Programmed cell death has been studied for decades in mammalian cells, but simpler organisms, including prokaryotes, plants, and fungi, also undergo regulated forms of cell death. We highlight the usefulness of the filamentous fungus Neurospora crassa as a model organism for the study of programmed cell death. In N. crassa, cell death can be triggered genetically due to hyphal fusion between individuals with different allelic specificities at het loci, in a process called “heterokaryon incompatibility.” Chemical induction of cell death can also be achieved upon exposure to death-inducing agents like staurosporine, phytosphingosine, or hydrogen peroxide. A summary of the recent advances made by our and other groups on the discovery of the mechanisms and mediators underlying the process of cell death in N. crassa is presented. 1. Neurospora crassa as a Model Organism Neurospora crassa is a nonpathogenic filamentous fungus, very easy to maintain, grow, and manipulate. N. crassa enjoys modest nutritional requirements: the common minimal medium (Vogel’s minimal medium) includes a sugar, a nitrogen source (ammonium and nitrate), phosphate, sulfate, potassium, magnesium, calcium, trace metals, and a small amount of the vitamin biotin [1]. Moreover, N. crassa is one of the fastest growing filamentous fungi (approximately 10？cm per day under optimal conditions), justifying its appearance among the first colonizers of recently burned vegetation [2]. It is prone to genetic experiments like the induction of mutations, genes, and mutants isolation, microscopic analysis, biochemical testing, and so on. Thus, Neurospora presents some features that turn it into a very attractive option to be used in the laboratory. N. crassa is a multicellular ascomycete. It was initially documented in 1843, when several Parisian bakeries were infested by cultures of an orange sporulating mould [3]. A century later, mycologists Cornelius Shear and Bernard Dodge moved it to the Neurospora genus, based on the discovery that this fungus possesses a sexual morphological structure called perithecia [4]. Literally translated, “Neurospora” means “nerve” plus “spore” and the explanation for this name resides in the fact that the fungal spores display longitudinal striations resembling animal axons which belong to the nervous system. In its natural habitat, Neurospora is found essentially in tropical and subtropical regions but also in temperate climates [2]. Figure 1 shows spots of N. crassa colonization that can be easily observed following a forest fire. During the 20th century, this fungus
References
[1]
H. J. Vogel, “A convenient growth medium for Neurospora (medium N),” Microbial Genetics Bulletin, vol. 13, pp. 42–43, 1956.
[2]
D. J. Jacobson, J. R. Dettman, R. I. Adams et al., “New findings of Neurospora in Europe and comparisons of diversity in temperate climates on continental scales,” Mycologia, vol. 98, no. 4, pp. 550–559, 2006.
[3]
A. Payen, “Extrain d'un rapport addressé à M. Le Maréchal Duc de Dalmatie, Ministre de la Guerre, Président du Conseil, sur une altération extraordinaire du pain de munition,” Annales de Chimie et de Physique, vol. 9, pp. 5–21, 1843.
[4]
C. L. Shear and B. O. Dodge, “Life histories and heterothallism of the red bread-mold fungi of the Monilia sitophila group,” Journal of Agricultural Research, vol. 34, pp. 1019–1042, 1927.
[5]
G. W. Beadle and E. L. Tatum, “Genetic control of biochemical reactions in Neurospora,” Proceedings of the National Academy of Sciences of the United States of America, vol. 27, no. 11, pp. 499–506, 1941.
[6]
A. M. Srb and N. H. Horowitz, “The ornithine cycle in Neurospora and its genetic control,” The Journal of Biological Chemistry, vol. 154, no. 1, pp. 129–139, 1944.
[7]
R. H. Davis and D. D. Perkins, “Neurospora: a model of model microbes,” Nature Reviews Genetics, vol. 3, no. 5, pp. 397–403, 2002.
[8]
J. E. Galagan, S. E. Calvo, K. A. Borkovich et al., “The genome sequence of the filamentous fungus Neurospora crassa,” Nature, vol. 422, no. 6934, pp. 859–868, 2003.
[9]
K. McCluskey, A. Wiest, and M. Plamann, “The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research,” Journal of Biosciences, vol. 35, no. 1, pp. 119–126, 2010.
[10]
A. Videira, “Complex I from the fungus Neurospora crassa,” Biochimica et Biophysica Acta, vol. 1364, no. 2, pp. 89–100, 1998.
[11]
A. Videira and M. Duarte, “On complex I and other NADH: ubiquinone reductases of Neurospora crassa mitochondria,” Journal of Bioenergetics and Biomembranes, vol. 33, no. 3, pp. 197–203, 2001.
[12]
A. Videira and M. Duarte, “From NADH to ubiquinone in Neurospora mitochondria,” Biochimica et Biophysica Acta, vol. 1555, no. 1–3, pp. 187–191, 2002.
[13]
P. Carneiro, M. Duarte, and A. Videira, “Characterization of apoptosis-related oxidoreductases from Neurospora crassa,” PLoS ONE, vol. 7, no. 3, Article ID e34270, 2012.
[14]
P. Carneiro, M. Duarte, and A. Videira, “Disruption of alternative NAD(P)H dehydrogenases leads to decreased mitochondrial ROS in Neurospora crassa,” Free Radical Biology and Medicine, vol. 52, no. 2, pp. 402–409, 2012.
[15]
A. Castro, C. Lemos, A. Falc？o, A. S. Fernandes, N. Louise Glass, and A. Videira, “Rotenone enhances the antifungal properties of staurosporine,” Eukaryotic Cell, vol. 9, no. 6, pp. 906–914, 2010.
[16]
A. Castro, C. Lemos, A. Falc？o, N. L. Glass, and A. Videira, “Increased resistance of complex I mutants to phytosphingosine-induced programmed cell death,” The Journal of Biological Chemistry, vol. 283, no. 28, pp. 19314–19321, 2008.
[17]
A. S. Fernandes, A. Castro, and A. Videira, “Reduced glutathione export during programmed cell death of Neurospora crassa,” Apoptosis, vol. 18, no. 8, pp. 940–948, 2013.
[18]
A. S. Fernandes, A. P. Gon？alves, A. Castro et al., “Modulation of fungal sensitivity to staurosporine by targeting proteins identified by transcriptional profiling,” Fungal Genetics and Biology, vol. 48, no. 12, pp. 1130–1138, 2011.
[19]
A. P. Gon？alves, V. Máximo, J. Lima, K. K. Singh, P. Soares, and A. Videira, “Involvement of p53 in cell death following cell cycle arrest and mitotic catastrophe induced by rotenone,” Biochimica et Biophysica Acta, vol. 1813, no. 3, pp. 492–499, 2011.
[20]
A. P. Gon？alves, A. Videira, V. Máximo, and P. Soares, “Synergistic growth inhibition of cancer cells harboring the RET/PTC1 oncogene by staurosporine and rotenone involves enhanced cell death,” Journal of Biosciences, vol. 36, no. 4, pp. 639–648, 2011.
[21]
A. P. Gon？alves, A. Videira, P. Soares, and V. Máximo, “Orthovanadate-induced cell death in RET/PTC1-harboring cancer cells involves the activation of caspases and altered signaling through PI3K/Akt/mTOR,” Life Sciences, vol. 89, no. 11-12, pp. 371–377, 2011.
[22]
A. Videira, T. Kasuga, C. Tian, C. Lemos, A. Castro, and N. L. Glass, “Transcriptional analysis of the response of Neurospora crassa to phytosphingosine reveals links to mitochondrial function,” Microbiology, vol. 155, no. 9, pp. 3134–3141, 2009.
[23]
Y. Fuchs and H. Steller, “Programmed cell death in animal development and disease,” Cell, vol. 147, no. 4, pp. 742–758, 2011.
[24]
L. Galluzzi, I. Vitale, J. M. Abrams et al., “Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012,” Cell Death and Differentiation, vol. 19, no. 1, pp. 107–120, 2012.
[25]
N. D. Fedorova, J. H. Badger, G. D. Robson, J. R. Wortman, and W. C. Nierman, “Comparative analysis of programmed cell death pathways in filamentous fungi,” BMC Genomics, vol. 6, article 177, 2005.
[26]
B. S. Strauss, “Cell death and ‘unbalanced growth’ in Neurospora,” Microbiology, vol. 18, no. 3, pp. 658–669, 1958.
[27]
N. S. Plesofsky, S. B. Levery, S. A. Castle, and R. Brambl, “Stress-induced cell death is mediated by ceramide synthesis in Neurospora crassa,” Eukaryotic Cell, vol. 7, no. 12, pp. 2147–2159, 2008.
[28]
J. Palma-Guerrero, I.-C. Huang, H.-B. Jansson, J. Salinas, L. V. Lopez-Llorca, and N. D. Read, “Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner,” Fungal Genetics and Biology, vol. 46, no. 8, pp. 585–594, 2009.
[29]
A. Munoz, J. F. Marcos, and N. D. Read, “Concentration-dependent mechanisms of cell penetration and killing by the de novo designed antifungal hexapeptide PAF26,” Molecular Microbiology, vol. 85, no. 1, pp. 89–106, 2012.
[30]
N. L. Glass and I. Kaneko, “Fatal attraction: nonself recognition and heterokaryon incompatibility in filamentous fungi,” Eukaryotic Cell, vol. 2, no. 1, pp. 1–8, 2003.
[31]
N. L. Glass and K. Dementhon, “Non-self recognition and programmed cell death in filamentous fungi,” Current Opinion in Microbiology, vol. 9, no. 6, pp. 553–558, 2006.
[32]
K. Dementhon, G. Iyer, and N. L. Glass, “VIB-1 is required for expression of genes necessary for programmed cell death in Neurospora crassa,” Eukaryotic Cell, vol. 5, no. 12, pp. 2161–2173, 2006.
[33]
E. A. Hutchison, J. A. Bueche, and N. L. Glass, “Diversification of a protein kinase cascade: IME-2 is involved in nonself recognition and programmed cell death in Neurospora crassa,” Genetics, vol. 192, no. 2, pp. 467–482, 2012.
[34]
E. Hutchison, S. Brown, C. Tian, and N. L. Glass, “Transcriptional profiling and functional analysis of heterokaryon incompatibility in Neurospora crassa reveals that reactive oxygen species, but not metacaspases, are associated with programmed cell death,” Microbiology, vol. 155, no. 12, pp. 3957–3970, 2009.
[35]
G. Wichmann, J. Sun, K. Dementhon, N. L. Glass, and S. E. Lindow, “A novel gene, phcA from Pseudomonas syringae induces programmed cell death in the filamentous fungus Neurospora crassa,” Molecular Microbiology, vol. 68, no. 3, pp. 672–689, 2008.
[36]
S. M. Marek, J. Wu, N. L. Glass, D. G. Gilchrist, and R. M. Bostock, “Nuclear DNA degradation during heterokaryon incompatibility in Neurospora crassa,” Fungal Genetics and Biology, vol. 40, no. 2, pp. 126–137, 2003.
[37]
A. Morales, H. Lee, F. M. Go？i, R. Kolesnick, and J. C. Fernandez-Checa, “Sphingolipids and cell death,” Apoptosis, vol. 12, no. 5, pp. 923–939, 2007.
[38]
J. Cheng, T.-S. Park, L.-C. Chio, A. S. Fischl, and X. S. Ye, “Induction of apoptosis by sphingoid long-chain bases in Aspergillus nidulans,” Molecular and Cellular Biology, vol. 23, no. 1, pp. 163–177, 2003.
[39]
L. Li, W. Gu, C. Liang, Q. Liu, C. C. Mello, and Y. Liu, “The translin-TRAX complex (C3PO) is a ribonuclease in tRNA processing,” Nature Structural & Molecular Biology, vol. 19, no. 8, pp. 824–830, 2012.
[40]
G. M. Jenkins, A. Richards, T. Wahl, C. Mao, L. Obeid, and Y. Hannun, “Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae,” The Journal of Biological Chemistry, vol. 272, no. 51, pp. 32566–32572, 1997.
[41]
M. Yamagata, K. Obara, and A. Kihara, “Unperverted synthesis of complex sphingolipids is essential for cell survival under nitrogen starvation,” Genes to Cells, vol. 18, no. 8, pp. 650–659, 2013.
[42]
M. M. Nagiec, E. E. Nagiec, J. A. Baltisberger, G. B. Wells, R. L. Lester, and R. C. Dickson, “Sphingolipid synthesis as a target for antifungal drugs: complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene,” The Journal of Biological Chemistry, vol. 272, no. 15, pp. 9809–9817, 1997.
[43]
M. S. Skrzypek, M. M. Nagiec, R. L. Lester, and R. C. Dickson, “Inhibition of amino acid transport by sphingoid long chain bases in Saccharomyces cerevisiae,” The Journal of Biological Chemistry, vol. 273, no. 5, pp. 2829–2834, 1998.
[44]
S. Omura, Y. Iwai, A. Hirano et al., “A new alkaloid AM-2282 of Streptomyces origin taxonomy, fermentation, isolation and preliminary characterization,” The Journal of Antibiotics, vol. 30, no. 4, pp. 275–282, 1977.
[45]
M. W. Karaman, S. Herrgard, D. K. Treiber et al., “A quantitative analysis of kinase inhibitor selectivity,” Nature Biotechnology, vol. 26, no. 1, pp. 127–132, 2008.
[46]
S. Yoshida, E. Ikeda, I. Uno, and H. Mitsuzawa, “Characterization of a staurosporine- and temperature-sensitive mutant, STT1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1,” Molecular and General Genetics, vol. 231, no. 3, pp. 337–344, 1992.
[47]
O. A. Gani and R. A. Engh, “Protein kinase inhibition of clinically important staurosporine analogues,” Natural Product Reports, vol. 27, no. 4, pp. 489–498, 2010.
[48]
M. B. Reeves, A. A. Davies, B. P. McSharry, G. W. Wilkinson, and J. H. Sinclair, “Complex I binding by a virally encoded RNA regulates mitochondria-induced cell death,” Science, vol. 316, no. 5829, pp. 1345–1348, 2007.
[49]
G. Huang, Y. Chen, H. Lu, and X. Cao, “Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-β and retinoic acid-induced cancer cell death,” Cell Death and Differentiation, vol. 14, no. 2, pp. 327–337, 2007.
[50]
J.-E. Ricci, C. Mu？oz-Pinedo, P. Fitzgerald et al., “Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain,” Cell, vol. 117, no. 6, pp. 773–786, 2004.
[51]
D. Martinvalet, D. M. Dykxhoorn, R. Ferrini, and J. Lieberman, “Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death,” Cell, vol. 133, no. 4, pp. 681–692, 2008.
[52]
J. S. Ladha, M. K. Tripathy, and D. Mitra, “Mitochondrial complex I activity is impaired during HIV-1-induced T-cell apoptosis,” Cell Death and Differentiation, vol. 12, no. 11, pp. 1417–1428, 2005.
[53]
A. Breier, M. Baran？ík, Z. Sulová, and B. Uhrík, “P-glycoprotein—implications of metabolism of neoplastic cells and cancer therapy,” Current Cancer Drug Targets, vol. 5, no. 6, pp. 457–468, 2005.
[54]
S. Yoshida and Y. Anraku, “Characterization of staurosporine-sensitive mutants of Saccharomyces cerevisiae: vacuolar functions affect staurosporine sensitivity,” Molecular and General Genetics, vol. 263, no. 5, pp. 877–888, 2000.
[55]
S. Yoshida, Y. Ohya, A. Nakano, and Y. Anraku, “Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae,” Molecular and General Genetics, vol. 242, no. 6, pp. 631–640, 1994.
[56]
S. Yoshida, Y. Ohya, M. Goebl, A. Nakano, and Y. Anraku, “A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae,” The Journal of Biological Chemistry, vol. 269, no. 2, pp. 1166–1172, 1994.
[57]
Y. Zhang, S. Muend, and R. Rao, “Dysregulation of ion homeostasis by antifungal agents,” Frontiers in Microbiology, vol. 3, article 133, 2012.
[58]
W. Li, L. Sun, Q. Liang, J. Wang, W. Mo, and B. Zhou, “Yeast AMID homologue Ndi1p displays respiration-restricted apoptotic activity and is involved in chronological aging,” Molecular Biology of the Cell, vol. 17, no. 4, pp. 1802–1811, 2006.
