been reported that PDE from another lower eukaryote, Saccharomyces cerevisiae, is also relatively insensitive to standard PDE inhibitors (Van Lookeren-Campagne et al., 1990
).
Characterization of the morphopoietic effect of cAMP analogues
M. rouxii sporangiospores grown in the presence of cAMP analogues or SQ 65,442 have several characteristics, besides the isodiametric growth, that are worth summarizing: 1, increased pigmentation that can be observed macroscopically (isodiametrically growing cultures are slightly yellow while control ones are white); 2, increased fragility, not prevented by osmotic stabilization of the growth medium (Pereyra et al., 1992 ); 3, loss of the typical adhesiveness that begins to be observed in the germling stage of control growth (compare Figs 1e and c
).
There are examples in the literature correlating all of these characteristics to cAMP signal-transduction pathways. Increased carotene pigmentation has already been described to occur upon addition of dbut-cAMP to Mucor mucedo and Mucor hiemalis (Jones & BuLock, 1977 ) and signal transduction by cAMP has been suggested to cross-talk with carotenogenesis in fungi (Dandekar & Modi, 1980
; Hohl et al., 1992
). Neurospora crassa cells, carrying a mutation in the regulatory subunit of PKA (Bruno et al., 1996
) are extremely fragile and show thick cell walls, as has been observed ultrastructurally for M. rouxii cells grown with cAMP analogues (to be published elsewhere). Regarding cell adhesiveness, it has been already reported that cAMP is an important intracellular modulator of cell adhesion in mammalian cells. Glass & Kreisberg (1993)
observed that cAMP elevation in cultures of mesangial cells caused loss of adhesion, shape change towards rounded cells, microfilament fragmentation and myosin-light-chain dephosphorylation. The diversity of cell types exhibiting similar changes suggested to these authors that there could be a common underlying mechanism regulating cytoskeletal structure and adhesion (Glass & Kreisberg, 1993
and references therein).
Effect of time of addition of compounds to the culture medium
The effects of cAMP analogues or PDE inhibitor on morphology described until now (Pereyra et al., 1992 and Table 1
) were observed upon addition of the compound 30 min after the inoculation of sporangiospores into the culture medium. To investigate the effect these compounds had on the germination process, experiments were performed in which 0·6 mM N6-benzoyl-cAMP or 0·15 mM dbut-cAMP were added to the culture medium at 0, 1, 3, 5, and 7 h of growth. The morphologies observed at 16 h of growth were identical to those of Fig. 1c
, no matter when the analogues were added, as long as it was before the germ-tube emergence (before 6 h). Once germination had begun (7 h; Fig. 1e
) a severe effect on morphology was also observed (Fig. 1f
); the images suggested that growth after the addition of the analogue tends to continue in a non-polarized form. The same results were observed when performing the experiment in the presence of 0·1 mM SQ 65,442 (data not shown). These results suggest that the process impaired by cAMP analogues or PDE inhibitor, leading to an isodiametric growth of the cells, occurs at a late stage in germination, immediately before the differentiation stage (germ-tube emergence). An additional observation was that the concentrations of dbut-cAMP, N6-benzoyl-cAMP and SQ 65,442 needed to promote the total effect upon morphology were lowered threefold, 1·5-fold and threefold respectively when the compounds were added immediately before germ-tube emergence.
Effect of cAMP analogues and SQ 65,442 under diverse nutritional and environmental conditions
The experiments described so far and by Pereyra et al. (1992) were performed with sporangiospores growing aerobically in a defined medium with glucose (DMG). We were interested in assaying whether the impairment of polarized growth promoted by cAMP analogues was also observed under different growth conditions. The media used for aerobic growth were the standard defined medium with glucose (DMG), the same medium with maltose as the carbon source (DMM), the Casamino acid-enriched medium with glucose (DMCG) and the classical rich medium (YPG). For anaerobic growth under CO2 atmosphere, only YPG medium was suitable, since yeast-like growth does not occur in defined medium (Bartnicki-García & Nickerson, 1962
) and spores germinating anaerobically in DMCG bud scarcely and therefore are an unsuitable model in which to detect isodiametric growth. Yeasthyphal phase transitions were performed by harvesting yeast-like cells coming from an anaerobic culture in YPG, and aerating them in defined DMG medium. The results, summarized in Table 2
, with representative morphologies being shown in Fig. 2
, indicate that under aerobic conditions, the addition of either dbut-cAMP or SQ 65,442 promotes isodiametric growth under all the nutritional conditions. It was observed that the richer the culture medium, the higher the concentration of dbut-cAMP needed to attain the morphological effect. With YPG, only partial effects (not shown) could be observed at the highest assayed concentration of dbut-cAMP (10 mM). Under anaerobic growth conditions in rich YPG medium, no effect was observed with 10 mM dbut-cAMP. However, in the presence of 0·1 mM SQ 65,442, growth was isodiametric, although with a slight impairment (Fig. 2b
). For the experiment where cultures were shifted from anaerobiosis to aerobiosis, shown in Table 2
, DMG medium was used for the aerobic condition in order to observe a good effect of the added cAMP analogue. The total effect was attained at 1 mM dbut-cAMP. The images shown in Fig. 2(c, d
) show that 24 h after the shift to aerobiosis, no more buds are emitted and cells continue growing in a non-polarized form (dry weight was increased 2·5-fold during the shift). An intense pigmentation of the cells was also observed.
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PKA and germination
We have previously measured and reported (Rossi & Moreno, 1994 ) the specific activity of PKA during germination of sporangiospores in YPG rich medium. The methodological considerations for validating the assay of PKA activity in the presence of an exogenous saturating concentration of cAMP as a measure of total PKA levels was also given. The results indicated that total activity of PKA in sporangiospores inoculated in YPG decreased immediately after the onset of germination, following a transient increase in cAMP levels (Rossi & Moreno, 1994
). After this stage, the total specific activity of PKA decreased in parallel with the increase in total protein and volume. A significant increase in the synthesis of regulatory and catalytic subunits to form the holoenzyme was not apparent until right before germ-tube emergence. From that time onwards the total specific activity for PKA, in the presence of cAMP, was more or less constant (Fig. 4c
). We were interested in investigating whether this variation in PKA total activity also occurred when the cells were grown in more defined media, to evaluate whether PKA activity could be correlated with growth or differentiation processes. Fig. 4 (a and b)
shows the variations in the levels of PKA total specific activity when spores were grown in DMM or DMG, respectively. The results show that a decrease in the activity of PKA is observed at the beginning of the germination process, independent of the growth medium used. This result was predictable, since it correlates with a transient peak of cAMP, occurring very shortly after inoculation of the spores in the growth medium. After this first decrease in PKA total specific activity, the extent of the decrease was inversely correlated with the growth of the culture. The poorer the medium, the less the PKA activity decreased. This result fits into the following interpretation: if during the first stages of growth the synthesis of PKA subunits does not go in parallel with the synthesis of total proteins, a decrease in the specific activity would be more severe in the cases where total growth is more evident (YPG>DMG>DMM). It is notable that in either DMG or YPG, total specific activity of PKA begins to increase significantly at germ-tube emergence. When cells were grown on DMM, a net increase is not observed, but shortly before germ-tube emergence, the specific activity stops decreasing and remains constant. Since, at this stage, growth begins to increase significantly, a constant level of PKA specific activity necessarily means an increase in the synthesis of the enzyme. It was surprising to observe that the specific activity of PKA in the three media at the time of germ-tube emergence was very similar. To analyse these data more carefully, an experiment was performed in which the cultures under the three growth conditions were performed in parallel, with the same batch of spores, and the extracts for enzymic assays were processed side by side, with cells harvested from each culture at the corresponding emergence time (as defined in Table 3
). The total specific activity of PKA attained at the time of germ-tube emergence under the three culture conditions was surprisingly alike (Table 3
). However, if we recalculate the data to express them as the total units of PKA per 106 cells (Table 3
) it can be seen that the variation is in accordance with the difference in size and growth of the cells grown in the different media; the total activity of PKA per cell unit is much less in a small cell (DMM) than in a large cell (YPG).
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We are therefore tempted to speculate that a threshold concentration of PKA must be attained in order to elicit the germ-tube emergence. If one divides the germination process in two the growth stage and the differentiation stage, that occur intermingled it seems that the PKA specific activity does not correlate with growth, as cell volume and total protein do, but with differentiation. Thus, the poorer the culture medium, the slower the growth process and the earlier the threshold specific activity of PKA is attained. In accordance with these results, it has been recently reported that the levels of another family of proteins involved in signal transduction, the Ras proteins in Mucor racemosus, do not correlate with growth but with differentiation (Roze et al., 1999 ).
Testing of the hypothesis
If the hypothesis that a threshold concentration of PKA must be attained in order to elicit germ-tube emergence is correct, we could predict the results to be observed when shifting a culture from a poor medium into a rich medium and vice versa, regarding time of germ-tube emergence and sensitivity to the morphological effect of dbut-cAMP. One of the predictions would be that when shifting a culture, before germ-tube emergence, from a poor medium into a rich medium the growth process should be favoured and therefore the specific activity of PKA should begin to decrease; this would have as a consequence a delay in the emergence of the germ tube and a necessity for higher concentrations of dbut-cAMP to promote isodiametric growth. Another prediction would be that, on the contrary, a shift from rich medium to poor medium should favour the differentiation process with an increase in PKA specific activity, an anticipation of the germination time and an increased sensitivity to dbut-cAMP. The results, shown in Table 4, are exactly the ones predicted. When shifting the culture from DMG to YPG the germination time was delayed and the sensitivity to dbut-cAMP was decreased, and exactly the opposite occurred when the shift was done from a rich medium (YPG) to a poorer medium (DMG).
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Conclusions
The results presented in this work, taken as a whole, suggest that PKA is involved in M. rouxii polarized growth, and that morphogenesis and growth are two unlinked processes. The evidence can be summarized as follows. Impairment of polarized growth was obtained with cAMP analogues that are good in vitro PKA activators such as the N6-cAMP analogues and by a very good M. rouxii PDE inhibitor. The lack of polarized growth observed in the presence of cAMP analogues was obtained under all the nutritional and environmental conditions studied, and was independent of the time of addition of the cAMP analogue to the culture medium. The effect was observed even when adding the compounds very shortly before germ-tube emergence. At this time the specific activity of regulatory and catalytic subunits is high, while being very low or almost undetectable at earlier times of germination. The time of germ-tube emergence correlated with the time of attainment of a threshold level of PKA specific activity. The concentration of dbut-cAMP needed to impair polarized growth correlated with the total amount of PKA to be activated per cell. All the observed effects, namely isodiametric growth, increased pigmentation, increased cell fragility and lack of adhesiveness, have been proposed to be related to the cAMP signal-transduction pathway in other systems.
The fact that an effect of cAMP analogue on morphology is still observed when added after germ-tube emergence suggests that polarized growth is not the result of a commitment decided in a previous step but that is a result of a regulatory process that must be maintained throughout and that PKA participates in this maintenance. Support for this statement is seen in the immediate reversion from isodiametric to polarized growth upon removal of the culture medium containing the cAMP analogue and replacement with fresh medium without analogue (Pereyra et al., 1992 ).
We have now established a model system in which to investigate the downstream targets of PKA in relation to polarized growth. Apical growth is a dynamic process involving the continual synthesis and deposition of cell wall at the apex, concomitant with the forward movement of cytoplasm and independent movement of vesicles, nuclei, mitochondria and other organelles, as they maintain their positions relative to the extending tip (Jackson & Heath, 1993 ). Over the last few years it has become increasingly clear that actin is involved in a variety of the processes that result in tip growth (Heath, 1994)
. Preliminary results obtained with M. rouxii (Sabanero, 1994
and our unpublished results) indicate that in the presence of cytochalasin, the morphology adopted by the cells regarding isodiametric growth and cell-wall width are similar to the results obtained with cAMP analogues. We are therefore tempted to speculate that PKA might be involved in the regulation of actin microfilament organization.
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ACKNOWLEDGEMENTS |
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Received 20 January 2000;
revised 3 May 2000;
accepted 15 May 2000.