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• W2045742010 abstract "We have studied the effects of phytosphingosine (PHS) on cells of the filamentous fungus Neurospora crassa. Highly reduced viability, impairment of asexual spore germination, DNA condensation and fragmentation, and production of reactive oxygen species were observed in conidia treated with the drug, suggesting that PHS induces an apoptosis-like death in this fungus. Interestingly, we found that complex I mutants are more resistant to PHS treatment than the wild type strain. This effect appears to be specific because it was not observed in mutants defective in other components of the mitochondrial respiratory chain, pointing to a particular involvement of complex I in cell death. The response of the mutant strains to PHS correlated with their response to hydrogen peroxide. The fact that complex I mutants generate fewer reactive oxygen species than the wild type strain when exposed to PHS likely explains the PHS-resistant phenotype. As compared with the wild type strain, we also found that a strain containing a deletion in the gene encoding an AIF (apoptosis-inducing factor)-like protein is more resistant to PHS and H2O2. In contrast, a strain containing a deletion in a gene encoding an AMID (AIF-homologous mitochondrion-associated inducer of death)-like polypeptide is more sensitive to both drugs. These results indicate that N. crassa has the potential to be a model organism to investigate the molecular basis of programmed cell death in eukaryotic species. We have studied the effects of phytosphingosine (PHS) on cells of the filamentous fungus Neurospora crassa. Highly reduced viability, impairment of asexual spore germination, DNA condensation and fragmentation, and production of reactive oxygen species were observed in conidia treated with the drug, suggesting that PHS induces an apoptosis-like death in this fungus. Interestingly, we found that complex I mutants are more resistant to PHS treatment than the wild type strain. This effect appears to be specific because it was not observed in mutants defective in other components of the mitochondrial respiratory chain, pointing to a particular involvement of complex I in cell death. The response of the mutant strains to PHS correlated with their response to hydrogen peroxide. The fact that complex I mutants generate fewer reactive oxygen species than the wild type strain when exposed to PHS likely explains the PHS-resistant phenotype. As compared with the wild type strain, we also found that a strain containing a deletion in the gene encoding an AIF (apoptosis-inducing factor)-like protein is more resistant to PHS and H2O2. In contrast, a strain containing a deletion in a gene encoding an AMID (AIF-homologous mitochondrion-associated inducer of death)-like polypeptide is more sensitive to both drugs. These results indicate that N. crassa has the potential to be a model organism to investigate the molecular basis of programmed cell death in eukaryotic species. The term apoptosis (apo-from, ptosis-falling) means “dropping off” of petals or leaves from plants or trees and was described as a particular mode of cell death that is characterized by rounding up of the cell, reduction of cell volume, condensation of chromatin, fragmentation of the nucleus, and maintenance of the intact plasma membrane until the very late stage of the death process (1Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12776) Google Scholar). Apoptosis is a form of programmed cell death (PCD) 2The abbreviations used are: PCD, programmed cell death; PHS, phytosphingosine; ROS, reactive oxygen species; AIF, apoptosis-inducing factor; AMID, AIF-homologous mitochondrion-associated inducer of death; GFS, glucose, fructose, sorbose; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. 2The abbreviations used are: PCD, programmed cell death; PHS, phytosphingosine; ROS, reactive oxygen species; AIF, apoptosis-inducing factor; AMID, AIF-homologous mitochondrion-associated inducer of death; GFS, glucose, fructose, sorbose; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. that plays a central role in tissue homeostasis and maintenance in multicellular organisms. Cytological phenotypes associated with apoptosis include chromatin condensation, DNA fragmentation, phosphatidylserine externalization, vacuolization, activation of caspases, and increased production of reactive oxygen species (ROS). Although the importance of apoptosis in stroke, neurodegenerative disorders, and cancer is increasingly evident, many details of its regulation and production of apoptotic phenotypes are poorly understood (for recent reviews, see Refs. 2Skulachev V.P. Apoptosis. 2006; 11: 473-485Crossref PubMed Scopus (322) Google Scholar, 3Madeo F. Herker E. Wissing S. Jungwirth H. Eisenberg T. Fröhlich K.-U. Curr. Opin. Microbiol. 2004; 7: 655-660Crossref PubMed Scopus (257) Google Scholar, 4Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2789) Google Scholar). In addition to the well established role of mitochondria in ATP production, regulation of cell death has emerged as a second major function of these organelles. This function appears to be related to the role of mitochondria as the major intracellular source of ROS, mainly generated at complexes I and III of the respiratory chain (5Andreyev A.Y. Kushnareva Y.E. Starkov A.A. Biochemistry (Mosc). 2005; 70: 200-214Crossref PubMed Scopus (783) Google Scholar). Enhanced ROS production has been associated with mtDNA mutations, aging, and cell death (6Orrenius S. Drug Metab. Rev. 2007; 39: 443-455Crossref PubMed Scopus (370) Google Scholar) and also with a wide variety of pathologies, including cancer, atherosclerosis, and neurodegenerative diseases (7Dröge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7413) Google Scholar). ROS overproduction induces an osmotic imbalance across the inner mitochondrial membrane. This leads to the swelling of the mitochondrial matrix and, consequently, the disruption of the outer mitochondrial membrane and release of proteins from the intermembrane space into the cytosol (2Skulachev V.P. Apoptosis. 2006; 11: 473-485Crossref PubMed Scopus (322) Google Scholar). Some of these released proteins, including cytochrome c, Smac/Diablo, and apoptosis-inducing factor (AIF), are proapoptotic regulators that subsequently activate cellular apoptotic programs (8Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2762) Google Scholar, 9Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (93) Google Scholar). The mitochondrial or intrinsic pathway is a major apoptotic pathway, where the release of cytochrome c from the mitochondrion and the production of ROS are the two main events proposed as integral control elements in the cell decision to die. In the second major pathway, the extrinsic pathway, caspases are activated directly (10Putcha G.V. Harris C.A. Moulder K.L. Easton R.M. Thompson C.B. Johnson Jr., E.M. J. Cell Biol. 2002; 157: 441-453Crossref PubMed Scopus (181) Google Scholar). Programmed cell death and apoptotic mechanisms are ubiquitous in both prokaryotic and eukaryotic species (11Koonin E.V. Aravind L. Cell Death Differ. 2002; 9: 394-404Crossref PubMed Scopus (311) Google Scholar). The complex regulatory network and the sometimes contradictory results obtained with human cell lines make it desirable to investigate cell death mechanisms in simpler model systems. For example, apoptotic-like programmed cell death has been observed in the unicellular yeast, Saccharomyces cerevisiae (12Ludovico P. Madeo F. Silva M. IUBMB Life. 2005; 57: 129-135Crossref PubMed Scopus (60) Google Scholar, 13Eisenberg T. Büttner S. Kroemer G. Madeo F. Apoptosis. 2007; 12: 1011-1023Crossref PubMed Scopus (181) Google Scholar), and in a number of filamentous fungi, including Aspergillus spp., Podospora anserina, and Candida albicans, following exposure to some environmental stimuli, such as acetic acid, hydrogen peroxide, sphingoid bases, viral killer toxins, and UV radiation (14Semighini C.P. Hornby J.M. Dumitru R. Nickerson K.W. Harris S.D. Mol. Microbiol. 2006; 59: 753-764Crossref PubMed Scopus (181) Google Scholar, 15Hamann A. Brust D. Osiewacz H.D. Mol. Microbiol. 2007; 65: 948-958Crossref PubMed Scopus (65) Google Scholar, 16Lu B.C.K. Kües U. Fischer R Growth, Differentiation and Sexuality. Springer, Berlin2006: 167-187Google Scholar). The fungal genomes contain a subset of metazoan apoptotic genes, although the genomes of filamentous fungi contain homologues of PCD genes of metazoans that are not present in S. cerevisiae (17Fedorova N.D. Badger J.H. Robson G.D. Wortman J.R. Nierman W.C. BMC Genomics. 2005; 6: 177-191Crossref PubMed Scopus (88) Google Scholar), thus making them attractive models for dissecting mechanisms of programmed cell death. The filamentous fungus Neurospora crassa is a morphologically complex multicellular organism with many differentiated cell types (18Davis R.H. Neurospora: Contributions of a Model Organism. University Press, Oxford2000Google Scholar). In addition, it is well studied, its genome was sequenced, and many molecular tools have been developed over the years to work with the organism (19Galagan J.E. Calvo S.E. Borkovich K.A. Selker E.U. Read N.D. Jaffe D. FitzHugh W. Ma L.J. Smirnov S. Purcell S. Rehman B. Elkins T. Engels R. Wang S. Nielsen C.B. Butler J. Endrizzi M. Qui D. Ianakiev P. Bell-Pedersen D. Nelson M.A. Werner-Washburne M. Selitrennikoff C.P. Kinsey J.A. Braun E.L. Zelter A. Schulte U. Kothe G.O. Jedd G. Mewes W. Staben C. Marcotte E. Greenberg D. Roy A. Foley K. Naylor J. Stange-Thomann N. Barrett R. Gnerre S. Kamal M. Kamvysselis M. Mauceli E. Bielke C. Rudd S. Frishman D. Krystofova S. Rasmussen C. Metzenberg R.L. Perkins D.D. Kroken S. Cogoni C. Macino G. Catcheside D. Li W. Pratt R.J. Osmani S.A. DeSouza C.P. Glass L. Orbach M.J. Berglund J.A. Voelker R. Yarden O. Plamann M. Seiler S. Dunlap J. Radford A. Aramayo R. Natvig D.O. Alex L.A. Mannhaupt G. Ebbole D.J. Freitag M. Paulsen I. Sachs M.S. Lander E.S. Nusbaum C. Birren B. Nature. 2003; 422: 859-868Crossref PubMed Scopus (1256) Google Scholar, 20Colot H.V. Park G. Turner G.E. Ringelberg C. Crew C.M. Litvinkova L. Weiss R.L. Borkovich K.A. Dunlap J.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10352-10357Crossref PubMed Scopus (853) Google Scholar, 21Davis R.H. Perkins D.D. Nat. Rev. Genet. 2002; 3: 397-403Crossref PubMed Scopus (151) Google Scholar). In addition to PCD induced by external factors, N. crassa and other filamentous fungi utilize a cell death pathway for non-self recognition that occurs when two genetically different colonies come in contact (22Dementhon K. Iyer G. Glass N.L. Eukaryot. Cell. 2006; 5: 2161-2173Crossref PubMed Scopus (71) Google Scholar). Thus, it has the potential to be very useful for research on the mechanisms and evolution of programmed cell death in eukaryotic species. The fact that phytosphingosine (PHS) induces apoptosis in the filamentous fungus Aspergillus nidulans (23Cheng J. Park T.-S. Chio L.-C. Fischl A.S. Ye X.S. Mol. Cell. Biol. 2003; 23: 163-177Crossref PubMed Scopus (124) Google Scholar) prompted us to initiate the characterization of the sphingolipid-induced PCD in wild type and respiratory mutants of N. crassa. In this work, we describe the programmed cell death phenotype of the fungal cells exposed to PHS and the finding that respiratory chain complex I is involved in the process. Strains and Growth Techniques—The N. crassa wild type strain 74-OR23-1A (74A) and mutants in the respiratory chain complex I genes nuo9.8, nuo14, nuo21, nuo21.3c, nuo30.4, nuo51, and nuo78 have been described previously (reviewed in Ref. 24Marques I. Duarte M. Assunção J. Ushakova A.V. Videira A. Biochim. Biophys. Acta. 2005; 1707: 211-220Crossref PubMed Scopus (52) Google Scholar). Deficient mutants in the mitochondrial complexes III (fes-1; FGSC11184), IV (cyt-12; FGSC4506), and V (oli; FGSC8738) and in cytochrome c (cyc-1; FGSC3558), as well as the AIF (ΔNCU05850, here called aif-1; FGSC11900) and AIF-homologous mitochondrion-associated inducer of death (AMID) (ΔNCU06061; FGSC12090) deletion strains (20Colot H.V. Park G. Turner G.E. Ringelberg C. Crew C.M. Litvinkova L. Weiss R.L. Borkovich K.A. Dunlap J.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10352-10357Crossref PubMed Scopus (853) Google Scholar), were obtained from the Fungal Genetics Stock Center (FGSC) (25McCluskey K. Adv. Appl. Microbiol. 2003; 52: 245-262Crossref PubMed Scopus (228) Google Scholar). General procedures for growth and handling of the fungal strains have been published (26Davis R.H. de Serres F.J. Methods Enzymol. 1970; 17A: 79-143Crossref Scopus (935) Google Scholar). When indicated, PHS from Avanti Polar Lipids (Alabaster, AL) was added to the media from a stock solution of 4 mg/ml in ethanol. The viability of conidia was determined by plating the cells on GFS agar medium, which induces colonial growth, and counting colony-forming units after incubation at 26 °C. Conidial germination in Vogel's minimal medium (27Vogel H.J. Microb. Genet. Bull. 1956; 13: 42-46Google Scholar) at 26 °C was evaluated by observation under a Nikon optical microscope. These types of experiments, as well as the following methods, were repeated at least three times. TUNEL Assay—DNA strand breaks were analyzed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) using the in situ cell death detection kit fluorescein (Roche Applied Science), as described previously (28Madeo F. Frolich E. Frolich K.-U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (679) Google Scholar). N. crassa conidia were preincubated in minimal medium for 30 min, washed in water, and then treated with phytosphingosine for 60 min and washed again. Spheroplasts were prepared (29Duarte M. Sousa R. Videira A. Genetics. 1995; 139: 1211-1221Crossref PubMed Google Scholar), and fixation was performed 5.5 h after treatment with the drug. Images were collected in a Zeiss Axiovert 200 M microscope using an Axiocam (Carl Zeiss, Germany). Flow cytometry analysis was performed in a FACSCalibur (BD Biosciences). Twenty-thousand cells per sample were analyzed. Data were acquired and analyzed with CELLQuest PRO 3.3 (BD Biosciences). Spot Assays—Conidia from different strains were harvested with distilled water, and the cell suspensions were adjusted to a concentration of 6 × 107 cells/ml. Three-fold serial dilutions of each strain were spotted on plates containing GFS medium containing various concentrations of the appropriate drugs. Images were taken by scanning the plates after about 3 days incubation at 26 °C. ROS Detection—ROS production by mitochondria was monitored by using dihydrorhodamine 123 from Sigma. The reduced form of dihydrorhodamine 123 does not fluoresce until entering an actively respiring cell, where it is oxidized by ROS to a green fluorescent compound that is sequestered in the mitochondria (30Li W. Sun L. Liang Q. Wang J. Mo W. Zhou B. Mol. Biol. Cell. 2006; 17: 1802-1811Crossref PubMed Scopus (115) Google Scholar). Conidial cells were harvested and suspended in minimal medium at a concentration of 5 × 106 cells/ml. Then, 50 μg/ml dihydrorhodamine 123 was added, and the cell suspensions were incubated at 26 °C during 30 min and washed before the addition of PHS. ROS production was evaluated using a FACSCalibur cytometer (BD Biosciences), and data were analyzed by CELLQuest version 3.3 (BD Biosciences) with excitation at 480 nm and emission at 530 nm. PHS Induces an Apoptosis-like Cell Death in N. crassa—The sphingolipid PHS has potent antifungal activity toward A. nidulans, inducing a caspase-independent apoptosis in this organism (23Cheng J. Park T.-S. Chio L.-C. Fischl A.S. Ye X.S. Mol. Cell. Biol. 2003; 23: 163-177Crossref PubMed Scopus (124) Google Scholar). In test effects in N. crassa, we evaluated the viability of asexual spores (conidia) upon exposure to PHS. Conidial cells were incubated in the presence of 10 μg/ml PHS, samples were withdrawn at different times and plated in GFS medium (which induces colonial growth of N. crassa), and the resulting colonies were counted. As shown in Fig. 1A, exposure of N. crassa wild type conidia to PHS resulted in cell death. The kinetics of cell viability, estimated by colony-forming unit counts, showed a dramatic decrease in the cell viability (around 60%) after a short period of incubation with the drug (5 min). Cell viability continued to decrease, reaching about 15% cell viability after a 120-min incubation with PHS. This effect was not seen in the control experiment where cell viability was nearly 100% and indicates that PHS possesses a potent activity against N. crassa. We also evaluated whether exposure to PHS impaired the germination of N. crassa conidia. Untreated wild type conidia progressively undergo germination when incubated in liquid medium; the majority of cells initiate germination within a couple of hours. In contrast, germination was inhibited in cells treated with PHS with less than 50% of the conidia initiating germination after a 4-h exposure (Fig. 1B). We observed that N. crassa conidia exposed to PHS underwent nuclear condensation (data not shown). Nuclear condensation is a hallmark of apoptosis, as is chromatin fragmentation. To determine whether chromatin fragmentation was also associated with cell death caused by PHS in N. crassa, we employed the TUNEL assay. TUNEL detects DNA fragmentation and has been used as a discriminating technique to detect programmed cell death at a single cell level (16Lu B.C.K. Kües U. Fischer R Growth, Differentiation and Sexuality. Springer, Berlin2006: 167-187Google Scholar). N. crassa wild type conidia were treated with PHS, and the TUNEL staining was subsequently performed. The results were visualized by fluorescence microscopy (Fig. 2A) and analyzed by flow cytometry (Fig. 2B). Control conidial cells, either treated with PHS but unlabeled or labeled but untreated, were TUNEL-negative. TUNEL-positive cells, indicative of DNA fragmentation, were only found in the positive control and among cells treated with PHS (Fig. 2A). Fluorescence-activated cell sorter analysis was performed for the quantification of TUNEL-positive conidia from PHS-treated cells by comparing with the labeled but untreated negative control cells. Under the conditions tested, about 12% of the cells exposed to PHS revealed DNA fragmentation (Fig. 2B). Taken together, our results indicate that PHS induces an apoptosis-like cell death in N. crassa, similarly to what has been observed in A. nidulans. Complex I Mutants Are More Resistant to PHS than the Wild Type Strain—Mitochondria are an essential mediator of apoptosis. To evaluate whether mitochondria are also involved in PHS-induced death as suggested (23Cheng J. Park T.-S. Chio L.-C. Fischl A.S. Ye X.S. Mol. Cell. Biol. 2003; 23: 163-177Crossref PubMed Scopus (124) Google Scholar), we analyzed the behavior of mitochondrial mutants of N. crassa when exposed to the sphingolipid. We started with our collection of complex I mutants by testing the viability of conidial cells from nuo9.8, nuo14, nuo21, nuo21.3c, nuo30.4, nuo51, and nuo78 mutant strains following PHS treatment. Interestingly, we found that all complex I mutants are more resistant to PHS than the wild type strain. Fig. 3 shows the results obtained with the nuo51 and nuo21 mutant strains. It can be seen that the nuo51 mutant is clearly more resistant to PHS. Roughly, more than half of the nuo51 cells remain viable after a 90-min exposure to PHS, whereas the viability of wild type cells dropped below 20% under the same conditions. The nuo51 mutant (which lacks the 51-kDa subunit) is still capable of assembling complex I. However, this complex I is non-functional in electron transfer because the 51-kDa protein harbors the FMN prosthetic group, which is the entry point of electrons into the respiratory chain (31Fecke W. Sled V.D. Ohnishi T. Weiss H. Eur. J. Biochem. 1994; 220: 551-558Crossref PubMed Scopus (83) Google Scholar). The resistance of the nuo21 mutant toward PHS lies between that of the nuo51 mutant and a wild type strain. This observation is in agreement with the fact that strains lacking the 21-kDa subunit assemble a functional complex but show lower amounts of complex I enzyme than wild type (32Ferreirinha F. Duarte M. Melo A.M. Videira A. Biochem. J. 1999; 342: 551-554Crossref PubMed Google Scholar). The complex I mutants were also more resistant than the wild type strain to inhibition of conidial germination by PHS (data not shown). To investigate whether the resistance to PHS is specific for complex I mutants, we tested the response to this sphingolipid in mutants that are defective in other components of the mitochondrial respiratory chain. In these experiments, we employed strain FGSC3558, which has a mutation in the cytochrome c structural gene, strain FGSC4506, which has a different mutation in cytochrome c but is also defective in complex IV, strain FGSC11184, which contains a deletion of the Rieske iron-sulfur subunit of complex III, strain FGSC8738, which contains a mutation in the dicyclohexylcarbodiimide-binding subunit of complex V and is defective in energy transduction (33Perkins D.D. Radford A. Sachs M.S. The Neurospora Compendium. Academic Press, London2001Google Scholar), plus the previous series of complex I mutants. Serial dilutions of conidial suspensions from each of these strains were spotted on media containing PHS, incubated at 26 °C, and the growth of the strains was qualitatively analyzed. Typical results are shown in Fig. 4. All strains grew similarly under control conditions (minimal medium) with the exception of the complex III defective mutant FGSC11184, which displayed reduced growth under all conditions. When PHS was included in the medium, the complex I mutants were more resistant to the drug than the wild type strain, as expected. In contrast, all of the other respiratory complex mutants were more sensitive to PHS than the wild type strain. These results point to a specific involvement of complex I in PHS-induced cell death. In addition to PHS, a number of other environmental stimuli induce apoptotic-like death in both filamentous fungi and unicellular yeasts. In particular, exposure to hydrogen peroxide has been reported to induce apoptotic-like death in Aspergillus fumigatus, Colletotrichum trifolii, C. albicans, and S. cerevisiae (34Phillips A.J. Sudbery I. Ramsdale M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14327-14332Crossref PubMed Scopus (334) Google Scholar, 35Mousavi S.A.A. Robson G.D. Microbiology (Read.). 2004; 150: 1937-1945Crossref PubMed Scopus (80) Google Scholar, 36Chen C. Dickman M.B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3459-3464Crossref PubMed Scopus (354) Google Scholar). We therefore evaluated whether mutations in the different respiratory complexes affected sensitivity of these mutants to hydrogen peroxide. We observed that complex I mutants showed a similar resistance to H202 exposure as they showed upon exposure to PHS (Fig. 4, see also below). However, strains containing mutations in other respiratory complexes (FGSC3558, cytochrome c mutant; FGSC4506, defective in complex IV; FGSC11184, defective in complex III; FGSC8738, defective in complex V) showed a similar sensitivity to PHS and H202 (Fig. 4). These data suggest that the response of N. crassa to PHS and hydrogen peroxide likely share common mechanisms. AIF and AMID are flavoproteins/oxidoreductases (similar to complex I) that promote caspase-independent apoptosis (37Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3434) Google Scholar, 38Wu M. Xu L.-G. Li X. Zhai Z. Shu H.-B. J. Biol. Chem. 2002; 277: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). In S. cerevisiae, mutations in the AIF homologue (AIF1) rescue cells from ROS stress and delays age-induced apoptosis (39Wissing S. Ludovico P. Herker E. Buttner S. Engelhardt S.M. Decker T. Link A. Proksch A. Rodrigues F. Corte-Real M. Frohlich K.-U. Manns J. Cande C. Sigrist S.J. Kroemer G. Madeo F. J. Cell Biol. 2004; 166: 969-974Crossref PubMed Scopus (321) Google Scholar). In N. crassa, there is one AIF-like homologue, NCU05850 (see supplemental Figs. S1 and S2), and two AMID-like predicted genes, NCU06016 and NCU11413, although NCU11413 is divergent. We therefore tested the sensitivity of deletion mutants in the AIF-like (FGSC11900) and an AMID-like (FGSC12090) predicted proteins of N. crassa toward both PHS and H2O2, using serial dilutions of conidia as outlined above. We observed that the N. crassa aif1 (FGSC11900) mutant was more resistant to exposure to both PHS and H202, whereas the mutant containing mutation in the predicted AMID homologue (FGSC12090) was more sensitive than the wild type strain to both drugs (Fig. 4). Complex I Mutants Generate Fewer ROS—As shown above, complex I mutants of N. crassa are more resistant to oxidative stress caused by hydrogen peroxide than the wild type strain (Fig. 4). We hypothesized that this resistance (and resistance to PHS) is related to the production of reactive oxygen species. ROS are involved in programmed cell death in many species, including filamentous fungi (2Skulachev V.P. Apoptosis. 2006; 11: 473-485Crossref PubMed Scopus (322) Google Scholar, 16Lu B.C.K. Kües U. Fischer R Growth, Differentiation and Sexuality. Springer, Berlin2006: 167-187Google Scholar). Exposure to hydrogen peroxide may elicit a “vicious cycle” of production of more ROS (40Zorov D.B. Filburn L.O. Klotz J.L. Zweier J.L. Slout S.J. J. Exp. Med. 2000; 192: 1001-1014Crossref PubMed Scopus (1105) Google Scholar, 41Zorov D.B. Juhaszova M. Sollott S.J. Biochim. Biophys. Acta. 2006; 1757: 509-517Crossref PubMed Scopus (828) Google Scholar). Thus, complex I mutants could produce more ROS than the wild type strain, resulting in up-regulation of defense mechanisms for scavenging ROS from the cell. In this case, they should be more resistant to exogenously applied drugs that induce ROS, such as PHS and H2O2. Alternatively, complex I mutants could be more resistant to exogenously added ROS because they produce fewer endogenous ROS than wild type. To discriminate between these two hypotheses, we measured by flow cytometry the production of ROS upon PHS treatment of wild type N. crassa versus the complex I mutant nuo14, using dihydrorhodamine 123 as a marker. Conidia from wild type and the nuo14 mutant were exposed to PHS, and the production of ROS was determined at different times (50–200 min) after drug treatment. This time course study showed a dramatic increase in ROS production in wild type cells treated with PHS, with a maximum production of ROS around 80 min (Fig. 5). More than half of the cells (about 60%) exhibited detectable production of ROS at this time point. In sharp contrast, ROS production of the complex I mutant nuo14 exposed to PHS under the same conditions was almost negligible. Only about 15% of the nuo14 cells displayed detectable ROS levels. We performed similar kinetics of ROS production with other complex I mutants. Fig. 6 shows the results obtained with these strains after an 80-min exposure to PHS. The results show that complex I mutants produced between ¼ and ⅓ of the ROS produced by the wild type strain under identical conditions. Thus, the inability to generate high levels of ROS upon exposure to PHS likely accounts for the increased resistance of complex I mutants toward both PHS and hydrogen peroxide. In this study, we evaluated whether the filamentous fungus N. crassa is a suitable model organism to investigate molecular mechanisms associated with PCD. Our results indicate that phytosphingosine exhibits a potent antifungal activity against Neurospora, as deduced by the dramatic decrease in viability of fungal cells upon treatment with the sphingolipid. We further observed that PHS induced rapid nuclear condensation, as revealed by 4′,6-diamidino-2-phenylindole dihydrocloride staining of DNA. Conidia with condensed nuclei did not take up propidium iodide (data not shown). This mitosis-independent nuclear condensation and propidium iodide exclusion are characteristic features of apoptosis in other eukaryotic cells (42Span L.F. Pennings A.H. Vierwinden G. Boezeman J.B. Raymakers R.A. de Witte T. Cytometry. 2002; 47: 24-31Crossref PubMed Scopus (77) Google Scholar). In addition, DNA fragmentation and ROS production are also hallmarks of apoptotic cells (16Lu B.C.K. Kües U. Fischer R Growth, Differentiation and Sexuality. Springer, Berlin2006: 167-187Google Scholar). Based on the TUNEL assay, we detected DNA fragmentation exclusively in N. crassa conidia exposed to PHS. Furthermore, we found that the sphingolipid stimulates the production of ROS by these cells. Altogether, these results indicate that PHS induces an apoptosis-like cell death in N. crassa, as shown previously in A. nidulans (23Cheng J. Park T.-S. Chio L.-C. Fischl A.S. Ye X.S. Mol. Cell. Biol. 2003; 23: 163-177Crossref PubMed Scopus (124) Google Scholar). Interestingly, we found that respiratory chain complex I mutants are less prone to die upon exposure to PHS than the wild type strain, clearly implicating mitochondria in the death process. The complex I involvement appears to be specific since mutants defective in other components of the oxidative phosphorylation system, such as complex III, IV, and V mutants, do not show a similar resistance to PHS (or hydrogen peroxide) as the complex I mutants. Although ATP is required for apoptosis (2Skulachev V.P. Apoptosis. 2006; 11:" @default.
• W2045742010 created "2016-06-24" @default.
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• W2045742010 creator A5032409837 @default.
• W2045742010 creator A5044849194 @default.
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• W2045742010 date "2008-07-01" @default.
• W2045742010 modified "2023-09-27" @default.
• W2045742010 title "Increased Resistance of Complex I Mutants to Phytosphingosine-induced Programmed Cell Death" @default.
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