Advertisement

Quantitative Analyses Using Video Bioinformatics and Image Analysis Tools During Growth and Development in the Multicellular Fungus Neurospora crassa

  • Ilva E. Cabrera
  • Asongu L. Tambo
  • Alberto C. Cruz
  • Benjamin X. Guan
  • Bir Bhanu
  • Katherine A. BorkovichEmail author
Chapter
Part of the Computational Biology book series (COBO, volume 22)

Abstract

Neurospora crassa (Neurospora) is a nonpathogenic multicellular fungus. Neurospora has many attributes that make it an ideal model organism for cell biology and genetic studies, including a sequenced genome, a predominantly haploid life cycle and the availability of knock-out mutants for the ~10,000 genes. Neurospora grows by polar extension of tube-like structures called hyphae. Neurospora has a complex life cycle, with two asexual sporulation pathways and a sexual cycle that produces meiotic progeny. This study analyzes stages during the formation of a colony, from asexual spore to mature hyphae with the use of video bioinformatics and image analysis tools. We are the first to analyze the asexual spore size, hyphal compartment size and hyphal growth rate in an automated manner, using video and image analysis algorithms. Quantitative results were obtained for all three phenotypic assays. This novel approach employs phenotypic parameters that can be utilized for streamlined analysis of thousands of mutants. This software, to be made publicly available in the future, eliminates subjectivity, and allows high-throughput analysis in a time saving manner.

Keywords

Filamentous Fungus Human Visual System Neurospora Crassa Phase Objective Pavement Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We thank Jhon Gonzalez for his insightful conversations and comments on this work. We also thank Alexander Carrillo and Caleb Hubbard for assistance in determining the optimal conditions for capture of microscopic images. I.E.C., A.L.T, A.C.C., and B.X.G. were supported by NSF IGERT Video Bioinformatics Grant DGE 0903667.

Supplementary material

Supplementary material 1 (AVI 44120 kb)

References

  1. 1.
    Agrios GN (1997) Plant Pathology, 4th edn. Academic Press, San DiegoGoogle Scholar
  2. 2.
    Latge JP (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12:310–350Google Scholar
  3. 3.
    Perkins DD (1992) Neurospora: the organism behind the molecular revolution. Genetics 130:687–701Google Scholar
  4. 4.
    Davis RH, Perkins DD (2002) Neurospora: a model of model microbes. Nat Rev Genet 3:397–403CrossRefGoogle Scholar
  5. 5.
    Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B, Elkins T, Engels R, Wang S, Nielsen CB, Butler J, Endrizzi M, Qui D, Ianakiev P, Bell-Pedersen D, Nelson MA, Werner-Washburne M, Selitrennikoff CP, Kinsey JA, Braun EL, Zelter A, Schulte U, Kothe GO, 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 RL, Perkins DD, Kroken S, Cogoni C, Macino G, Catcheside D, Li W, Pratt RJ, Osmani SA, DeSouza CP, Glass L, Orbach MJ, Berglund JA, Voelker R, Yarden O, Plamann M, Seiler S, Dunlap J, Radford A, Aramayo R, Natvig DO, Alex LA, Mannhaupt G, Ebbole DJ, Freitag M, Paulsen I, Sachs MS, Lander ES, Nusbaum C, Birren B (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859–868CrossRefGoogle Scholar
  6. 6.
    Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, Read ND, Seiler S, Bell-Pedersen D, Paietta J, Plesofsky N, Plamann M, Goodrich-Tanrikulu M, Schulte U, Mannhaupt G, Nargang FE, Radford A, Selitrennikoff C, Galagan JE, Dunlap JC, Loros JJ, Catcheside D, Inoue H, Aramayo R, Polymenis M, Selker EU, Sachs MS, Marzluf GA, Paulsen I, Davis R, Ebbole DJ, Zelter A, Kalkman ER, O’Rourke R, Bowring F, Yeadon J, Ishii C, Suzuki K, Sakai W, Pratt R (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68:1–108CrossRefGoogle Scholar
  7. 7.
    Riquelme M, Yarden O, Bartnicki-Garcia S, Bowman B, Castro-Longoria E, Free SJ, Fleissner A, Freitag M, Lew RR, Mourino-Perez R, Plamann M, Rasmussen C, Richthammer C, Roberson RW, Sanchez-Leon E, Seiler S, Watters MK (2011) Architecture and development of the Neurospora crassa hypha—a model cell for polarized growth. Fungal Biol 115:446–474CrossRefGoogle Scholar
  8. 8.
    Bruno KS, Aramayo R, Minke PF, Metzenberg RL, Plamann M (1996) Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase. EMBO J 15:5772–5782Google Scholar
  9. 9.
    Fleissner A, Simonin AR, Glass NL (2008) Cell fusion in the filamentous fungus, Neurospora crassa. Methods Mol Biol 475:21–38CrossRefGoogle Scholar
  10. 10.
    Kolmark G, Westergaard M (1949) Induced back-mutations in a specific gene of Neurospora crassa. Hereditas 35:490–506CrossRefGoogle Scholar
  11. 11.
    Barratt RW, Garnjobst L (1949) Genetics of a colonial microconidiating mutant strain of Neurospora crassa. Genetics 34:351–369Google Scholar
  12. 12.
    Perkins DD (1996) Details for collection of asci as unordered groups of eight projected ascospores. Neurospora Newsl. 9:11Google Scholar
  13. 13.
    Lindegren CC (1932) The genetics of Neurospora I. The inheritance of response to heat-treatment. Bull Torrey Bot Club:85–102Google Scholar
  14. 14.
    Horowitz NH (1991) Fifty years ago: the Neurospora revolution. Genetics 127:631–635Google Scholar
  15. 15.
    Perkins DD, Davis RH (2000) Neurospora at the millennium. Fungal Genet Biol 31:153–167CrossRefGoogle Scholar
  16. 16.
    Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 101:12248–12253CrossRefGoogle Scholar
  17. 17.
    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 USA 103:10352–10357CrossRefGoogle Scholar
  18. 18.
    Nichols CB, Fraser JA, Heitman J (2004) PAK kinases Ste20 and Pak1 govern cell polarity at different stages of mating in Cryptococcus neoformans. Mol Biol Cell 15:4476–4489CrossRefGoogle Scholar
  19. 19.
    Castillo-Lluva S, Alvarez-Tabares I, Weber I, Steinberg G, Perez-Martin J (2007) Sustained cell polarity and virulence in the phytopathogenic fungus Ustilago maydis depends on an essential cyclin-dependent kinase from the Cdk5/Pho85 family. J Cell Sci 120:1584–1595CrossRefGoogle Scholar
  20. 20.
    Harris SD, Read ND, Roberson RW, Shaw B, Seiler S, Plamann M, Momany M (2005) Polarisome meets spitzenkorper: microscopy, genetics, and genomics converge. Eukaryot Cell 4:225–229CrossRefGoogle Scholar
  21. 21.
    Bartnicki-Garcia S, Bartnicki DD, Gierz G, Lopez-Franco R, Bracker CE (1995) Evidence that Spitzenkorper behavior determines the shape of a fungal hypha: a test of the hyphoid model. Exp Mycol 19:153–159CrossRefGoogle Scholar
  22. 22.
    Reynaga-Pena CG, Gierz G, Bartnicki-Garcia S (1997) Analysis of the role of the Spitzenkorper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger. Proc Natl Acad Sci USA 94:9096–9101CrossRefGoogle Scholar
  23. 23.
    Riquelme M, Reynaga-Pena CG, Gierz G, Bartnicki-Garcia S (1998) What determines growth direction in fungal hyphae? Fungal Genet Biol 24:101–109CrossRefGoogle Scholar
  24. 24.
    Konzack S, Rischitor PE, Enke C, Fischer R (2005) The role of the kinesin motor KipA in microtubule organization and polarized growth of Aspergillus nidulans. Mol Biol Cell 16:497–506CrossRefGoogle Scholar
  25. 25.
    Takeshita N, Higashitsuji Y, Konzack S, Fischer R (2008) Apical sterol-rich membranes are essential for localizing cell end markers that determine growth directionality in the filamentous fungus Aspergillus nidulans. Mol Biol Cell 19:339–351CrossRefGoogle Scholar
  26. 26.
    Harlow GJ, Cruz AC, Li S, Bhanu B, Yang Z (2013) Pillars of plant cell polarity: 3-D automated microtubule order & asymmetric cell pattern analysis. NSF IGERT Video and Poster Competition, Washington, DCGoogle Scholar
  27. 27.
    Vogel HJ (1964) Distribution of lysine pathways among fungi: evolutionary implications. Am Nat 98:435–446CrossRefGoogle Scholar
  28. 28.
    Elorza MV, Rico H, Sentandreu R (1983) Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. J Gen Microbiol 129:1577–1582Google Scholar
  29. 29.
    Hickey PC, Swift SR, Roca MG, Read ND (2004) Live-cell Imaging of filamentous fungi using vital fluorescent dyes and confocal microscopy. Meth Microbiol 34:63–87CrossRefGoogle Scholar
  30. 30.
    (2013) Excel. Microsoft, Redmond, WashingtonGoogle Scholar
  31. 31.
    Gopinath S, Wen Q, Thakoor N, Luby-Phelps K, Gao JX (2008) A statistical approach for intensity loss compensation of confocal microscopy images. J Microsc 230:143–159CrossRefMathSciNetGoogle Scholar
  32. 32.
    Pound MP, French AP, Wells DM, Bennett MJ, Pridmore TP (2012) CellSeT: novel software to extract and analyze structured networks of plant cells from confocal images. Plant Cell 24:1353–1361CrossRefGoogle Scholar
  33. 33.
    Su H, Yin Z, Huh S, Kanade T (2013) Cell segmentation in phase contrast microscopy images via semi-supervised classification over optics-related features. Med Image Anal 17:746–765CrossRefGoogle Scholar
  34. 34.
    Grigorescu C, Petkov N, Westenberg MA (2003) Contour detection based on nonclassical receptive field inhibition. IEEE Trans Image Process 12:729–739CrossRefGoogle Scholar
  35. 35.
    Ryan FJ, Beadle GW, Tatum EL (1943) The tube method of measuring the growth rate of Neurospora. Amer J Bot 30:784–799CrossRefGoogle Scholar
  36. 36.
    (2010) MATLAB software .7.10.0.584 Natick, MAGoogle Scholar
  37. 37.
    Li C, Xu C, Gui C, Fox MD (2010) Distance regularized level set evolution and its application to image segmentation. IEEE Trans Image Process 19:3243–3254CrossRefMathSciNetGoogle Scholar
  38. 38.
    Lopez-Franco R, Bartnicki-Garcia S, Bracker CE (1994) Pulsed growth of fungal hyphal tips. Proc Natl Acad Sci USA 91:12228–12232CrossRefGoogle Scholar
  39. 39.
    Springer ML, Yanofsky C (1989) A morphological and genetic analysis of conidiophore development in Neurospora crassa. Genes Dev 3:559–571CrossRefGoogle Scholar
  40. 40.
    Davis RH, deSerres FJ (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol 71A:79–143CrossRefGoogle Scholar
  41. 41.
    Davis R (2000) Neurospora: contributions of a model organism. Oxford University Press, New YorkGoogle Scholar
  42. 42.
    Mehrotra KN, Namuduri KR, Ranganathan N (1992) Gabor filter-based edge detection. Pattern Recognit 25:1479–1492CrossRefGoogle Scholar
  43. 43.
    Otsu N (1979) A threshold selection method from gray-level histograms. IEEE Trans Syst Man Cybern SMC-9: 62–66Google Scholar
  44. 44.
    Haralick RM, Shapiro LG (1991) Computer and robot vision. Addison-Wesley Longman Publishing Co. Inc., BostonGoogle Scholar
  45. 45.
    van den Boomgard R, van Balen R (1992) Methods for fast morphological image transforms using bitmapped binary images. CVGIP: Graph Models Image Process 54:252–258Google Scholar
  46. 46.
    Soille P (1999) Morphological image analysis: principles and applications. Springer, BerlinGoogle Scholar
  47. 47.
    Li ZH, Huang FG, Liu YM (2006) A method of motion segmentation based on region shrinking. In: Proceedings of intelligent data engineering and automated learning—Ideal 2006, vol 4224, pp 275–282Google Scholar
  48. 48.
    Torralba S, Raudaskoski M, Pedregosa AM, Laborda F (1998) Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans. Microbiology 144(Pt 1):45–53CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Ilva E. Cabrera
    • 1
  • Asongu L. Tambo
    • 2
  • Alberto C. Cruz
    • 2
  • Benjamin X. Guan
    • 2
  • Bir Bhanu
    • 2
  • Katherine A. Borkovich
    • 1
    Email author
  1. 1.Department of Plant Pathology and MicrobiologyUniversity of CaliforniaRiversideUSA
  2. 2.Center for Research in Intelligent SystemsUniversity of CaliforniaRiversideUSA

Personalised recommendations