Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca 2+ gradient is essential for hyphal growth and that one mechanism responsible for the Ca 2+ cellular concentration starts with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2) by receptor-regulated forms of phosphoinositide-specific phospholipase C (PI-PLC). In the present study the levels of calcium in Aspergillus nidulans wild type (A26) and plcA-deficient mutant (AP27) growing in a carbon source readily assimilated, as glucose or pectin a non-readily assimilated carbon source was investigated. Intracellular calcium levels in A26 were higher in the presence of glucose than in pectin, but lower in AP27 independently of the carbon source and in AP27 the vesicular calcium distribution occurred mainly at the apex of the hyphae. Delay in nuclear division was also observed if A26 and AP27 were grown in pectin presence when compared with growth in glucose. For the first time, it is demonstrated that the levels of intracellular Ca 2+ were higher when A. nidulans was growing in glucose than in a non readily assimilated carbon source as pectin. Further, it also showed that the plcA gene, although not essential, may be responsible for high-molecular weight carbon source recongnation, for the intracellular Ca 2+ levels maintenance and consequently by the nuclear division in A. nidulans.
Calcium ions act as second messengers in the signal transduction pathways in eukaryotic organisms from fungi to humans. The key features of the Ca2+ machinery in fungi are conserved as multicellular eukaryotes and underlie the diverse fungal physiological processes [
Very little is known about the mechanisms, which coordinate morphogenesis, growth and nuclear division in fungi, mostly when they are growing in different carbon sources. Our previous results showed that two phospholipase C (E.3.1.4.3) (PLC) inhibitors delayed the first nuclear division in the A. nidulans cultures growing in the glucose presence, but stimulated it in a medium with pectin [
A. nidulans, FGSC A26 (biA1, veA1) designated A26 here and the mutant transformed with plcA gene rupture referred as AP27 [
To determine the spatial distribution of the Ca2+-containing organelles, the mycelium pre-grown for 30 h at 30˚C were fixed as described by Kawano and Said [
Intracellular Ca2+ was determined with the Ca2+-sensitive fluorescence dye Fluo-3/AM, dissolved in DMSO and stored in aliquots protected from light at 4˚C. Conidia suspensions (106) from A26 and AP27 strains pre-incubated for 8 h in liquid MM containing 1 mM of ethylene glycol tetra acetic acid (EGTA), to chelate extracellular calcium, were centrifuged at 472× g for 5 minutes and resuspended in 1 mL water. Conidia and germlings were transferred to individual tubes (0.5 mL), loaded with Fluo-3/AM (25 μM) and incubated for 35 min at 25˚C. During incubation Pluronic F-127, a nonionic poliol, was added to a final concentration of 1% to facilitate dye penetration. DMSO and Pluronic F-127 were also added to the controls. Analyses were performed on a FACSCanto (BD San Jose-USA) and the FACSDiva software was used for the acquisition of 10,000 events collected for each sample on FACSArray. The Fluo-3 fluorescence was detected using a 530/30 nm filter. Two groups of cells were detected by flow cytometer; P8 cells with Fluo-3 fluorescence signal negative and P9 cells with Fluo-3 fluorescence signal positive.
Experiments were done in duplicate or triplicate and independently repeated. The bars in the figures indicate the mean values ± standard deviation (SD). Data were statistically analyzed by one-way ANOVA followed by Bonferroni’s t test and considered significantly different when P < 0.05 was obtained.
CTC fluorescence, indicating the Ca2+ containing vesicles, was observed after 30 h of incubation in glucose or pectin. CTC fluorescence was detected in subapical hyphal region and mainly in apex of A26 strain grown in glucose (
Fluo-3/AM was used for the first time in A. nidulans to detect intracellular calcium by flow cytometry. In conidia and germlings of A26 incubated in media supplemented with glucose or pectin, fluorescence was detected in approximately 22% and 4% cells, respectively, while in AP27 the percentages were approximately 11 and 3.5, respectively. Thus, it was demonstrated that A26 had two times more intracellular Ca2+ than AP27 when growing in glucose, but in pectin-supplemented media, the strains had similar values, which were about 2 to 5 times smaller than those detected in glucose presence (
No significant differences were detected on conidial germination when A26 or AP27 was being cultivated in pectin or glucose presence but the percentage of cells contained four nuclei was bigger in both cultures growing in glucose than those in pectin (
number of nuclei present.
The nuclear division might be activated by plcA when glucose is available and a polymeric carbon source as pectin or polypectate would lead to an opposite response [
medium the plcA gene would be not activated conesquently the low Ca2+ levels would be not enough to realize the nuclear duplication and others activities at same rate observed in glucose. In AP27 mutant when PLC inhibitors spermine and C48/80 were used [
The results of the present study clearly showed that in A. nidulans, plcA gene and carbon source are not the unique but they are involved in maintenance of intracellular Ca2+ levels and consequently in other functions of cellular germination as on nuclear duplication. Other experiments have to be conducted using other different carbon sources and in other fungi to investigate if this is a general mechanism of regulation.
The present study is part of a thesis presented by J.A.R. to the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, in partial fulfillment of the requirements for the Doctor’s degree. J.A.R. received a Doctoral Fellowship from Coordenação de Apoio de Pessoal de Nível Superior (CAPES). The authors are grateful to Dr. A. C. Tedesco for the Pluronic F-127 reagent, to F. R. Morais for assistance in flow cytometry analysis.