Current Microbiology

, 63:100 | Cite as

Characterization of Neurospora crassa α-Actinin

  • Marta Cotado-Sampayo
  • Ruben Ortega Pérez
  • Mukti Ojha
  • Carole Seum
  • François Barja
Article
  • 134 Downloads

Abstract

α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crassa.

Notes

Acknowledgments

We gratefully acknowledge financial support from the Academic Society of Geneva (F. B.). Thanks are due to R. Strasser for interest in the project and encouragement, M.-L. Chappuis for technical assistance and A. Fehr for secretarial assistance.

References

  1. 1.
    Addis MF, Rappelli P, Delogu G, Carta F, Cappuccinelli P, Fiori PL (1998) Cloning and molecular characterization of a cDNA clone coding for Trichomonas vaginalis alpha-actinin and intracellular localization of the protein. Infect Immun 66:4924–4931PubMedGoogle Scholar
  2. 2.
    Araujo-Bazan L, Penalva MA, Espeso EA (2008) Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans. Mol Microbiol 67:891–905PubMedCrossRefGoogle Scholar
  3. 3.
    Barja F, Chappuis ML, Turian G (1993) Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa. FEMS Microbiol Lett 107:261–266PubMedCrossRefGoogle Scholar
  4. 4.
    Berepiki A, Lichius A, Shoji JY, Tilsner J, Read ND (2010) F-actin dynamics in Neurospora crassa. Eukaryot Cell 9:547–557PubMedCrossRefGoogle Scholar
  5. 5.
    Broderick MJ, Winder SJ (2005) Spectrin, α-actinin, and dystrophin. Adv Protein Chem 70:203–246PubMedCrossRefGoogle Scholar
  6. 6.
    Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, Weiss RL, Borkovich KA, Dunlap JC (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci 103:10352–10357PubMedCrossRefGoogle Scholar
  7. 7.
    Cotado-Sampayo M, Ojha M, Ortega Perez R, Chappuis ML, Barja F (2006) Proteolytic cleavage of a spectrin-related protein by calcium-dependent protease in Neurospora crassa. Curr Microbiol 53:311–316PubMedCrossRefGoogle Scholar
  8. 8.
    Cotado-Sampayo M, Okenve Ramos P, Ortega Perez R, Ojha M, Barja F (2008) Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: cross-reaction with proteins other than spectrin. Fungal Genet Biol 45:1008–1015PubMedCrossRefGoogle Scholar
  9. 9.
    Davis RH, De Serres FJ (1970) Genetic and microbial research techniques for Neurospora crassa. Methods Enzymol 17:79–143CrossRefGoogle Scholar
  10. 10.
    Delgado-Álvarez DL, Callejas-Negrete OA, Gómez N, Freitag M, Roberson RW, Smith LG, Mouriño-Pérez RR (2010) Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa. Fungal Genet Biol 47:573–586PubMedCrossRefGoogle Scholar
  11. 11.
    Dominguez R (2004) Actin-binding proteins: a unifying hypothesis. Trends Biochem Sci 29:572–578PubMedCrossRefGoogle Scholar
  12. 12.
    Freitag M, Hickey PC, Raju NB, Selker EU, Read ND (2004) GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genet Biol 41:897–910PubMedCrossRefGoogle Scholar
  13. 13.
    Harris SD, Hamer L, Sharpless KE, Hamer JE (1997) The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity. EMBO J 16:3474–3483PubMedCrossRefGoogle Scholar
  14. 14.
    Heath IB, Gupta G, Bai S (2000) Plasma membrane-adjacent actin filaments, but not microtubules, are essential for both polarization and hyphal tip morphogenesis in Saprolegnia ferax and Neurospora crassa. Fungal Genet Biol 30:45–62PubMedCrossRefGoogle Scholar
  15. 15.
    Lee SH, Dominguez R (2010) Regulation of actin cytoskeleton dynamics in cells. Mol Cells 29:311–325PubMedCrossRefGoogle Scholar
  16. 16.
    Lek M, North KN (2010) Are biological sensors modulated by their structural scaffolds? The role of the structural muscle proteins alpha-actinin-2 and alpha-actinin-3 as modulators of biological sensors. FEBS Lett 584:2974–2980PubMedCrossRefGoogle Scholar
  17. 17.
    McGoldrick CA, Gruver C, May CS (1995) myoA of Aspergillus nidulans encodes myosin I required for secretion and polarized growth. J Cell Biol 128:577–587PubMedCrossRefGoogle Scholar
  18. 18.
    Otey CA, Carpen O (2004) Alpha-actinin revisited: a fresh look at an old player. Cell Motil Cytoskeleton 58:104–111PubMedCrossRefGoogle Scholar
  19. 19.
    Rasmussen CG, Glass NL (2007) Localization of RHO-4 indicates differential regulation of conidial versus vegetative septation in the filamentous fungus Neurospora crassa. Eukaryot Cell 6:1097–1107PubMedCrossRefGoogle Scholar
  20. 20.
    Riquelme M, Reynaga-Pena CG, Gierz G, Bartnicki-Garcia S (1998) What determines growth direction in fungal hyphae? Fungal Genet Biol 24:101–109PubMedCrossRefGoogle Scholar
  21. 21.
    Rivero F, Furukawa R, Fechheimer M, Noegel AA (1999) Three actin cross-linking proteins, the 34 kDa actin-bundling protein, α-actinin and gelation factor (ABP-120), have both unique and redundant roles in the growth and development of Dictyostelium. J Cell Sci 112:2737–2751PubMedGoogle Scholar
  22. 22.
    Sharpless KE, Harris SD (2002) Functional characterization and localization of the Aspergillus nidulans formin SEPA. Mol Biol Cell 13:469–479PubMedCrossRefGoogle Scholar
  23. 23.
    Sjöblom B, Salmazo A, Djinović-Carugo K (2008) Alpha-actinin structure and regulation. Cell Mol Life Sci 65:2688–2701PubMedCrossRefGoogle Scholar
  24. 24.
    Taheri-Talesh N, Horio T, Araujo-Bazan L, Dou X, Espeso EA, Peñalva MA, Osmani AS, Oakley BR (2008) The tip growth apparatus of Aspergillus nidulans. Mol Biol Cell 19:1439–1449PubMedCrossRefGoogle Scholar
  25. 25.
    Upadhyay S, Shaw BD (2008) The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans. Mol Microbiol 68:609–705CrossRefGoogle Scholar
  26. 26.
    Virag A, Griffiths AJ (2004) A mutation in the Neurospora crassa actin gene results in multiple defects in tip growth and branching. Fungal Genet Biol 41:213–225PubMedCrossRefGoogle Scholar
  27. 27.
    Virag A, Harris SD (2006) Functional characterization of Aspergillus nidulans homologues of Saccharomyces cerevisiae Spa2 and Bud6. Eukaryot Cell 5:881–895PubMedCrossRefGoogle Scholar
  28. 28.
    Virel A, Backman L (2004) Molecular evolution and structure of α-actinin. Mol Biol Evol 21:1024–1031PubMedCrossRefGoogle Scholar
  29. 29.
    Virel A, Backman L (2006) Characterization of Entamoeba histolytica alpha-actinin. Mol Biochem Parasitol 145:11–17PubMedCrossRefGoogle Scholar
  30. 30.
    Virel A, Backman L (2007) A comparative and phylogenetic analysis of the α-actinin rod domain. Mol Biol Evol 10:2254–2265CrossRefGoogle Scholar
  31. 31.
    Virel A, Addario B, Backman L (2007) Characterization of Entamoeba histolytica alpha-actinin 2. Mol Biochem Parasitol 154:82–89PubMedCrossRefGoogle Scholar
  32. 32.
    Vogel HJ (1956) A convenient growth medium for Neurospora (medium N). Microbiol Genet Bull 13:42–43Google Scholar
  33. 33.
    Wang J, Hu H, Wang S, Shi J, Chen S, Wei H, Xu X, Lu L (2009) The important role of Actinin-like protein (AcnA) in cytokinesis and apical dominance of hyphal cells in Aspergillus nidulans. Microbiology 155:2714–2725PubMedCrossRefGoogle Scholar
  34. 34.
    Wu JQ, Bahler J, Pringle JR (2001) Roles of a fimbrin and an α-actinin like protein in fission yeast cell polarization and cytokinesis. Mol Biol Cell 12:1061–1077PubMedGoogle Scholar
  35. 35.
    Yamashita RA, Osherov N, May GS (2000) Localization of wild type and mutant class I myosin proteins in Aspergillus nidulans using GFP-fusion proteins. Cell Motil Cytoskeleton 45:163–172PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Marta Cotado-Sampayo
    • 1
  • Ruben Ortega Pérez
    • 1
  • Mukti Ojha
    • 2
  • Carole Seum
    • 2
  • François Barja
    • 1
  1. 1.Department of Botany and Plant BiologyUniversity of GenevaJussy, GenevaSwitzerland
  2. 2.Department of BiochemistryUniversity of GenevaGeneva 4Switzerland

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