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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ATM is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of DNA damage response in eukaryotes. In humans, mutations in ATM cause the devastating neurodegenerative disease Ataxia-Telangiectasia. Previously, we characterized the homologue of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Our results suggested that AtmA probably regulates the function and/or localization of landmark proteins required for the formation of a polarity axis. Here, we extended these studies by investigating which pathways are influenced by AtmA during proliferation and polar growth by comparatively determining the transcriptional profile of A. nidulans wild-type and ΔatmA mutant strains in different growth conditions. Our results indicate an important role of the pentose phosphate pathway in the fungal proliferation during endogenous DNA damage and polar growth monitored by the AtmA kinase. Furthermore, we identified several genes that have decreased mRNA expression in the ΔatmA mutant that are involved in the formation of a polarized hyphae and control of polar growth; in the synthesis of phosphatidic acid (e.g. phospholipase D); in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts); and in intracellular trafficking.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ATM is a phosphatidyl-3-kinase-related (PI-3 kinase) protein kinase (PIKK) that functions as a central regulator of the DNA damage response in eukaryotic cells (Shiloh, 2001; 2006; McKinnon, 2004). In humans, mutations in ATM cause the devastating neurodegenerative disease Ataxia-Telangiectasia (A-T; summarized by Gatti, 1998; Chun and Gatti, 2004). Like its homologue ATR, ATM responds to damage such as double-strand breaks (DSBs) by phosphorylating multiple targets that collectively act to maintain genome integrity. Key targets include proteins that direct chromatin modification (histone H2AX), promote DNA repair (BRCA1, BLM), activate cell cycle checkpoints (Chk1, Chk2, Nbs1, FancD2, Mdm2) and trigger apoptosis (p53) (summarized by McKinnon, 2004). Many of these proteins contain consensus ATM phosphorylation sites whose importance has been demonstrated in functional studies (O'Neill et al., 2000; Matsuoka et al., 2007). Notably, studies in yeast have shown that the ATM homologue Tel1 is one of the first proteins recruited to DSBs, where it appears to mediate formation of protein complexes involved in checkpoint activation and repair (Lisby et al., 2004). Accordingly, it is generally thought that the primary function of ATM is to choreograph the response to DNA damage.

The filamentous fungus Aspergillus nidulans possesses a sophisticated DNA damage response that ensures the maintenance of genome integrity (reviewed by Goldman et al., 2002; Goldman and Kafer, 2004). The ATR homologue UvsBATR is a central component of this response, where it controls the activation of multiple checkpoints, regulates damage-induced gene expression and also promotes DNA repair (DeSouza et al., 1999; Hofmann and Harris, 2000). UvsBATR displays an extensive web of genetic interactions with other proteins involved in the DNA damage response, including the Mre11 complex (ScaANBS1, MreAMRE11 and SldIRAD50), the cdc2-related kinase NpkA, and SepBCTF4 (Fagundes et al., 2004; 2005; Gygax et al., 2005; Malavazi et al., 2005). Recently, we characterized the homologue of ATM (AtmA) in A. nidulans (Malavazi et al., 2006). In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. We demonstrated that an atmA mutant failed to generate a stable axis of hyphal polarity. Here, we extended these studies by investigating which pathways are influenced by AtmA during proliferation and polar growth (i.e. lack of germ tube emergence) by comparatively determining the transcriptional profile of A. nidulans wild-type and ΔatmA mutant strains in different growth conditions. Our results show an important involvement of the pentose phosphate pathway in the ΔatmA mutant phenotype. Furthermore, we identified several genes that have decreased mRNA expression in the ΔatmA mutant involved in the formation of a polarized hyphae and control of polar growth, in the synthesis of phosphatidic acid and phosphatidylinositol, and in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts), and intracellular trafficking, secretion and vesicular transport.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

To gain an insight on which pathways are influenced by AtmA during proliferation and polar growth, we determined the transcriptional profile of A. nidulans wild-type and ΔatmA mutant strains in different growth conditions. First, we compared the accelerated rate of proliferation, i.e. increased nuclear kinetics (number of nuclei per cell compartment), of the ΔatmA mutant with the wild type (see Table 1). We have incubated both A. nidulans conidia in the presence of 50 mM HU for 5 h; the germlings were washed to release the HU and grown again for different periods of time. The HU blockage synchronized the polar growth, and the kinetics of growth after HU release represents the process of emergence of the polar tube from the swollen conidia. Table 2 shows that the ΔatmA mutant strain has a decreased percentage of polar growth when compared with the wild-type strain as it was previously shown by Malavazi et al. (2006). Thus, total RNA extracted from these cultures was used to synthesize fluorescent-labelled cDNAs for competitive microarray hybridizations. In the first set of experiments, we have compared the mRNA expression of the ΔatmA mutant with the wild-type strain at 60, 90 and 120 min germling growth. In the second set of experiments, we first verified the mRNA expression for each strain after HU release (i.e. 60, 90 and 120 min against the reference time zero), and then we compared the transcriptional profile of the wild type with that of the ΔatmA mutant strain. In these experiments, the main aim was to focus on genes that have increased or decreased mRNA expression in the absence of AtmA. The full data set was deposited in the Gene Expression Omnibus (GEO) from the National Center of Biotechnology Information (NCBI) with the number GSE8529 (http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE8529).

Table 1.  Nuclear kinetics during germling growth in the wild-type and ΔatmA mutant strains.
StrainsaOne nucleusTwo nucleiFour nuclei
  • a.

    Conidia were grown for different periods of time at 37°C. Cells were DAPI-stained and the number of germlings with different number of nuclei counted. The results are expressed as the average ± standard deviation of three experiments analysing 100 germlings each.

Wild type (60 min)100.0 ± 0.00.0 ± 0.00.0 ± 0.0
Wild type (90 min)98.0 ± 0.02.0 ± 0.00.0 ± 0.0
Wild type (120 min)95.5 ± 0.74.5 ± 0.70.0 ± 0.0
Wild type (150 min)94.0 ± 1.46.0 ± 1.40.0 ± 0.0
ΔatmA (60 min)93.0 ± 1.47.0 ± 1.40.0 ± 0.0
ΔatmA (90 min)91.5 ± 2.18.5 ± 2.10.0 ± 0.0
ΔatmA (120 min)87.5 ± 0.711.5 ± 0.71.0 ± 0.0
ΔatmA (150 min)81.0 ± 2.816.5 ± 2.12.5 ± 0.7
Table 2. Aspergillus nidulans polar growth upon HU release.
StrainsaPolar growth (%)
0 min60 min90 min120 min150 min
  • a.

    Conidia were grown in the presence of HU (50 mM) for 5 h at 37°C. The cells were washed to release the HU and grown again at 37°C for different periods of time. Results are expressed as percentage of germlings that showed polar growth after HU release; they are the average ± standard deviation of three experiments analysing 100 germlings each.

Wild type1.0 ± 0.042.0 ± 1.467.0 ± 2.873.5 ± 2.182.5 ± 3.5
ΔatmA1.0 ± 0.014.5 ± 0.721.5 ± 2.139.5 ± 2.145.0 ± 1.4

Genes involved in DNA replication and in the pentose phosphate pathway (ppp) are more expressed in the ΔatmA mutant

We compared the mRNA expression of the ΔatmA mutant with the wild-type strains grown for 60, 90 and 120 min. We were able to observe 1034 genes modulated in at least one time point in the mutant when compared with the wild-type strain. These genes are involved with a variety of cellular processes and A. nidulans atmA absence is likely to be implicated with their increased or decreased mRNA expression (Tables 3 and 4 show the genes with log ratios ≥ 1 or ≤ 1 respectively). These genes were classified into COG functional categories (http://www.ncbi.nlm.nih.gov/COG/). As it can be observed in Table 3, there is an increased expression of genes encoding proteins involved in cell cycle control, cell division, chromosome partitioning, nucleotide transport and metabolism, carbohydrate transport and metabolism, DNA replication, recombination and repair, noticeably, most of the genes of the pentose phosphate pathway (ppp), nucleotide metabolism (such as both subunits of the ribonucleotide reductase) and DNA replication-licensing factors.

Table 3.  Genes more expressed at mRNA level in the A. nidulansΔatmA mutant.
Cell cycle control, cell division, chromosome partitioning
 AN4597.3KOG0996Structural maintenance of chromosome protein 4 (Condensin, subunit C)
 AN7440.3KOG4563Cell cycle-regulated histone H1-binding protein
 AN6895.3KOG0987DNA helicase PIF1/RRM3
 AN6868.3KOG2687Spindle pole body protein, contains UNC-84 domain
 AN3941.3KOG3772M-phase inducer phosphatase
 AN6300.3KOG2035Replication factor C, subunit RFC3
 AN1016.3KOG0082G-protein alpha subunit (small G protein superfamily)
Nucleotide transport and metabolism
 AN4380.3KOG1112Ribonucleotide reductase, alpha subunit
 AN0067.3KOG1567Ribonucleotide reductase, beta subunit
 AN6856.3KOG2056Equilibrative nucleoside transporter protein
 AN4603.3KOG2584Dihydroorotase and related enzymes
 AN0271.3KOG3370dUTPase
Carbohydrate transport and metabolism
 AN2981.3KOG0563Glucose 6-phosphate 1-dehydrogenase
 AN1246.3KOG13673-Phosphoglycerate kinase
 AN5604.3KOG1458Fructose-1,6-bisphosphatase
 AN4988.3KOG1616Protein involved in Snf1 protein kinase complex assembly
 AN1015.3KOG2099Glycogen phosphorylase
 AN6286.3KOG2161Glucosidase I
 AN6824.3KOG2178Predicted sugar kinase
 AN6037.3KOG2446Glucose 6-phosphate isomerase
 AN9148.3KOG2638UDP-glucose pyrophosphorylase
 AN3954.3KOG26536-Phosphogluconate dehydrogenase
 AN8010.3KOG3742Glycogen synthase
 AN2875.3KOG4153Fructose 1,6-bisphosphate aldolase
 AN4674.3KOG4157Beta-1,6-N-acetylglucosaminyltransferase, contains WSC domain
 AN5123.3KOG2741Dimeric dihydrodiol dehydrogenase
Lipid transport and metabolism
 AN7625.3KOG0693Myo-inositol-1-phosphate synthase
 AN6014.3KOG1180Acyl-CoA synthetase
 AN0981.3KOG3072Long-chain fatty acid elongase
 AN2953.3KOG4338Predicted lipoprotein
 AN4094.3KOG1435Sterol reductase/lamin B receptor
Transcription
 AN7174.3KOG0048Transcription factor, Myb superfamily
 AN10263.3KOG0260RNA polymerase II, large subunit
 AN8858.3KOG0262RNA polymerase I, large subunit
 AN5115.3KOG0384Chromodomain-helicase DNA-binding protein
 AN2020.3KOG0773Transcription factor MEIS1 and related HOX domain proteins
 AN4034.3KOG0869CCAAT-binding factor, subunit A (HAP3)
 AN5056.3KOG1874KEKE-like motif-containing transcription regulator (Rlr1)/suppressor of sin4
 AN8858.3KOG2294Transcription factor of the Forkhead/HNF3 family
Replication, recombination and repair
 AN1708.3KOG0217Mismatch repair ATPase MSH6 (MutS family)
 AN0855.3KOG0390DNA repair protein, SNF2 family
 AN2491.3KOG0477DNA replication licensing factor, MCM2 component
 AN6070.3KOG0478DNA replication licensing factor, MCM4 component
 AN10497.3KOG0479DNA replication licensing factor, MCM3 component
 AN5992.3KOG0482DNA replication licensing factor, MCM7 component
 AN10755.3KOG0970DNA polymerase alpha, catalytic subunit
 AN10855.3KOG1625DNA polymerase alpha-primase complex, polymerase-associated subunit B
 AN6303.3KOG1968Replication factor C, subunit RFC1 (large subunit)
 AN6692.3KOG2093Translesion DNA polymerase – REV1 deoxycytidyl transferase
 AN0872.3KOG2475CDC45 (cell division cycle 45)-like protein
 AN3035.3KOG25185′−3′ exonuclease
 AN2764.3KOG25195′−3′ exonuclease
 AN5676.3KOG2227Pre-initiation complex, subunit CDC6, AAA+ superfamily ATPase
Post-translational modification, protein turnover, chaperones
 AN0170.3KOG0907Thioredoxin
 AN5895.3KOG1439RAB proteins geranylgeranyltransferase component A
 AN4450.3KOG2699Predicted ubiquitin regulatory protein
 AN5442.3KOG1282Serine carboxypeptidases (lysosomal cathepsin A)
Inorganic ion transport and metabolism
 AN5918.3KOG0047Catalase
 AN0566.3KOG3599Ca2+-modulated non-selective cation channel polycystin
 AN4249.3KOG3599Ca2+-modulated non-selective cation channel polycystin
General function prediction only
 AN6004.3KOG0118FOG: RRM domain
 AN2989.3KOG0118FOG: RRM domain
 AN9295.3KOG0254Predicted transporter (major facilitator superfamily)
 AN1677.3KOG0725Reductases with broad range of substrate specificities
 AN0229.3KOG1134Uncharacterized conserved protein
 AN8145.3KOG1721FOG: Zn-finger
 AN0096.3KOG1721FOG: Zn-finger
 AN4542.3KOG1721FOG: Zn-finger
 AN0885.3KOG1721FOG: Zn-finger
 AN0977.3KOG1721FOG: Zn-finger
 AN1247.3KOG1944Peroxisomal membrane protein MPV17 and related proteins
 AN4830.3KOG2728Similarity to phosphopantothenoylcysteine synthetase/decarboxylase
 AN7710.3KOG2914Predicted haloacid-halidohydrolase and related hydrolases
 AN1089.3KOG3780Thioredoxin-binding protein TBP-2/VDUP1
 AN4880.3KOG4525Jacalin-like lectin domain-containing protein
Function unknown
 AN1695.3KOG0290Conserved WD40 repeat-containing protein AN11
 AN6828.3KOG2654Uncharacterized conserved protein
 AN1022.3KOG4539Uncharacterized conserved protein
 AN10847.3KOG4562Uncharacterized conserved protein (tumour-rejection antigen MAGE in humans)
 AN10789.3KOG4735Extracellular protein with conserved cysteines
 AN8339.3KOG4744Uncharacterized conserved protein
Signal transduction mechanisms
 AN4483.3KOG0032Ca2+/calmodulin-dependent protein kinase, EF-Hand protein superfamily
 AN4113.3KOG0519Sensory transduction histidine kinase
 AN6207.3KOG0787Dehydrogenase kinase
 AN9449.3KOG0902Phosphatidylinositol 4-kinase
 AN2877.3KOG2210Oxysterol-binding protein
 AN7690.3KOG2243Ca2+ release channel (ryanodine receptor)
 AN4940.3KOG3543Ca2+-dependent activator protein
 AN10737.3KOG3557Epidermal growth factor receptor kinase substrate
 AN2795.3KOG3575FOG: hormone receptors
 AN8236.3KOG4363Putative growth response protein
 AN8564.3KOG4658Apoptotic ATPase
 AN6578.3KOG4476Gluconate transport-inducing protein
 AN6696.3KOG0998Synaptic vesicle protein EHS-1 and related EH domain proteins
 AN2952.3KOG1225Teneurin-1 and related extracellular matrix proteins, contain EGF-like repeats
Intracellular trafficking, secretion and vesicular transport
 AN2815.3KOG3103Rab GTPase-interacting factor, Golgi membrane protein
 AN5217.3KOG3251Golgi SNAP receptor complex member
Cytoskeleton
 AN5803.3KOG0046Ca2+-binding actin-bundling protein (fimbrin/plastin), EF-Hand protein superfamily
 AN0306.3KOG2826Actin-related protein Arp2/3 complex, subunit ARPC2
Table 4.  Genes less expressed at mRNA level in the A. nidulansΔatmA mutant.
Chromatin structure and dynamics
 AN8717.3KOG3105DNA-binding centromere protein B (CENP-B)
Lipid transport and metabolism
 AN11065.3KOG0497Oxidosqualene-lanosterol cyclase and related proteins
 AN3817.3KOG24803-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase
Transcription
 AN1984.3KOG1474Transcription initiation factor TFIID, subunit BDF1
 AN7011.3KOG1952Transcription factor NF-X1, contains NFX-type Zn2+-binding and R3H domains
 AN8803.3KOG2169Zn-finger transcription factor
 AN3810.3KOG2424Protein involved in transcription start site selection
 AN0719.3KOG3902Histone acetyltransferase PCAF/SAGA, subunit SUPT3H/SPT3
 AN7526.3KOG2773Apoptosis antagonizing transcription factor/protein transport protein
Replication, recombination and repair
 AN8794.3KOG0851Single-stranded DNA-binding replication protein A (RPA), large (70 kDa) subunit
 AN7546.3KOG1275PAB-dependent poly(A) ribonuclease, subunit PAN2
 AN10955.3KOG22493′−5′ exonuclease
Post-translational modification, protein turnover, chaperones
 AN6193.3KOG2004Mitochondrial ATP-dependent protease PIM1/LON
 AN5477.3KOG2628Farnesyl cysteine-carboxyl methyltransferase
General function prediction only
 AN0421.3KOG0110RNA-binding protein (RRM superfamily)
 AN2624.3KOG0254Predicted transporter (major facilitator superfamily)
 AN8122.3KOG0255Synaptic vesicle transporter SVOP (major facilitator superfamily)
 AN10114.3KOG0490Transcription factor, contains HOX domain
 AN2263.3KOG1424Predicted GTP-binding protein MMR1
 AN5782.3KOG1535Predicted fumarylacetoacetate hydralase
 AN7064.3KOG1535Predicted fumarylacetoacetate hydralase
 AN4292.3KOG2945Predicted RNA-binding protein
 AN11423.3KOG3552FERM domain protein FRM-8
 AN3415.3KOG3656FOG: 7 transmembrane receptor
 AN5449.3KOG3867Sulphatase
 AN4029.3KOG4246Predicted DNA-binding protein, contains SAP domain
 AN3713.3KOG4684Uncharacterized conserved protein, contains C4-type Zn-finger
Function unknown
 AN3008.3KOG1426FOG: RCC1 domain
 AN5720.3KOG2913Predicted membrane protein
 AN1670.3KOG3017Defence-related protein containing SCP domain
 AN4296.3KOG3444Uncharacterized conserved protein
 AN3983.3KOG3594FOG: Cadherin repeats
Signal transduction mechanisms
 AN9108.3KOG3514Neurexin III-alpha
 AN5011.3KOG3979FGF receptor-activating protein
 AN6249.3KOG4019Calcineurin-mediated signalling pathway inhibitor DSCR1
 AN4689.3KOG0998Synaptic vesicle protein EHS-1 and related EH domain proteins
Intracellular trafficking, secretion and vesicular transport
 AN10878.3KOG3103Rab GTPase-interacting factor, Golgi membrane protein
 AN5211.3KOG0230Phosphatidylinositol-4-phosphate 5-kinase, FAB1 homologue
Nuclear structure
 AN8823.3KOG2992Nucleolar GTPase/ATPase p130
Cytoskeleton
 AN7149.3KOG4462WASP-interacting protein VRP1/WIP, contains WH2 domain

We were also able to observe a decreased mRNA expression of several genes involved in ergosterol metabolism [such as AN11065.3 and AN3817.3 encoding oxidosqualene-lanosterol cyclase and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase] and genes involved in signal transduction mechanisms and in the synthesis of phosphatidic acid (1,2-diacyl-sn-glycerol 3-phosphate) (such as AN7787.3 encoding phosphatidylinositol-4-phosphate 5-kinase) in the ΔatmA mutant strain (Table 4). As it will be seen later, other genes from these pathways are also observed as less expressed in the ΔatmA mutant strain upon HU release (see Table 7).

Table 7.  Genes less expressed at mRNA level in the A. nidulansΔatmA mutant strain upon HU release.
Chromatin structure and dynamics
 AN5899.3KOG0933Structural maintenance of chromosome protein 2 (condensiin, subunit E)
 AN4894.3KOG1472Histone acetyltransferase SAGA/ADA, catalytic subunit PCAF/GCN5
 AN5795.3KOG1080Histone H3 (Lys4) methyltransferase complex, subunit SET1
Cell cycle control, cell division, chromosome partitioning
 AN5822.3KOG0601Cyclin-dependent kinase WEE1
 AN5100.3KOG2952Cell cycle control protein
 AN6616.3KOG3999Checkpoint 9-1-1 complex, HUS1 component
 AN1016.3KOG0082G-protein alpha subunit (small G protein superfamily)
 AN8270.3KOG2655Septin family protein (P-loop GTPase)
Nucleotide transport and metabolism
 AN0919.3KOG1377Uridine 5′ monophosphate synthase/orotate phosphoribosyltransferase
 AN5613.3KOG0430Xanthine dehydrogenase
Carbohydrate transport and metabolism
 AN7152.3KOG2366Alpha-d-galactosidase (melibiase)
 AN7750.3KOG2175Protein predicted to be involved in carbohydrate metabolism
 AN7454.3KOG4123Putative alpha 1,2 mannosyltransferase
 AN3020.3KOG2246Galactosyltransferases
 AN6985.3KOG2517Ribulose kinase and related carbohydrate kinases
 AN0830.3KOG0224Aquaporin (major intrinsic protein family)
 AN8639.3KOG1050Trehalose 6-phosphate synthase component TPS1 and related subunits
 AN10844.3KOG0234Fructose 6-phosphate 2-kinase/fructose-2,6-biphosphatase
 AN5523.3KOG1050Trehalose 6-phosphate synthase component TPS1 and related subunits
 AN4674.3KOG4157BEta-1,6-N-acetylglucosaminyltransferase, contains WSC domain
Lipid transport and metabolism
 AN5107.3KOG1362Choline transporter-like protein
 AN7211.3KOG4826C-8,7 sterol isomerase
 AN8283.3KOG0684Cytochrome P450
 AN1901.3KOG0684Cytochrome P450
 AN6433.3KOG0831Acyl-CoA
 AN10527.3KOG3889Predicted gamma-butyrobetaine,2-oxoglutarate dioxygenase
 AN5646.3KOG13893-Oxoacyl CoA thiolase
 AN10413.3KOG1329Phospholipase D1
 AN0913.3KOG3240Phosphatidylinositol synthase
 AN3796.3KOG1737Oxysterol-binding protein
 AN2720.3KOG1455Lysophospholipase
 AN10807.3KOG2208Vigilin
 AN2277.3KOG1118Lysophosphatidic acid/acyltransferase endophilin/synaptic vesicle formation
Transcription
 AN4026.3KOG1081Transcription factor NSD1 and related SET domain proteins
 AN8168.3KOG0384Chromodomain-helicase DNA-binding protein
 AN1995.3KOG4210Nuclear localization sequence-binding protein
 AN2009.3KOG0849Transcription factor PRD and related proteins, contain PAX and HOX domains
 AN1217.3KOG4577Transcription factor LIM3, contains LIM and HOX domains
 AN9303.3KOG0015Regulator of arginine metabolism and MADS box-containing transcription factors
 AN10822.3KOG2169Zn-finger transcription factor
 AN7038.3KOG1601GATA-4/5/6 transcription factors
 AN0825.3KOG0260RNA polymerase II, large subunit
 AN9087.3KOG3598Thyroid hormone receptor-associated protein complex, subunit TRAP230
 AN4938.3KOG4167Predicted DNA-binding protein, contains SANT and ELM2 domains
 AN4878.3KOG4091Transcription factor
 AN2911.3KOG1414Transcriptional activator FOSB/c-Fos and related bZIP transcription factors
 AN0817.3KOG4124Putative transcriptional repressor regulating G2/M transition
 AN4210.3KOG2043Signalling protein SWIFT and related BRCT domain proteins
Replication, recombination and repair
 AN2304.3KOG0011Nucleotide excision repair factor NEF2, RAD23 component
 AN7651.3KOG4141DNA repair and recombination protein RAD52/RAD22
 AN6740.3KOG0012DNA damage inducible protein
 AN7712.3KOG3041Nucleoside diphosphate-sugar hydrolase of the MutT (NUDIX) family
 AN3186.3KOG25205′−3′ exonuclease
 AN4555.3KOG1956DNA topoisomerase III alpha
 AN10415.3KOG0250DNA repair protein RAD18 (SMC family protein)
Cell wall/membrane/envelope biogenesis
 AN6318.3KOG2571Chitin synthase/hyaluronan synthase (glycosyltransferases)
 AN5586.3KOG1322GDP-mannose pyrophosphorylase/mannose-1-phosphate guanylyltransferase
 AN4411.3KOG2571Chitin synthase/hyaluronan synthase (glycosyltransferases)
Post-translational modification, protein turnover, chaperones
 AN4863.3KOG3496Cytochrome c oxidase assembly protein/Cu2+ chaperone COX17
 AN2302.3KOG1724SCF ubiquitin ligase, Skp1 component
 AN3022.3KOG2512Beta-tubulin folding cofactor C
 AN6686.3KOG1339Aspartyl protease
 AN1339.3KOG0940Ubiquitin protein ligase RSP5/NEDD4
 AN11102.3KOG1872Ubiquitin-specific protease
 AN9304.3KOG0867Glutathione S-transferase
 AN2072.3KOG1868Ubiquitin C-terminal hydrolase
 AN7713.3KOG4265Predicted E3 ubiquitin ligase
 AN10461.3KOG0779Protease, Ulp1 family
 AN4557.3KOG0731AAA+-type ATPase containing the peptidase M41 domain
 AN10339.3KOG0800FOG predicted E3 ubiquitin ligase
 AN6049.3KOG4628Predicted E3 ubiquitin ligase
 AN0226.3KOG0425Ubiquitin-protein ligase
 AN8269.3KOG0019Molecular chaperone (HSP90 family)
 AN3918.3KOG2195Transferrin receptor and related proteins with the protease-associated domain
Secondary metabolites biosynthesis, transport and catabolism
 AN5335.3KOG0156Cytochrome P450 CYP2 subfamily
 AN10641.3KOG0061Transporter, ABC superfamily (breast cancer resistance protein)
 AN3564.3KOG1199Short-chain alcohol dehydrogenase/3-hydroxyacyl-CoA dehydrogenase
 AN1623.3KOG0029Amine oxidase
 AN3247.3KOG0065Pleiotropic drug resistance proteins (PDR1-15), ABC superfamily
General function prediction only
 AN3387.3KOG3656FOG 7 transmembrane receptor
 AN3789.3KOG3086Predicted dioxygenase
 AN10373.3KOG3605Beta amyloid precursor-binding protein
 AN3434.3KOG0395Ras-related GTPase
 AN2522.3KOG0391SNF2 family DNA-dependent ATPase
 AN10036.3KOG0255Synaptic vesicle transporter SVOP (major facilitator superfamily)
 AN0979.3KOG4339RPEL repeat-containing protein
 AN4569.3KOG4061DMQ mono-oxygenase/Ubiquinone biosynthesis protein COQ7/CLK-1/CAT5
 AN4743.3KOG0393Ras-related small GTPase, Rho type
 AN4312.3KOG4443Putative transcription factor HALR/MLL3, involved in embryonic development
 AN4310.3KOG0504FOG Ankyrin repeat
 AN0976.3KOG0956PHD finger protein AF10
 AN1251.3KOG1721FOG Zn-finger
 AN1203.3KOG4300Predicted methyltransferase
 AN0895.3KOG1196Predicted NAD-dependent oxidoreductase
 AN0467.3KOG3175Protein phosphatase 4 regulatory subunit 2-related protein
 AN7083.3KOG1552Predicted alpha/beta hydrolase
 AN7864.3KOG2383Predicted ATPase
 AN7300.3KOG4299PHD Zn-finger protein
 AN9281.3KOG1862GYF domain-containing proteins
 AN9498.3KOG4234TPR repeat-containing protein
 AN6008.3KOG4532WD40-like repeat containing protein
 AN4292.3KOG2945Predicted RNA-binding protein
 AN4170.3KOG3780Thioredoxin-binding protein TBP-2/VDUP1
 AN4072.3KOG0504FOG Ankyrin repeat
 AN5568.3KOG1947Leucine-rich repeat proteins, some proteins contain F-box
 AN10046.3KOG3665ZYG-1-like serine/threonine protein kinases
 AN7138.3KOG2563Permease of the major facilitator superfamily
 AN4889.3KOG3943THUMP domain-containing proteins
Function unknown
 AN0693.3KOG3528FOG PDZ domain
 AN8984.3KOG2922Uncharacterized conserved protein
 AN8237.3KOG4149Uncharacterized conserved protein
 AN8850.3KOG0396Uncharacterized conserved protein
 AN8339.3KOG4744Uncharacterized conserved protein
 AN1001.3KOG2306Uncharacterized conserved protein
 AN6015.3KOG3661Uncharacterized conserved protein
 AN6058.3KOG2342Uncharacterized conserved protein
 AN1022.3KOG4539Uncharacterized conserved protein
 AN4070.3KOG3200Uncharacterized conserved protein
 AN0062.3KOG2594Uncharacterized conserved protein
 AN1330.3KOG4306Uncharacterized conserved protein
 AN7101.3KOG3854SPRT-like metalloprotease
 AN9473.3KOG2428Uncharacterized conserved protein
 AN5718.3KOG4674Uncharacterized conserved coiled-coil protein
 AN2567.3KOG4267Predicted membrane protein
 AN2751.3KOG4592Uncharacterized conserved protein
 AN3053.3KOG3594FOG Cadherin repeats diacylglycerol acyltransferase (DGAT)
 AN0424.3KOG4674Uncharacterized conserved coiled-coil protein
Signal transduction mechanisms
 AN4745.3KOG1450Predicted Rho GTPase-activating protein
 AN6982.3KOG0789Protein tyrosine phosphatase
 AN3834.3KOG3751Growth factor receptor-bound proteins (GRB7, GRB10, GRB14)
 AN6046.3KOG4225Sorbin and SH3 domain-containing protein
 AN4515.3KOG4641FOG Toll/interleukin receptor and related proteins with LRR and TIR repeats
 AN2766.3KOG0229Phosphatidylinositol-4-phosphate 5-kinase, MSS4 homologue
 AN6305.3KOG0616cAMP-dependent protein kinase catalytic subunit (PKA)
 AN6315.3KOG0583Serine/threonine protein kinase
 AN4935.3KOG0583Serine/threonine protein kinase
 AN6618.3KOG2197Ypt/Rab-specific GTPase-activating protein GYP7 and related proteins
 AN10031.3KOG3653Growth factor beta/activin receptor subfamily of serine/threonine kinases
 AN3746.3KOG4269Rac GTPase-activating protein BCR/ABR
 AN0988.3KOG0671LAMMER dual specificity kinases
 AN1099.3KOG0197Tyrosine kinases
 AN4940.3KOG3543Ca2+-dependent activator protein
 AN5213.3KOG2052Activin A type IB receptor, serine/threonine protein kinase
 AN10150.3KOG1786Lysosomal trafficking regulator LYST and related BEACH and WD40 repeats
 AN4169.3KOG2122Beta-catenin-binding protein APC, contains ARM repeats
 AN5973.3KOG0598Serine/threonine protein kinase
Intracellular trafficking, secretion and vesicular transport
 AN3751.3KOG1013Synaptic vesicle protein rabphilin-3 A
 AN5920.3KOG2319Vacuolar assembly/sorting protein VPS9
 AN1179.3KOG4635Vacuolar import and degradation protein
 AN10849.3KOG2063Vacuolar assembly/sorting proteins VPS39/VAM6/VPS3
 AN2979.3KOG0860Synaptobrevin/VAMP-like protein
 AN5219.3KOG3724Negative regulator of COPII vesicle formation
 AN2692.3KOG4809Rab6 GTPase-interacting protein involved in endosome-to-TGN transport
 AN5579.3KOG1809Vacuolar protein sorting-associated protein
 AN6120.3KOG0929Guanine nucleotide exchange factor
Nuclear structure
 AN0085.3KOG2992Nucleolar GTPase/ATPase p130
 AN10996.3KOG2171Karyopherin (importin) beta 3
Cytoskeleton
 AN3437.3KOG0516Dystonin, GAS (growth arrest-specific protein), and related proteins
 AN3547.3KOG1840Kinesin light chain
 AN1558.3KOG0162Myosin class I heavy chain
 AN8244.3KOG0516Dystonin, GAS (growth arrest-specific protein), and related proteins
 AN4513.3KOG0242Kinesin-like protein
 AN2480.3KOG0161Myosin class II heavy chain
 AN7256.3KOG1702Nebulin repeat protein
 AN1156.3KOG0161Myosin class II heavy chain
 AN7484.3KOG0613Projectin/twitchin and related proteins
 AN4706.3KOG0161Myosin class II heavy chain
 AN5942.3KOG4568Cytoskeleton-associated protein and related proteins

Transaldolase is important for increased nuclear kinetics and decreased polar growth in the ΔatmA mutant strain

The ppp generates NADPH and ribose 5-phosphate in the cytosol. NADPH is used in reductive biosyntheses, whereas ribose 5-phosphate is used in the synthesis of RNA, DNA and nucleotide coenzymes (Nelson and Cox, 2005). Mutants of the ppp have been isolated in A. nidulans (Hankinson, 1974). These fail to grow on a variety of carbohydrates that are catabolized through the ppp, such as xylose.

As there is an increased mRNA expression in the ΔatmA mutant of several genes from the ppp pathway, we decided to investigate the NADPH/NADP levels in the wild-type and mutant strains. There is a decrease (about two to three times at 60 and 90 min) in the NADPH/NADP+ ratio in the mutant when compared with the wild strain (Table 5). There is a proportional increase in the NADP+ and in total NADPT (NADPH+NADP+) (Table 5). NADPH is essential in many biosynthetic pathways and also protects cells from oxidative damage by hydrogen peroxide and superoxide-free radicals (Nelson and Cox, 2005). Thus, these results suggest the ΔatmA germlings are possibly under oxidative stress conditions, producing increasing amounts of NADP+ that are converted to NADPH and immediately consumed by cell, aiming to detoxify reactive oxygen species (ROS).

Table 5.  NADPH/NADP+ levels in A. nidulans wild-type and ΔatmA mutant strains.
StrainaNADPH/NADP+NADPH (nmol per mg of protein)NADP+ (nmol per mg of protein)NADPT
  • a.

    The results are expressed as the means ± standard deviation of three experiments.

  • ND, not detected.

Wild type (60 min)0.12 ± 0.016.62 ± 0.6250.59 ± 4.7757.23 ± 5.38
Wild type (90 min)0.20 ± 0.044.97 ± 0.8120.94 ± 2.9625.53 ± 4.15
Wild type (120 min)0.10 ± 0.012.74 ± 0.1723.69 ± 1.5126.42 ± 1.68
Wild type (150 min)0.04 ± 0.001.80 ± 0.1838.18 ± 3.4639.70 ± 3.93
ΔatmA (60 min)0.04 ± 0.004.96 ± 0.47101.27 ± 9.52106.23 ± 10.0
ΔatmA (90 min)0.07 ± 0.013.23 ± 0.5336.96 ± 6.0240.20 ± 6.54
ΔatmA (120 min)0.08 ± 0.013.08 ± 0.2036.99 ± 2.1735.89 ± 3.56
ΔatmA (150 min)ND53.79 ± 5.33

In A. nidulans, the ppp mutations have been assigned to two unlinked genes. Mutants with lesions in the pppB locus have reduced activities of four enzymes of the ppp, of glucose-phosphate isomerase and of mannitol-1-phosphate dehydrogenase. The pppA- mutants have elevated activities of these same enzymes except for transaldolase, for which they have much reduced activity. It was shown by the method of haploidization that the pppA is located on linkage group VIII. The position of pppA1 on linkage group VIII was determined by carrying out appropriate crosses. The pppA1 mutation is located about 32.8 kb downstream from uZ4 (gene ureD, AN0232.3) and about 47.4 kb upstream from palB7 (gene palB, AN0256.3). There are 28 open reading frames (ORFs; AN0231.3 to AN0235.3; AN0237.3 to AN0256.3; AN10048.3, AN10029.3, AN10040.3 and 10030.3) in this interval. Interestingly, one of these ORFs (AN0240.3) encodes a transladolase. This ORF, as well as about 1 kb up- and downstream non-coding sequences, was PCR-amplified by high-fidelity Taq-polymerase and transformed into the pppA- strain, selecting for transformants that could grow in the presence of xylose as single carbon source. Several transformants were obtained and two of them that had the pppA DNA fragment integrated at the pppA locus as verified by Southern blot (Fig. S1) were chosen for further analysis (see Fig. 1A). We crossed them to a wild-type strain and found no progeny unable to grow on xylose. We therefore conclude that we have complemented the inability to grow on xylose phenotype with the wild-type copy of the pppA gene.

image

Figure 1. The pppA mutant strain is unable to grow on xylose as single carbon source and it can be complemented by the transladolase gene. A. The GR5 (wild type), G840 (pppA1) and IM-TR20 (pppA1 complemented with the transaldolase gene) strains were grown in YAG (first row), Cove's MM+xylose 2% w/v (second row) and Cove's MM+glucose 1% w/v (third row) for 48 h at 37°C. B. Conidia were inoculated onto coverslips and DAPI-stained after 2–6 h incubation at 37°C. The material was photographed using a Zeiss epifluorescence microscope. C. Aspergillus nidulans conidia were grown in the presence of 50 mM HU for 5 h, and the cells were washed to release the HU and grown again for different periods of time. The HU blockage synchronized the polar growth, and the kinetics of growth after HU release represents the process of emergence from the swollen conidia. The results were expressed by the average of three independent experiments (100 germlings each) and means ± standard deviation are shown.

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The genomic sequence of the pppA gene from the wild-type and mutant pppA- strains was determined. A single base (C to T) transition was identified in the pppA- mutant 131 bases downstream of the predicted translational start site. This transition changes the predicted leucine residue (aa 43) into a phenylalanine residue. As this mutation is close to the transaldolase signature 1 (between residues 32 and 40), it could potentially impair substrate binding, causing a decrease in the enzymatic activity. This could help to explain the observed phenotype of reduction of the transaldolase activity in this mutant by Hankinson (1974). Taken together, these data indicate that the pppA- mutant can be complemented by the pppA gene that encodes a transaldolase.

To validate the involvement of the ppp in the phenotype of the ΔatmA mutant, we constructed a double mutant strain ΔatmA pppA- and investigated its nuclear kinetics and polar growth (Fig. 1B–C). The double mutant has a significantly reduced nuclear kinetics when compared with the ΔatmA mutant strain (about 40%; Fig. 1B). Interestingly, the pppA- mutant strain also showed a defect in polar growth comparable to the ΔatmA and the double mutant strains (Fig. 1C). These results suggest that there is epistasis between atmA and pppA during polar growth. We have not observed any increase of sensitivity to DNA-damaging agents in the pppA1 and pppA1ΔatmA mutant strains, and most probably due to the defective transaldolase, we were not able to detect NADPH in these mutants (data not shown). Furthermore, they indicate an important role of the ppp in the fungal proliferation during endogenous DNA damage and polar growth monitored by the AtmA kinase.

Juhnke et al. (1996) showed that mutants in genes from Saccharomyces cerevisiae that encode enzymes of the ppp (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide. Furthermore, the ATM gene maintains genome stability by activating a key cell cycle checkpoint not only in response to DNA damage and telomeric instability, but also oxidative stress (for a review, see Barzilai et al., 2002). Thus, we evaluated if the pppA-, ΔatmA and ΔatmA pppA- mutants are sensitive to hydrogen peroxide by verifying their survival when incubated in the presence of this oxidative stressing agent (Fig. 2A). The wild-type strain survival was not affected by incubation in the presence of hydrogen peroxide (Fig. 2A). The ΔatmA, pppA- and ΔatmA pppA- mutants are more sensitive to hydrogen peroxide than the wild type (about 50%, 10% and 9% survival respectively). Furthermore, the transaldolase deficiency has a more dramatic effect on the sensitivity to hydrogen peroxide. However, the AtmA and PppA do not seem to be interacting when germlings are incubated in the presence of hydrogen peroxide.

image

Figure 2. The ΔatmA mutant has decreased survival in the presence of hydrogen peroxide. A. Five-hour-old germlings were incubated in 50 mM hydrogen peroxide for 20 min at 37°C (Noventa-Jordão et al., 1999). In all cases appropriate dilutions were made and 100 μl aliquots spreaded on YAG plates. Viability was determined as the percentage of colonies on treated plates compared with untreated controls. The results were expressed by the average of four independent experiments and means ± standard deviation are shown. Statistical differences were determined by one-way analysis of variance (anova) followed, when significant, by Newman–Keuls multiple comparison test, using GraphPad Prism statistical software (GraphPad Software, version 3, 2003). The ΔatmA, pppA and ΔatmA pppA strains were significantly different from the wild type (< 0.01). B. The wild type, ΔatmA, pppA1 and ΔatmA pppA1 were grown for 12 h at 44°C in MM-G, and germlings were stained with H2DCFDA for 30 min at 30°C, and incubated for further 30 min at the same temperature, before confocal microscopic analysis. Bars, 10 μm.

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To investigate the production of ROS in the ΔatmA mutant, we used 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), a cell-permeable ROS indicator that is non-fluorescent until acetate groups are removed (H2DCF) by intracellular esterases and oxidation occurs within the cell. Although H2DCF can be oxidized by different ROS, it is mainly used to detect superoxides. Germlings of the wild-type and the ΔatmA mutant strains were incubated with H2DCFDA and examined by confocal microscopy (Fig. 2B). The DCFDA-dependent green fluorescence was barely detected in the wild-type strain while the ΔatmA germlings displayed intense fluorescence vastly distributed along the hyphae. These results indicate the AtmA gene could be involved in monitoring and/or repairing the oxidative stress caused by ROS.

Transcriptome analysis for A. nidulans polar growth upon HU release

Recently, we demonstrated that AtmAATM plays a role in the formation of stable axes of polarized hyphal growth (Malavazi et al., 2006). Thus, to investigate which genes have their expression influenced by AtmA during the polar growth process, we compared the mRNA expression of the ΔatmA mutant with the wild-type strain after HU release. We were able to observe 2190 genes modulated in at least one time point in the mutant when compared with the wild-type strain (Fig. 3; Tables 6 and 7 show the genes with log ratio ≥ 1 or ≤1, respectively). Again, these genes were classified into COG functional categories (http://www.ncbi.nlm.nih.gov/COG/). As it can be observed in Table 6, there is an increased expression of genes encoding proteins involved in post-translational modification and protein turnover (such as chaperones) and translation, ribosomal structure and biogenesis (such as several ribosomal proteins and translation factors) in the ΔatmA mutant. Interestingly, there is also an increased expression of genes involved in rRNA gene transcription (AN3944.3 and AN10316.3 that encode a predicted regulator of rRNA gene transcription and an RNA polymerase III, large subunit); this suggests a need by the ΔatmA mutant of correctly assemblying proteins and synthesizing rRNA molecules for accelerated growth.

image

Figure 3. Hierarchical clustering comparing the pattern of expression of A. nidulansΔatmA mutant and wild-type strain after the HU release. The colour code displays the Log2 (Cy5/Cy3) ratio for each time point, having Cy3 as the reference value (time point = 0). Clusters I, III and VI, and clusters II, IV, V and VIII show genes more and less expressed in the ΔatmA mutant strain, respectively, when compared with wild-type strain. Tables 6 and 7 show genes that belong to these clusters.

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Table 6.  Genes more expressed at mRNA level in the A. nidulansΔatmA mutant strain upon HU release.
Cell cycle control, cell division, chromosome partitioning
 AN1221.3KOG0964Structural maintenance of chromosome protein 3 (Cohesin, SMC3)
Nucleotide transport and metabolism
 AN2185.3KOG3721Mitochondrial endonuclease
 AN2494.3KOG1097Adenine deaminase/adenosine deaminase
 AN6541.3KOG0237Glycinamide ribonucleotide synthetase
 AN6711.3KOG1448Ribose-phosphate pyrophosphokinase
Lipid transport and metabolism
 AN7008.3KOG23043-Hydroxyacyl-CoA dehydrogenase
 AN2573.3KOG0873C-4 sterol methyl oxidase
 AN1036.3KOG1202Animal-type fatty acid synthase and related proteins
 AN10538.3KOG2926Malonyl-CoA ACP transacylase
Translation, ribosomal structure and biogenesis
 AN10195.3KOG0432Valyl-tRNA synthetase
 AN6563.3KOG1627Translation elongation factor EF-1 gamma
 AN3172.3KOG083040S ribosomal protein SA (P40)/Laminin receptor 1
 AN9151.3KOG2102Exosomal 3′−5′ exoribonuclease complex, subunit Rrp44/Dis3
 AN4802.3KOG173260S ribosomal protein L21
 AN2743.3KOG2072Translation initiation factor 3, subunit a (eIF-3a)
 AN1013.3KOG087560S ribosomal protein L5
 AN2997.3KOG0643Translation initiation factor 3, subunit i (eIF-3i)
Transcription
 AN3944.3KOG1926Predicted regulator of rRNA gene transcription (MYB-binding protein)
 AN10316.3KOG0261RNA polymerase III, large subunit
Post-translational modification, protein turnover, chaperones
 AN0381.3KOG0363Chaperonin complex component, TCP-1 beta subunit (CCT2)
 AN4025.3KOG2157Predicted tubulin-tyrosine ligase
 AN0401.3KOG0868Glutathione S-transferase
 AN5111.3KOG4400E3 ubiquitin ligase interacting with arginine methyltransferase
 AN3481.3KOG0909Peptide N-glycanase
 AN2918.3KOG0358Chaperonin complex component, TCP-1 delta subunit (CCT4)
 AN1851.3KOG0362Chaperonin complex component, TCP-1 theta subunit (CCT8)
 AN10526.3KOG1499Protein arginine N-methyltransferase PRMT1 and related enzymes
General function prediction only
 AN2801.3KOG1577Aldo/keto reductase family proteins
 AN3836.3KOG0254Predicted transporter (major facilitator superfamily)
 AN11463.3KOG4299PHD Zn-finger protein
 AN8394.3KOG0254Predicted transporter (major facilitator superfamily)
 AN7458.3KOG2055WD40 repeat protein
 AN10848.3KOG0118FOG RRM domain
 AN4816.3KOG2325Predicted transporter/transmembrane protein
 AN5283.3KOG0118FOG RRM domain
 AN5865.3KOG1490GTP-binding protein CRFG/NOG1 (ODN superfamily)
 AN3833.3KOG1552Predicted alpha/beta hydrolase
 AN3771.3KOG3272Predicted coiled-coil protein
Function unknown
 AN6584.3KOG4828Uncharacterized conserved protein
 AN2370.3KOG2521Uncharacterized conserved protein
 AN10231.3KOG0606Microtubule-associated serine/threonine kinase and related proteins
Intracellular trafficking, secretion and vesicular transport
 AN9250.3KOG2060Rab3 effector RIM1 and related proteins, contain PDZ and C2 domains
 AN3871.3KOG2319Vacuolar assembly/sorting protein VPS9
 AN6412.3KOG3764Vesicular amine transporter
 AN0958.3KOG1983Tomosyn and related SNARE-interacting proteins
Nuclear structure
 AN6006.3KOG1991Nuclear transport receptor RANBP7/RANBP8 (importin beta superfamily)
Cytoskeleton
 AN4111.3KOG3565Cdc42-interacting protein CIP4

We observed a decreased mRNA expression of several genes involved in cell cycle control and chromosome partitioning (such as AN5822.3 and AN6616.3 encoding the cyclin-dependent kinase WEE1 and the checkpoint 9-1-1 complex, HUS1 component respectively). Genes involved in ergosterol biosynthesis, such as AN7211.3 (C-8,7 sterol isomerase), and AN8283.3 (CYP51A, Cytochrome P450) and AN1901.3 (CYP51B, Cytochrome P450), have also decreased mRNA expression. Several genes that play a role in the inositol and signalling pathways, such as AN10413.3, AN0913.3, AN2720.3, AN2277.3 and AN2766.3 encoding phospholipase D1, phosphatidylinositol synthase, lysophospholipase, lysophosphatidic acid/acyltransferase endophilin/synaptic vesicle formation and phosphatidylinositol-4-phosphate 5-kinase, respectively (Table 7), are also less expressed in the atmA inactivation mutant. A great number of genes that act on intracellular trafficking, secretion and vesicular transport were also less expressed in the ΔatmA mutant strain (Table 7).

Furthermore, genes involved in polar growth, such as the homologues of Rho GTPase Rac1 (AN4743.3) and the Rho GTPase-activating protein Bem2p (AN4745.3), the homologues of the β-tubulin folding cofactor C (AN3022.3) and a serine/threonine protein kinase (AN5973.3), shown by Seiler and Plamann (2003) to be involved in cellular morphogenesis, and genes encoding motor proteins as kinesin and myosin (AN3547.3, AN1558.3, AN4513.3, AN2480.3, AN1156.3 and AN4706.3), previously shown as supporting many cellular processes including polar growth (McGoldrick et al., 1995; Steinberg, 2000; Schuchardt et al., 2005), were also seen as less expressed in the ΔatmA mutant strain (Table 7).

To assess the reliability of the microarray hybridizations and validate the expression of some of these genes, we chose seven genes from clusters II, IV, V and VIII (Fig. 3): AN0913.3 (phosphatidylglycerophosphate synthase), AN3443.3 (Ras family GTPase), AN5100.3 (CDC50, endosomal protein that regulates cell polarity), AN1016.3 (G protein alpha subunit), AN10413.3 (phospholipase D), AN2766.3 (phosphatidylinositol-4-phosphate 5-kinase) and AN4743.3 (RacA Rho GTPase CDC42), designed Lux fluorescent probes (Invitrogen) and used real-time reverse transcription polymerase chain reaction (RT-PCR) analysis to quantify their expression in the wild-type and ΔAtmA mutant strain 120 min after HU release (Fig. 4). The results were expressed as the number of times the genes have decreased expression in the ΔatmA mutant when compared with the wild-type strain. Our results strongly indicate that the genes described here might have their mRNA expression decreased when the ΔAtmA mutant strain is released from the HU treatment and is in the process of polar growth (Fig. 4). Thus, it seems that our microarray hybridization approach is capable of providing information about A. nidulans gene expression modulation with a considerably high level of confidence

image

Figure 4. Fold decrease in RNA levels after the HU release in the ΔatmA mutant strain. Real-time RT-PCR was the method used to quantify the mRNA 120 min after the HU release. The measured quantity of the AN0913.3 (phosphatidylglycerophosphate synthase), AN3443.3 (Ras family GTPase), AN5100.3 (CDC50, endosomal protein that regulates cell polarity), AN1016.3 (G protein alpha subunit), AN10413.3 (phospholipase D), AN2766.3 (phosphatidylinositol-4-phosphate 5-kinase) and AN4743.3 (RacA Rho GTPase CDC42) mRNA in each of the treated samples was normalized using the Ct values obtained for the tubC RNA amplifications run in the same plate. The relative quantification of all the genes and tubulin gene expression was determined by a standard curve (i.e. Ct values plotted against logarithm of the DNA copy number). The results are the means ± standard deviation of four sets of experiments. The values represent the number of times the genes have decreased expression in the ΔatmA mutant when compared with the wild-type strain.

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We decided to investigate in more detail some of the pathways mentioned above with decreased mRNA expression in the ΔatmA mutant. The main hypothesis is that the mRNA of several of the genes that encode proteins involved in these pathways is less expressed in the ΔatmA mutant strain than in the wild-type because somehow the AtmA can affect directly or indirectly their mRNA expression. As a initial strategy to investigate this hypothesis, we applied two approaches: (i) the use of fluorescent subcellular and pharmacological probes aiming to investigate several aspects of the polar growth metabolism of the ΔatmA mutant strain, and (ii) the construction of double mutants with ΔatmA mutation and other mutations and GFP protein fusions also involved in several pathways above mentioned. These genes could be summarized in three main pathways: (i) genes involved in the formation of a polarized hyphae (e.g. cdc42) and control of polar growth, (ii) genes involved in the synthesis of phosphatidic acid (e.g. phospholipase D); and (iii) genes involved in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts), and intracellular trafficking, secretion, and vesicular transport.

Genes involved in the formation of a polarized hyphae and control of polar growth.  To investigate genes involved in polar growth, we performed several crosses with mutants and gfp fusions of genes that play a role in this process, such as the β-tubulin TubA (Konzack et al., 2005), the tropomyosin TpmA (Pearson et al., 2004), the kinesin motor KipA (Konzack et al., 2005), the proposed scaffold protein SpaA (Sharpless and Harris, 2002), modACDC42· (A. Virag and S.D. Harris, unpubl. results) and proteins important for the formation of the polarisome, such as SepA and BudA (Virag and Harris, 2006) (see Table 8 and Table S1).

Table 8. Aspergillus nidulans strains used in this work
StrainsGenotypesReferences
  • a.

    Fungal Genetics Stock Center (http://www.fgsc.net).

  • b.

    FGSC A690.

  • c.

    G840-complemented strains.

A26biA1FGSCa
A4Glasgow wild type (veA+)FGSC
AAV1pyroA4 pyrG89; spaA::gfp::pyr4Virag and Harris (2006)
AAV11pyroA4 pyrG89; wA3;ΔspaA::pyr4Virag and Harris (2006)
AAV2pyroA4 pyrG89; wA3; alcA::gfp::budA::pyr4Virag and Harris (2006)
ACP115pyrG89; wA3; tpmA::gfp::pyr4Pearson et al. (2004)
AKS70pyrG89 pabaA1 yA2; sepA:gfp:pyr4Sharpless and Harris (2002)
AML37pyrG89 argB2; alcA::modAG14V::argBS.D. Harris (unpublished)
G840byA2; pyroA4; lacA1; pppA1Hankinson (1974)
GR5pyroA4 pyrG89, wA3FGSC A773
IM69pyroA4 pyrG89, wA3;ΔatmA::pyrGMalavazi et al. (2006)
IM69-37wA3; alcA::modAG14V::argB;ΔatmAThis work
IM69-78wA3; calC2;ΔatmA::pyrGThis work
IM69-840chaA1; pppA1;ΔatmAThis work
IM69-ACPwA3; tpmA::gfp::pyr4;ΔatmAThis work
IM69-AV1chaA1; spaA::gfp;ΔatmAThis work
IM69-AV11AwA3;ΔspaA::pyr4;ΔatmAThis work
IM69-AV2wA3; alcA::gfp::budA::pyr4;ΔatmAThis work
IM69-K13wA3; ΔkipA;ΔatmAThis work
IM69-K100wA3; alcA::gfp::kipA::pyr4;ΔatmAThis work
IM69-KS70yA2; sepA::gfp::pyr4;ΔatmAThis work
IM69-W100alcA::gfp::tubA,ΔatmAThis work
IM-TR20cyA2; pyroA4; lacA1; pppA1; pppA+This work
IM-TR21cyA2; pyroA4; lacA1; pppA1; pppA+This work
R78pyroA4 pyrG89, wA3; calC2Teepe et al. (2007)
SJW100wA3; pyroA4;ΔargB::trpCΔB; alcA::gfp::tubA; veA; alcA::stuA(NLS)::DsRedKonzack et al. (2005)
SSK13pabaA1; wA3;ΔkipA::pyr4; veA1Konzack et al. (2005)
SSK99pyroA4 pyrG8; wA3; alcA::grp::kipA::pyr4; gpd::stuA(NLS)::DsRedKonzack et al. (2005)

Wild-type hyphae featured longitudinal arrays of cytoplasmic microtubules that terminated at a discrete site within the hyphal tip (Malavazi et al., 2006). In contrast, in the ΔatmA alcA::gfp::tubA mutant, microtubule arrays typically did not terminate at a discrete site and instead appeared to be splayed out in a random manner (Fig. S2). Quantification of these phenotypes revealed that about 50% of ΔatmA hyphae possessed cytoplasmic microtubules that failed to converge at a discrete site, compared with none of the wild-type hyphae (n = 100; two independent experiments). These observations are consistent with what was previously observed by Malavazi et al. (2006) using immunofluorescence microscopy with anti-β-tubulin antibodies. We were unable to see any difference in terms of protein localization between tpmA::gfp and the double mutant ΔatmA tpmA::gfp (Table S1).

Konzack et al. (2005) identified a kinesin motor protein, KipA, as required for growing microtubules in A. nidulans. In a ΔkipA mutant, microtubule plus ends reach the tip but shows continuous lateral movement. Hyphae lose directionality and grow in curves, apparently due to mislocalization of Spitzenkörper in the apex. The GFP::KipA protein accumulates at microtubule plus ends, suggesting that KipA requires an intrinsic motor activity to reach the microtubule plus ends. The double mutant ΔatmAΔkipA behaves like ΔatmA mutant in terms of nuclear kinetics and polar growth. However, a double ΔatmA gfp::kipA showed different results: approximately 70% of the germlings behaved as the wild type, i.e. KipA localizes to the hyphal tip; in about 30% of the germlings, either KipA is not located in the hyphal tip or its content is significantly decreased (Fig. 5). These 30% germlings also displayed abnormal hyphal tip and/or germling development (Fig. 5).

image

Figure 5. KipA is either not located in the hyphal tip or its content is significantly decreased in the atmA inactivation mutant. (A) Conidiospores from the wild-type and (B) ΔatmA inactivation mutant strains were grown in glass-bottom dishes (Mattek Corporation, USA) in 2 ml of MM-G and/or ethanol + supplements for 18 h at 30°C (n = 100; two independent experiments). The arrows show the wild-type and atmA inactivation mutant hyphal tip. Images were analysed using the Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) (Laboratory of Confocal Microscopy, FMRP-USP, Brazil) using 63× magnification water immersion objective lens using laser line 488 nm for gfp. Images were captured by direct acquisition with Z step ranging from 0.5 to 2 μm with the Leica LAS AF software (Leica Microsystems). DIC and fluorescence images are maximum projection of Z stacks. Bars, 5 μm.

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The double mutant ΔatmA sepA::gfp and ΔatmA alcA::gfp::budA behaved like the single sepA::gfp and alcA::gfp::budA, i.e. they showed protein fluorescence localized with the same intensity to the hyphal tip (data not shown).

We also constructed double mutants with Δatm and ΔspaA and the conditional mutant alcA::modAG14V and evaluated their polarization after HU release. The SpaA functions exclusively at the hyphal tip and it is believed to be interacting with numerous proteins, including the other polarisome components (Virag and Harris, 2006). Upon HU release, the wild-type strain showed 75.5 ± 2.1% germlings with polar growth while the ΔatmA, ΔspaA and ΔatmΔspaA mutant strains displayed 29.5 ± 0.7%, 73.5 ± 9.2% and 17.0 ± 2.8% respectively (Table S1). The modA is the A. nidulans homologue of cdc42 and the G14V is a dominant active mutation. This construct is under the control of the alcA promoter that is induced to high levels by glycerol, ethanol and l-threonine, and repressed by glucose (Flipphi et al., 2002). When induced to high levels this mutated protein causes polarity maintenance defects that partially overlap with those of the atmA deletion mutant, i.e. failure to maintain a stable axis of hyphal polarity (S.D. Harris, pers. comm.). After derepression with ethanol, the alcA::modAG14V presented 25.0 ± 1.5% while the ΔatmA and ΔatmA alcA::modAG14V mutant strains showed 40.0 ± 0.0% and 10.5 ± 0.7% polarization as measured by germ tube emergence from germinating conidia respectively (Table S1).

Taken together, these data emphasize the importance of AtmA in the organization of cytoplasmic microtubules at the hyphal tip, and its impact on the organization of the polarisome.

Genes involved in the synthesis of phosphatidic acid.  As it can be seen in Fig. 6A, we have observed phospholipase D (AN10413.3), phosphatidylinositol synthase (AN0913.3) and phosphatidylinositol-4-phosphate 5-kinase (AN2766.3) as lower expressed genes in the ΔatmA mutant strain. These genes are important for the synthesis of phosphatidic acid and the activation of protein kinase C (for a review, see Wang et al., 2006). To investigate the involvement of this pathway in polar growth, we performed experiments of polarization by indirectly titrating phosphatidic acid out by using 1-butanol. We also constructed a double mutant with the mutant calC2 that is complemented by the protein kinase C (Teepe et al., 2007) (see Table 8).

image

Figure 6. Production of phosphatidic acid is involved in the polarization of the ΔatmA mutant. (A) Simplified scheme of the synthesis of phosphatidic acid and diacylglycerol. A. nidulans conidia were incubated in the presence of 50 mM HU for 5 h, and the cells were washed to release the HU and grown again in different concentrations of 1-butanol (B) or myriocin (D) for 150 min. After this period, polar growth was assessed. The results are related to the evaluation of 200 germlings in each strain. (C) Conidia from A. nidulans wild type, ΔatmA, calC2 and ΔatmA calC2 mutants were incubated in the presence of 50 mM HU for 5 h, and the cells were washed to release the HU and grown for additional 150 min in fresh medium. After this period, polar growth was assessed. The results are related to the evaluation of 200 germlings in each strain.

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Phospholipase D has a unique catalytic activity, termed transphosphatidylation that can be used to more directly test for phospholipase D involvement in a system (Ritchie et al., 2002). If provided with a primary (but not secondary or tertiary) alcohol, phospholipase D will preferentially transphosphatidylate the alcohol to produce a phosphatidyl-alcohol, and as a consequence, less of its normal product phosphatidic acid (Fig. 6B). Thus the application of, for example, 1-butanol to cells can be used to inhibit signalling pathways or cellular events which are mediated by phospholipase D. We took advantage of this property to investigate if the ΔatmA mutant polarization upon HU release would be affected by a reduction in the concentration of phosphatidic acid (Fig. 6B). As it can be seen, the ΔatmA mutant has significantly reduced polarization when compared with the wild-type strain after 1-butanol addition (< 0.05 for every concentration; Fig. 6B).

The reduced mRNA expression of phosphatidylinositol synthase (AN0913.3) and phosphatidylinositol-4-phosphate-5-kinase (AN2766.3) suggested that diacylglycerol (DAG) production via phospholipase C is impaired, possibly causing a reduction in the activation of protein kinase C (Fig. 6A). Accordingly, we constructed a double mutant ΔatmA calC2 and evaluated its polarization upon HU release (Fig. 6C). Interestingly, the double mutant ΔatmA calC2 has very much reduced polarization when compared with the parental strains, suggesting a possible interaction between AtmA and CalC (< 0.05).

Recently, Kobayashi et al. (2005) showed that disturbance of sphingolipid biosynthesis abrogates the signalling of Mss4, the yeast homologue of phosphatidylinositol-4-phosphate 5-kinase. Thus, we also analysed the effects of myriocin, a specific inhibitor of serine palmitoyltransferase (SPT) that catalyses the first committed step in sphingolipid biosynthesis (Miyake et al., 1995) in the polarization of the ΔatmA mutant strain. There was a significant reduction of polar growth in the ΔatmA mutant when A. nidulans conidia were exposed to different concentrations of myriocin for 150 min (< 0.05 for every concentration; Fig. 6D).

Taken together, these data thus suggest that polarity defects in the atmA inactivation mutant were probably caused by reduced levels of phosphatidic acid, indicating an important role of this pathway in hyphal polarity in the ΔatmA mutant.

Genes involved in the ergosterol biosynthesis and intracellular trafficking, secretion and vesicular transport.  To investigate genes involved in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts) and intracellular trafficking, secretion and vesicular transport, we performed assays with the ergosterol-depletion agent lovastatin that inhibits the key enzyme in the ergosterol biosynthesis, HMG-CoA reductase (Charlton-Menys and Durrington, 2007), the fluorescent probes filipin, to detect the major membrane sterol, ergosterol (Martin and Konopka, 2004), and FM4-64 to follow membrane internalization and transport to the vacuolar system and endomembranes in A. nidulans (Peñalva, 2005). To determine whether membrane lipids are polarized during polar growth in A. nidulans wild-type and ΔatmA mutant strains, germlings were stained with filipin (Fig. 7A). Filipin is a polyene antibiotic that is similar in structure to amphotericin B in that it binds sterols. It has been used to visualize membrane sterols in a wide range of cell types because it has fluorescent properties that can be observed by microscopy (Ghannoum and Rice, 1999). During polar growth in the wild type, germlings showed intense filipin staining at the hyphal tip (Fig. 7A, left) while the ΔatmA mutant germlings display uniform filipin staining throughout the membrane (Fig. 7A, right). Polar growth in the ΔatmA mutant strain was also considerably more affected by lovastatin than in the wild-type mutant strain (< 0.05 for every concentration; Fig. 7B).

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Figure 7. Polarization of membrane ergosterol and influence of lovastatin during hyphal growth of A. nidulans. A. The images show filipin staining of wild-type and ΔatmA mutant strains grown at 44°C in MM-G for 12 h. The cells were stained with filipin (25 μg ml−1) for 5 min and then analysed by fluorescence microscopy. The bottom panels show sterol-rich membrane domains that fail to form a discrete patch at the tips of the ΔatmA mutant. Randomly distributed sterol-rich domains can be seen on the surface of the hyphal tips with bright spots that appear occasionally at both apical and subapical sites (bottom, white arrows). B. A. nidulans conidia were incubated in the presence of 50 mM HU for 5 h, and the cells were washed to release the HU-blockage and grown again in different concentrations of lovastatin for 150 min. After this period, polar growth was assessed. The graph shows means ± standard deviation of three experiments with 100 germlings each.

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FM4-64 staining of wild-type strain revealed that the Spitzenkörper is present at the hyphal tip and also structures that probably represent mature endosomes/vacuoles (see Peñalva, 2005 for a description; Fig. 8A). In contrast, the Spitzenkörper cannot be visualized in the ΔatmA mutant and there is a significant decrease of the endosome/vacuole structures (Fig. 8B).

image

Figure 8. The ΔatmA mutation can affect endocytosis and vacuolar distribution in A. nidulans. Conidiospores were grown in glass-bottom dishes (Mattek Corporation, USA) in 2 ml of MM-G + supplements for 12 h at 44°C and exposed to pre-warmed media containing 10 μM FM4-64 for 2 min. The coverslips were briefly rinsed in fresh media and incubated in MM-G without FM4-64 for dye internalization and vesicle trafficking during 3 h. The living cells were visualized at room temperature in confocal microscope. The arrows show the wild-type and atmA inactivation mutant hyphal tip. Images were analysed using the Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) (Laboratory of Confocal Microscopy, FMRP-USP, Brazil) using 63× magnification water immersion objective lens using laser line 514 nm for FM4-64. Images were captured by direct acquisition with Z step ranging from 0.5 to 2 μm with the Leica LAS AF software (Leica Microsystems). DIC and fluorescence images are maximum projection of Z stacks. Bar, 5 μm.

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Taken together, these results suggest that membrane domains may play a special role in hyphal morphogenesis, and membrane lipid polarization is deficient in the ΔatmA mutant strain. Furthermore, it also suggests that the ΔatmA mutation could affect endocytosis and vacuolar distribution in A. nidulans.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We have provided several evidences about pathways involved in the AtmA gene function. Mutants of human ATM show the symptoms of cerebellar ataxia, telangiectases (visibly dilated blood vessel on the skin or mucosal surface), immune defects and a predisposition to malignancy (OMIM at http://www.ncbi.nlm.nih.gov). The ATM gene is very pleiotropic and a central question about pleiotropy is whether the pleiotropic effects of a gene are conferred by multiple molecular functions of the gene or by multiple consequences of a single molecular function (Dudley et al., 2005; Van de Peppel and Holstege, 2005; He and Zhang, 2006). We have previously characterized the A. nidulans ATM homologue, AtmA (Malavazi et al., 2006), and shown that in addition to its expected role in the DNA damage response, AtmA is also required for polarized hyphal growth. Taking into consideration the pleiotropic nature of the atmA, we used transcriptional profiling of the atmA inactivation strain as a preliminary strategy to gain insight on pathways that are influenced by AtmA. ATM could affect the mRNA expression of these genes in a direct or indirect manner: it may directly regulate genes that encode proteins involved in DNA damage response and polar growth or modify the activity of such proteins via post-translational modifications. Such modifications could be carried out by ATM itself (i.e. phosphorylations) or by other enzymes subjected to ATM-mediated post-translational modifications. There are very few studies that tried to identify genes regulated by ATM kinase through microarray hybridization analysis (Cheng et al., 2004; Jang et al., 2004; Elkon et al., 2005).

Genes involved in DNA replication and in the ppp are more expressed in the ΔatmA mutant

There is an increased nuclear kinetics in the ΔatmA mutant when compared with the wild-type strain (Malavazi et al., 2006; and this work). This could reflect that the checkpoint controls that deal with the genome integrity in the mutant strain are impaired and consequently there is a faster DNA replication and segregation in this mutant. Accordingly, our microarray analysis experiments comparing early growth of germlings with the ΔatmA mutant strains indicated that a large number of genes involved in DNA replication were more expressed in the ΔatmA mutant than in the wild-type strain. In contrast, several genes related to DNA damage checkpoint and cell cycle progression, such as the homologues of HUS1 and WEE1, were less expressed in the ΔatmA mutant strain. ATM is definitively involved in the inhibition of cell cycle progression upon DSB (Kastan and Lim, 2000). AT cells are defective in the inhibition of DNA synthesis upon ionizing radiation (a cellular phenotype named radioresistant DNA synthesis, RDS; Painter, 1981). The same phenotype was observed in the ΔatmA mutant strain (Malavazi et al., 2006). This DSB-dependent inhibition of DNA synthesis results from activation of the S-phase checkpoint, and RDS occurs as a consequence of checkpoint failure. Is ATM activating directly or indirectly the firing of new replication origins? While ATM most clearly responds to DSBs by halting the cell cycle and initiating DNA repair, ATR is primarily activated by DNA replication intermediates and thus monitors the progress of DNA replication forks (Hurley and Bunz, 2007). However, it appears that ATM may play a role in this process as well as in Xenopus cell-free systems, localized ATM and ATR activation during DNA replication inhibit S-phase kinases and prevent neighbouring origins from firing (Shechter et al., 2004; Shechter and Gautier, 2005). ATM is activated at the site of a DSB and, via phosphorylation of key substrates, triggers a cascade of signals that activates multiple downstream pathways, including those that modulate DNA repair and cell cycle checkpoints (Shiloh, 2006; Hurley and Bunz, 2007). ATM tightly controls this response by phosphorylating important proteins in the G1/S, S-phase and G2/M checkpoint pathways (reviewed in Shiloh, 2006). Thus, the nuclear kinetics and the microarray hybridization analysis indicate that ΔatmA mutant looks completely ‘blind’ for endogenous DNA damage, not properly activating the cell cycle checkpoints for arresting nuclear division and activating DNA repair and/or apoptosis.

Considering this increased nuclear kinetics, the germlings will need increasing amounts of precursors such as riboses and desoxiriboses. We have observed that the genes encoding proteins from the ppp pathway and two subunits of the ribonucleotide reductase have increased mRNA expression. We tested the involvement of the ppp by constructing double mutants with existing A. nidulans mutations in genes of the ppp, such as pppA1. This mutation also confers decreased polar growth and was complemented by transaldolase. Interestingly, the double mutant ΔatmA pppA1 showed a reduction in the proliferation rate when compared with the parental strains, strongly suggesting ppp is somehow influenced by AtmA. Furthermore, the polar growth defect in the ΔatmA mutation was epistastic to the pppA1 mutation, suggesting a role for the ppp in the control of polar growth by AtmA. Previously, it was observed that mutations in other genes of the ppp also conferred morphological defects, like, for example, the Neurospora crassa morphological mutant, known as a colonial, in which mutations at the col-2 locus altered the structure of the glucose 6-phosphate dehydrogenase, leading to an in vivo accumulation of glucose-6-phosphate, and ultimately to a striking change in the growth pattern of N. crassa (Brody and Tatum, 1966). This mutant contained only 40% as much NADPH in extracts as did the wild type, emphasizing the pleiotropic effects of the NADPH deficiency (Brody and Tatum, 1970).

There is evidence showing that AT cells may chronically suffer from increased oxidative stress and that ATM deficiency is directly involved in this phenotype (for a review, see Barzilai et al., 2002). Accordingly, we investigated the influence of oxidative stress on ΔatmA growth. When compared with the wild-type strain, the ΔatmA growth was more affected by hydrogen peroxide, and ROS are accumulated in the ΔatmA gemlings as demonstrated by H2DCFDA fluorescence. The cells prevent oxidative damage to proteins by maintaining a reducing atmosphere, i.e. a high ratio of NADPH to NADP+ (Nelson and Cox, 2005). A. nidulansΔatmA mutant strain has a reduced ratio of NADPH/NADP+ and increasing concentrations of NADPT and NADP+. Stern et al. (2002) showed a perturbation in the steady-state levels of pyridine nucleotides in the cerebellum of Atm-deficient mice. There is a significant decrease in NADPT levels and NADP+ in the cerebella of 4-month-old Atm (–/–) mice. This change was the result of a decrease in NADP+ with no change in NADPH. In contrast to the Atm-deficient mice, it is conceivable that the increased NADP+ production in the ΔatmA mutant strain reflects the fact that the ppp is producing more NADPH that is immediately consumed by germlings aiming to attenuate the oxidative stress caused by the atmA inactivation mutation. Interestingly, the catalase (AN5918.3) and thioredoxin genes are more expressed in the ΔatmA mutant strain (see Table 3).

Evidence implicates AtmA in A. nidulans control of polar growth by multiple pathways

ATM function has never been linked to the establishment or maintenance of cell polarity in yeast cells. However, recently Enserink et al. (2006) and Shi et al. (2007) showed that Rad53CHK2 was involved in polar growth in S. cerevisiae and Candida albicans respectively. We were not able to observe any polar growth defect in A. nidulans uvsBATR, chkACHK1 and chkBCHK2 inactivation strains (I. Malavazi et al., in preparation). In contrast, we have also observed a role in these morphogenetic events for C. albicans Mec1 but not for Atm inactivation strains (I. Malavazi and G.H. Goldman, unpubl. results). Thus, it seems that these checkpoint proteins are important for co-ordinating morphogenetic events with DNA replication and DNA damage response during growth. Our results revealed that several genes involved in different aspects of polar growth have decreased mRNA expression in the ΔatmA mutant strain. These genes were arranged into four distinct groups: (i) those directly involved in the formation of a polarized hyphae and control of polar growth; (ii) in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts); (iii) in the synthesis of phosphatidic acid and phosphatidylinositol (e.g. phospholipase D); and (iv) intracellular trafficking, secretion and vesicular transport.

One of most accepted models for hyphal growth is the vesicle supply centre model of Bartnicki-Garcia (Bartnicki-Garcia et al., 1989). This model is based on the acummulation of vesicles within the hyphal apex named Spitzenkorper (for a review, see Steinberg, 2007). It is thought that the Spitzenkorper – that is important for hyphal growth and determines the directionality of growth – receives Golgi-derived vesicles that release exocytic vesicles in a controlled manner, thereby generating an exocytosis gradient that determines the shape of the hyphal apex (Bartnicki-Garcia et al., 1989). The integrity of the Spitzenkörper depends on the polarisome and there are some parallels between them (Harris et al., 2005; Steinberg, 2007). Notably, interactions between cytoplasmic microtubules and the cell cortex appear to play an important role in maintaining the position of the Spitzenkörper within the middle of the growth zone (Konzack et al., 2005). We had previously proposed that AtmA-dependent phosphorylation may influence these interactions, thereby helping to define a discrete zone for the cortical recruitment of cytoplasmic microtubules (Malavazi et al., 2006). The absence of this zone in the ΔatmA mutant would presumably lead to random interactions between microtubules and the cortex at hyphal tips, which would in turn affect Spitzenkörper position and the maintenance of a stable polarity axis. Consistent to this hypothesis, Horio and Oakley (2005) demonstrated that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. Here, we have strengthened this idea by showing that a kinesin, KipA, already shown to regulate Spitzenkörper position (Konzack et al., 2005), fails to properly localize to the hyphal tip in the ΔatmA.

We also investigated possible interactions between AtmA and the homologues of Spa2 (SpaA) and Bud6 (BudA) (Virag and Harris, 2006), the single A. nidulans formin SepA, and ModACDC42. We have observed no interaction between AtmA and BudA and SepA. However, there is an increased defect in polar growth in the double mutant ΔatmAΔspaA. Virag and Harris (2006) have shown that SpaA functions exclusively at hyphal tips and is not required for the assembly or maintenance of Spitzenkörper. Additional genetic evidence of interaction between AtmA and polar growth comes from the double mutant ΔatmA alcA::modAG14V. There is an increase in the polar growth defect when the dominant active protein ModA is expressed in the background of the ΔatmA mutant.

Apical extension at hyphal tips demands an appropriate supply of precursors and removing excess materials for recycling and/or destruction. The endocytic pathway plays a key role in nutrient acquisition, regulation of membrane receptors, transporters and ion channels as well as in membrane recycling (Peñalva, 2005). There is also evidence suggesting that the hyphal apex is not only a site of exocytosis but also participates in membrane recycling processes that support tip growth (Steinberg, 2007). FM4-64 staining of the ΔatmA mutant has shown that the Spitzenkörper cannot be visualized and there is a significant decrease of the endosome/vacuole structures.

These results strongly suggest an interaction between AtmA and the machinery involved in Spitzenkörper and polarisome assembly, and accordingly a large imbalance in the traffic of endocytic/exocytic vesicles in the ΔatmA mutant strain.

The sterol composition of animal and yeast membranes influences the polar localization of proteins. Lipid rafts are specialized membrane domains rich in sphingolipids and ergosterol that are thought to provide platforms that anchor proteins at the plasma membrane and mediate their biosynthetic or endocytic transport (Alvarez et al., 2007). These sterol- and sphingolipid-rich raft domains are thought to play important roles in dynamic processes including protein sorting, cell polarity and signal transduction (Alvarez et al., 2007). We have seen that polar growth was more affected in the mutant strain by a further reduction of ergosterol pools by inhibiting ergosterol biosynthesis through the inhibition of HMG-CoA reductase by lovastatin. Furthermore, lipid rafts – that are rich in sphingolipids and can therefore be visualized by using the polyene antibiotic filipin – are not organized at the hyphal tips in the mutant strain, strongly indicating that the AtmA can influence the production and deposition of sterol-rich domains at the plasma membrane. Inhibition of sphingolipid biosynthesis by myriocin in C. albicans and A. nidulans reduces tip growth and increases branching (Cheng et al., 2001; Martin and Konopka, 2004). We also observed that polar growth is more affected in the ΔatmA mutant when sphingolipids biosynthesis is inhibited by myriocin. This loss of polarity is accompanied by a rearrangement of actin patches, which suggests that lipid rafts are essential for a polarized F-actin cytoskeleton (Cheng et al., 2001). We were unable to observe any differences between the wild-type and the ΔatmA mutant strains in the tropomyosin gfp localization. However, one of the genes that showed to have reduced mRNA expression in the ΔatmA mutant strain is the MSS4 homologue, a phosphatidylinositol-4-phosphate 5-kinase (AN2766.3). Studies with S. cerevisiae have demonstrated that actin polarization and actin cable formation require MSS4 (Desrivières et al., 1998). In addition, Kobayashi et al. (2005) showed that disturbance of sphingolipid biosynthesis abrogates the signalling of Mss4. Yorek et al. (1999) investigated how AT affects myo-inositol metabolism and phospholipid synthesis using cultured human fibroblasts. They observed as consistent finding that the proportion of 32P in total labelled phospholipid that was incorporated into phosphatidylglycerol was greater in AT than in normal fibroblasts, whereas the fraction of radioactivity in phosphatidic acid was decreased. Furthermore, these authors showed turnover studies revealing that AT cells exhibit a less active phospholipid metabolism as compared with normal cells.

We have also observed a reduction in the ΔatmA mutant of the expression of several genes involved in the synthesis of phosphatidic acid, and consequently the production of DAG. The role of DAG in activating mammalian PKC is well understood (for a review, see Colon-Gonzalez and Kazanietz, 2006), however, in fungi is much less clear (for a review, see Shea and Del Poeta, 2006). In Cryptococcus neoformans, the purified recombinant cryptococcal Pkc1 protein showed increased kinase activity in the presence of several subspecies of DAG and phosphatidylserine (Shea and Del Poeta, 2006). In mammalian models, DAG binds to the C1 (cysteine-rich) domain of PKCs (Hurley et al., 1997). In Cryptococcus neoformans, the purified recombinant cryptococcal Pkc1 protein showed increased kinase activity in the presence of several subspecies of DAG and phosphatidylserine, and the C1 domain of this enzyme shows key consensus residues required for DAG binding and activation (Shea and Del Poeta, 2006). A. nidulans has two putative Pkc-encoding genes, which were designated pkcA and pkcB (Hermann et al., 2006). The gene that complements the mutant calC2 is pkcA, and cysteine-rich regions, usually involved in the binding of DAG or phorbol esters, could not be found in both A. nidulans PkcA and PkcB (Hermann et al., 2006). However, we were able to show that the reduction of phosphatidic acid production by a primary alcohol affected more the polar growth in the ΔatmA than in the wild type, suggesting that the mutant strain is already producing low levels of phosphatidic acid. The protein kinase C PkcA plays a role in polarized growth in A. nidulans (Teepe et al., 2007). Interestingly, A. nidulans polar growth was considerably reduced in the double mutant ΔatmA calC2. Although it remains to be determined if DAG can activate A. nidulans protein kinase C, it is tempting to speculate that AtmA can cross-talk with the protein kinase C via activation of the phosphatidic acid pathway.

These data suggest that AtmA is influencing the phosphatidic acid pathway, and affecting the sphingolipid biosynthesis. The misregulation of these pathways will surely contribute to a reduction of the polar growth via a decrease of sphingolipids in sterol-rich domains and/or a modulation of protein kinase C activity. We are currently investigating these hypotheses.

Taken together our data suggest that AtmA can control a complex interplay of cytoplasmic microtubules, lipid kinases, phosphatidic acid and sphingolipids synthesis and protein kinase C activity, and the machinery for actin polarization within apical lipid rafts of fungi. This complex web of interactions co-ordinates not only cell cycle progression upon DNA damage but also morphogenetic checkpoints.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains, media and culture methods

Aspergillus nidulans strains used are described in Table 8. The media used were of two basic types, i.e. complete and minimal. The complete media comprised the following three variants: YAG (2% w/v glucose, 0.5% w/v yeast extract, 2% w/v agar, trace elements), YUU (YAG supplemented with 1.2 g l−1[each] of uracil and uridine) and liquid YG or YG+UU medium with the same composition (but without agar). The minimal media were a modified minimal medium (MM; 1% w/v glucose, original high-nitrate salts, trace elements, 2% w/v agar, pH 6.5). Expression of tagged genes under the control of alcA promoter was regulated by carbon source: repression on glucose 4% w/v, derepression on glycerol and induction on ethanol. Therefore, MM-G and MM-E were identical to MM, except that glycerol (2% v/v) and/or ethanol (2% v/v) were used, respectively, in place of glucose as the sole carbon source. Trace elements, vitamins and nitrate salts were included as described by Kafer (1977). Growth tests for the gene impaired in the pentose phosphate pathway (pppA1 mutant) were performed on Cove's MM (Cove, 1966) containing urea (5 mM) as sole nitrogen source and glucose (1% w/v) or xylose (1% w/v) as carbon sources and solidified with 1.5% agar. Strains were grown at 37°C unless indicated otherwise.

For the oxidative stress viability assay (Noventa-Jordão et al., 1999), 1 × 106 conidia ml−1 were incubated in YG+UU for 5 h at 30°C in a reciprocal shaker (250 r.p.m.). After this period, 50 mM of hydrogen peroxide was added to the germlings and they were allowed to grow for additional 20 min at the same conditions. Conidia were conveniently diluted and plated in YAG+UU plates. Plates were incubated at 37°C for 48 h. Viability was determined as the percentage of colonies on treated plates compared with untreated controls.

For the measurement of nuclear division kinetics, conidia were incubated in YG at 37°C for 2, 4 and 6 h before staining and counting. To access the polarization kinetics, conidia were incubated in YG+50 mM HU for 5 h at 37°C. Germlings were released from cell cycle arrest by three sequential washes in pre-warmed YG, followed by addition of fresh pre-warmed YG. Following release, samples were taken at 30 min intervals over the next 150 min. A spore was counted as polarized if it possessed a germ tube readily detectable as small protuberances on the spherical shape of spore surface.

To evaluate the differences among the strains, we used one-way anova and Student Newman Keuls post hoc test. The data shown are the average of two or three independent experiments and means ± standard deviation are shown. Analyses were performed using the software package Sigma Stat (SysStat Corporation, USA) and the statistical significance was set at α = 0.05.

Staining and microscopy

For nuclear staining of the germlings, conidia were inoculated on coverslips. After incubation at the appropriate conditions for each experiment, coverslips with adherent germlings were transferred to fixative solution (3.7% v/v formaldehyde, 50 mM w/v sodium phosphate buffer pH 7.0, 0.2% v/v Triton X-100) for 30 min at room temperature. Then, they were briefly rinsed with PBS buffer (140 mM NaCl, 2 mM KCl, 10 mM NaHPO4, 1.8 mM KH2PO4, pH 7.4) and incubated for 5 min in a solution with 100 ng ml−1 DAPI (4′,6-diamino-2-phenyylindole, Sigma Chemical). After 5 min of incubation with the dyes, they were washed with PBS buffer for 5–10 min at room temperature and then rinsed in distilled water, mounted and visualized in epifluorescence microscope. To verify the pattern of sterol distribution in the ΔatmA mutant, strains were grown overnight at 44°C in MM-G and stained with filipin at a concentration 25 μg ml−1 in pre-warmed media for 5 min, washed in growth media, mounted and viewed in epifluorescence microscope. Slides were viewed with a Carl Zeiss (Jena, Germany) microscope using 100× magnification oil immersion objective lens (EC Plan-Neofluar, NA 1.3) equipped with a 100 W HBO mercury lamp epifluorescence module. Phase contrast for the brightfield images and fluorescent images were captured with a AxioCam camera (Carl Zeiss), processed using the AxioVision software version 3.1 and saved as TIFF files. Further processing was performed using Adobe Photoshop 7.0 (Adobe Systems Incorporated, CA).

For membrane staining and to verify the endocytyc machinery in the ΔatmA mutant, the styryl dye FM4-64 [N-(3-thiethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)] pyridinium dibromide was used (Invitrogen Molecular Probes) based on the protocol previously described by Peñalva (2005). Conidiospores were grown in glass-bottom dishes (Mattek Corporation, USA) in 2 ml of MM-G +  supplements for 12 h at 44°C and exposed to pre-warmed media containing 10 μM FM4-64 for 2 min. The coverslips were briefly rinsed in fresh media and incubated in MM-G without FM4-64 for dye internalization and vesicle trafficking during 3 fours. The living cells were visualized at room temperature in confocal microscope. In order to verify the accumulation of ROS in the ΔatmA and wild-type strains, cells were stained with H2DCFDA. Conidiospores were grown in glass-bottom dishes (Mattek Corporation, USA) in 2 ml of MM-G + supplements for 12 h at 44°C and exposed to pre-warmed PBS buffer containing 20 μM H2DCFDA (Invitrogen Molecular Probes) for 30 min in the dark at 30°C. The coverslips were washed with PBS and pre-warmed fresh medium was added. The germilings were further incubated for 30 min at 30°C and images were analysed by confocal microscopy at room temperature. For live cell imaging of proteins fused to gfp, conidiospores were grown in glass-bottom dishes (Mattek Corporation, USA) in 2 ml of MM-G and/or ethanol + supplements for 15 h at 30°C. All the confocal images were analysed using the Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) (Laboratory of Confocal Microscopy, FMRP-USP, Brazil) using 63× magnification water immersion objective lens using laser lines 488 nm for gfp and H2DCFDA; and 514 nm for FM4-64. Images were captured by direct acquisition with Z step ranging from 0.5 to 2 μm with the Leica LAS AF software (Leica Microsystems) and additional processing was carried out using Adobe Photoshop 7.0 (Adobe Systems Incorporated, CA).

Molecular techniques

Standard genetic techniques for A. nidulans were used for all strain constructions and transformations (Kafer, 1977). DNA and RNA analysis were performed as described by Sambrook and Russel (2001). For PCR experiments standard protocols were applied using a PTC100 96-well thermal cycler (MJ Research, Watertown, MA) for reaction cycles. Gene-specific primers were used for single-run sequencing using the dideoxy chain termination method and dye termination chemistry (Applied Biosystems) to fully sequence the genomic region of the transaldolase gene (AN0240.3) in the mutant (G840) and wild-type strains (A26). The transaldolase gene was PCR amplified with the primers Transaldolase F: 5′-CTATCTCTCATGCTCAAAGTCC-3′ and Transaldolase R: 5′-GACAATCACAACTTACCAGCCT-3′. Three independent reactions for both the wild-type and mutant strains were performed. The PCR products were gel-purified and used in the sequencing reactions to identify the mutation pppA1.

RNA isolation

For the time-course microarray experiments, two sets of experiments were designed. In the first one, 1.0 × 1010 conidia per ml of A. nidulans wild-type (GR5) or IM69 (ΔatmA mutant) strains were used to inoculate 400 ml of pre-warmed liquid cultures (YG) in 1000 ml erlenmeyer flasks that were incubated in a reciprocal shaker (250 r.p.m.) at 37°C for 60, 90 and 120 min. At each time point the germlings were harvested by centrifugation and frozen in liquid nitrogen. For the second experiment, the same amount of conidia were identically inoculated in 400 ml of liquid cultures (YG) containing 50 mM hydroxyurea (HU) and allow to grow for 5 h to synchronize the germlings in the S-phase of cell cycle. After this time, the cultures were centrifuged and washed, and pre-warmed drug-free fresh medium was aseptically added to the cultures. They were allowed to grow for additional 60, 90 and 120 min after the HU release. At each time point the germlings were also harvested by centrifugation and quickly frozen in liquid nitrogen. For total RNA isolation, the germlings were disrupted by grinding in liquid nitrogen and total RNA was extracted with Trizol reagent (Invitrogen, USA). Ten micrograms of RNA from each treatment was then fractionated in 2.2 M formaldehyde, 1.2% w/v agarose gel, stained with ethidium bromide, and then visualized with UV-light. The presence of intact 25S and 17S ribosomal RNA bands was used as a criterion to assess the integrity of the RNA. RNase-free DNase treatment was performed as previously described (Semighini et al., 2002).

Real-time PCR reactions

All the PCR and RT-PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystem, USA). Taq-Man™EZ RT-PCR kits (Applied Biosystems, USA) were used for RT-PCR reactions. Taq-Man™ Universal PCR Master Mix kit was used for PCR reactions. The thermal cycling conditions comprised an initial step at 50°C for 2 min, followed by 10 min at 95°C, and 40 cycles at 95°C for 15 s and 60°C for 1 min. The reactions and calculations were performed according to Semighini et al. (2002). Table 9 describes the primers and Lux™ fluorescent probes (Invitrogen) used in this work.

Table 9.  Primers and fluorescent probes used in the real-time RT-PCR reactions.
Primers and probesSequencesGeneb
tubC_525FLa5′-CACTTTATGCCGTCGCCGAAAG[FAM]G-3′AN6838.3
tubC_525FL_583RU5′-GCAGAATGTCTCGTCCGAATG-3′ 
AN0913.3791RLa5′-CGGCTAACTGGGAACGAGACTGC[FAM]G-3′AN0913.3
AN0913.3791RL/773FU5′-GGGCCAACAAGATTGACAGCTT-3′ 
AN2766.31897FLa5′-CGGGTAATCCAGAAGGAGCGTCC[FAM]G-3′AN2766.3
AN2766.31897FL/1919RU5′-AGGATCTCGTCTGGCATCTTTG-3′ 
AN1016.3737FLaCGGTTGAAGAGGGAAACCAGAAC[FAM]GAN1016.3
AN1016.3737FL/769RUTGAACCATCGAGAGTTGACGAC 
AN5100.3765FLaCGGAAAACCAGGTGCGGTAGTTC[FAM]GAN5100.3
AN5100.3765FL/798RUCAGATTTGGGATTCCACTGTCG 
AN4743.3315FLaCGGGCGTCAAGTCTAAGTGGTTCC[FAM]GAN4743.3
AN4743.3315FL/347RUAGCTTGGTGCCAACTAGGATGA 
AN3434.3325FLaCGGCGATATGATCGAGATGGAAGC[FAM]GAN3434.3
AN3434.3325FL/341RUTTCTCTCATCAACCGCAAGTTGT 
Phosph_D_3176RLaCGGATAGACCGAGAGCTTCTAAATC[FAM]GAN10413.3
Phosph_D_3176RL/3155FUCCCGACGCCTTTCCTACTACC 

Gene expression methods

We have used the A. nidulans oligonucleotide slides version 1 for microarray hybridizations (for details see http://pgfrc.tigr.org/slide_html/microarray_descriptions.shtml). The RNA samples extracted with Trizol, as described above, were further purified with the RNA easy kit (Qiagen, Germany) and directly labelled by incorporation of Cy3- or Cy5-dUTP (GE Health Care). Briefly, 30 μg of total RNA were mixed with 20 μg of Randon Primer Hexamers (GE Health Care), 100 pmol of oligo dTV (Invitrogen) and 40 U of RNase Out™ (Invitrogen) in a volume of 13 μl and heated to 70°C for 5 min. The tubes were briefly chilled to 4°C for 5 min and mixed with 5 μl of 5× first-strand buffer (Invitrogen), 2 μl of DTT (100 mM Invitrogen), 2 μl of dNTP (5 mM dATP, 5 mM dCTP, 5 mM dGTP, 2 mM dTTP), 2 μl of Superscript II™ (Invitrogen) and 1 μl of Cy™3-dUTP (25 nmol) or Cy™5-dUTP (25 nmol). The reaction was then incubated at 25°C for 5 min and at 42°C for 3 h. The RNA was degraded by adding 2.5 μl of EDTA (0.5 M, pH 8) and 5 μl of NaOH (1 M) following an incubation at 37°C for 40 min. The resulting first-strand cDNA was purified and concentrated using a Microcon YM-50 cartridge. Labelled cDNAs were mixed with 8 μl of Liquid block RPN3601 Batch 24 (GE Health Care), 5.5 μl of SDS 2% v/v and 19 μl of SSC (20×) and the volume adjusted to 110 μl. Slides were hybridized overnight (42°C) in a Gene-Tac Hybridization Station (Genomic Solutions) and washed in 2× SSC 0.5% v/v SDS, 0.5× SSC and 0.05× SSC. All washing steps consisted of 1 min of flow, followed by 5 min of incubation. Slides were then dried and subjected to fluorescent detection with a GMS 418 Array Scanner (Affymetrix, Santa Clara, CA) and the TIFF images generated were analysed using TIGR Spotfinder (http://www.tigr.org/software/microarray.shtml/) to obtain relative transcript levels. Data were normalized using a local regression technique LOWESS (LOcally WEighted Scatterplot Smoothing) for hybridizations with RNA-based samples and SD regularization of the Cy5/Cy3 ratio across all sectors (blocks) of the array using a software tool MIDAS (http://www.tigr.org/software/microarray.shtml). The resulting data were averaged from duplicate genes on each array, from dye-swap hybridizations for each experiment and from two biological replicates, taking a total of four intensity data points for each gene. Differentially expressed genes at the 95% confidence level were determined using intensity-dependent Z-scores (with Z = 1.96) as implemented in MIDAS and the union of all genes identified at each time point was considered significant in this experiment. The resulting data were organized and visualized based on similar expression vectors in genes using Euclidean distance and hierarchical clustering with average linkage clustering method to view the whole data set and k-means to group the genes in 60 clusters with TIGR MEV (multiexperiment viewer), also available at http://www.tigr.org/software/microarray.shtml.

NADPH/NADP measurements

For NADPH and NADP quantification, 1 × 1010 conidia per ml of ΔatmA and wild-type strains were incubate in YG + supplements for 60, 90, 120 and 150 min in order to reproduce the time point experiment design used for the transcriptional profiling experiment (see above). At each time point, the germlings were harvested by centrifugation and frozen in liquid nitrogen. For NADPH and NADP extraction, the germlings were disrupted by grinding in liquid nitrogen and extraction was performed using 400 μl of extraction buffer supplied by the kit NADP+/NADPH quantification kit (BioVision, USA). Samples were mixed by vortex for 1 min and centrifuged at 14 000 r.p.m. for 5 min. The supernatant was recovered, centrifuged for 2 min at 14 000 r.p.m. and used to NADPH, NADP and protein determination. The protein concentration was determined by use of a modified Bradford assay (Bio-Rad). NADPH and NADP were quantified by high-performance liquid chromatography (HPLC). HPLC system consisted of two LC-10AD solvent pumps, an SLC-10A system controller, a CTO-10AS column oven set at 24°C, a 7125 Rheodyne injector with a 20 μl loop and a diode array detector (SPD-M10A) set at 254 nm (Shimadzu, Kyoto, Japan). Separations were carried out on a Lichrospher C18 column (125 × 4.6 mm inner diameter, 5 μm particle size, Merck, Darmstadt, Germany). A C18 guard column (4 × 4 mm inner diameter, Merck) of the same material was used to protect the analytical column. The mobile phase consisted of NaH2PO4 0.2 M-Methanol (96:4, v/v), at a flow-rate of 1 ml min−1.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We would like to thank Dr Steven D. Harris for providing several mutant strains used in this work; we also want to thank Drs Terry Hill, John Clutterbuck and Reinhard Fischer for providing the protein kinase C, some of the ppp mutant strains and the kipA mutants respectively. We also thank the Fungal Genetics Stock Center for providing some of the ppp mutant strains, José Luiz Cappelaro for technical assistance in some of the A. nidulans crosses, the two anonymous reviewers for their suggestions, Drs. Eliana G.M. Lemos and Lúcia M.L. Alves (FCAV-UNESP, Brazil) for their assistance in the microarray hibridizations and the Laboratory of Confocal Microscopy, FMRP-USP, Brazil, for the use of the confocal microscope. This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

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  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
MMI+5885+Supplementary+Figure+1.ppt425KSupporting info item
MMI+5885+Supplementary+Figure+2.ppt2211KSupporting info item
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