1887

Abstract

Creating and maintaining cell polarity are complex processes that are not fully understood. Fungal hyphal tip growth is a highly polarized and dynamic process involving both F-actin and microtubules (MTs), but the behaviour and roles of the latter are unclear. To address this issue, MT dynamics and subunit distribution were analysed in a strain of expressing GFP–-tubulin. Apical MTs are the most dynamic, the bulk of which move tipwards from multiple subapical spindle pole bodies, the only clear region of microtubule nucleation detected. MTs populate the apex predominantly by elongation at rates about three times faster than tip extension. This polymerization was facilitated by the tipward migration of MT subunits, which generated a tip-high gradient. Subapical regions of apical cells showed variable tubulin subunit distributions, without tipward flow, while subapical cells showed even tubulin subunit distribution and low MT dynamics. Short MTs, of a similar size to those reported in axons, also occasionally slid into the apex. During mitosis in apical cells, MT populations at the tip varied. Cells with less distance between the tip and the first nucleus were more likely to loose normal MT populations and dynamics. Reduced MTs in the tip, during mitosis or after exposure to the MT inhibitor carbendazim (MBC), generally correlated with reduced, but continuing growth and near-normal tip morphology. In contrast, the actin-disrupting agent latrunculin B reduced growth rates much more severely and dramatically distorted tip morphology. These results suggest substantial independence between MTs and hyphal tip growth and a more essential role for F-actin. Among MT-dependent processes possibly contributing to tip growth is the transportation of vesicles. However, preliminary ultrastructural data indicated a lack of direct MT–organelle interactions. It is suggested that the population of dynamic apical MTs enhance migration of the ‘cytomatrix’, thus ensuring that organelles and proteins maintain proximity to the constantly elongating tip.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27750-0
2005-05-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/5/mic1511543.html?itemId=/content/journal/micro/10.1099/mic.0.27750-0&mimeType=html&fmt=ahah

References

  1. Ahringer J. 2003; Control of cell polarity and mitotic spindle positioning in animal cells. Curr Opin Cell Biol 15:73–81 [CrossRef]
    [Google Scholar]
  2. Akashi T., Kanbe T., Tanaka K. 1994; The role of the cytoskeleton in the polarized growth of the germ tube in Candida albicans. Microbiology 140:271–280 [CrossRef]
    [Google Scholar]
  3. Akashi T., Yoon Y., Oakley B. R. 1997; Characterization of gamma-tubulin complexes in Aspergillus nidulans and detection of putative gamma-tubulin interacting proteins. Cell Motil Cytoskeleton 37:149–158 [CrossRef]
    [Google Scholar]
  4. Alexopoulos C., Mims C., Blackwell M. 1996 Introductory Mycology , 4th edn. New York: Wiley;
  5. Allaway W. G., Ashford A. E., Heath I. B., Hardham A. R. 1997; Vacuolar reticulum in oomycete hyphal tips: an additional component of the Ca2+ regulatory system?. Fungal Genet Biol 22:209–220 [CrossRef]
    [Google Scholar]
  6. Bachewich C., Heath I. B. 1998; Radial F-actin arrays precede new hypha formation in Saprolegnia: implications for establishing polar growth and regulating tip morphogenesis. J Cell Sci 111:2005–2016
    [Google Scholar]
  7. Bachewich C., Heath I. B. 1999; Cytoplasmic migrations and vacuolation are associated with growth recovery in hyphae of Saprolegnia, and are dependent on the cytoskeleton. Mycol Res 103:849–858 [CrossRef]
    [Google Scholar]
  8. Collinge A. J., Markham P. 1985; Woronin bodies rapidly plug septal pores of severed Penicillium chrysogenum hyphae. Exp Mycol 9:80–85 [CrossRef]
    [Google Scholar]
  9. deLucas J., Monistrol M., Laborda F. 1993; Effect of antimicrotubular drugs on the secretion process of extracellular proteins in Aspergillus nidulans. Mycol Res 97:961–966 [CrossRef]
    [Google Scholar]
  10. Dustin P. 1984 Microtubules, 2nd edn. Berlin, Heidelberg & New York: Springer;
    [Google Scholar]
  11. Fiddy C., Trinci A. P. J. 1976; Mitosis, septation, branching and duplication cycle in Aspergillus nidulans. J Gen Microbiol 97:169–184 [CrossRef]
    [Google Scholar]
  12. Geitmann A., Emons A. M. 2000; The cytoskeleton in plant and fungal cell tip growth. J Microsc 198:218–245 [CrossRef]
    [Google Scholar]
  13. Han G., Liu B., Zhang J., Zuo W., Morris N. R., Xiang X. 2001; The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics. Curr Biol 11:719–724 [CrossRef]
    [Google Scholar]
  14. Heath I. B. 1981; Nucleus associated organelles of fungi. Int Rev Cytol 69:191–221
    [Google Scholar]
  15. Heath I. B. 1988; Evidence against a direct role for cortical actin arrays in saltatory organelle motility in hyphae of the fungus Saprolegnia ferax. J Cell Sci 91:41–47
    [Google Scholar]
  16. Heath I. 1990; The roles of actin in tip growth of fungi. Int Rev Cytol 123:95–127
    [Google Scholar]
  17. Heath I. B. 1994; The cytoskeleton in hyphal growth, organelle movements, and mitosis. In The Mycota. I. Growth, Differentiation and Sexuality pp 43–65 Edited by Wessels J. G. H., Meinhardt F. Berlin: Springer;
    [Google Scholar]
  18. Heath I. B. 2000; Organisation and functions of actin in hyphal tip growth. In Actin: a Dynamic Framework for Multiple Plant Cell Functions pp 275–300 Edited by Staiger C., Baluska F., Volkmann D., Barlow P. Dordrecht, Boston & London: Kluwer Academic;
    [Google Scholar]
  19. Heath I. B., Heath M. C. 1978; Microtubules and organelle movements in the rust fungus Uromyces phaseoli var. vignae. Cytobiologie 16:393–411
    [Google Scholar]
  20. Heath I. B., Kaminskyj S. G. W. 1989; The organization of tip-growth related organelles and microtubules revealed by quantitative analysis of freeze-substituted oomycete hyphae. J Cell Sci 93:41–52
    [Google Scholar]
  21. Heath I. B., Steinberg G. 1999; Mechanisms of hyphal tip growth: tube dwelling amebae revisited. Fungal Genet Biol 28:79–93 [CrossRef]
    [Google Scholar]
  22. Heath I. B., 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 30:45–62 [CrossRef]
    [Google Scholar]
  23. Heath I. B., Bonham M., Akram A., Gupta G. D. 2003; The interrelationships of actin and hyphal tip growth in the ascomycete Geotrichum candidum. Fungal Genet Biol 38:85–97 [CrossRef]
    [Google Scholar]
  24. Heitz M. J., Petersen J., Valovin S., Hagan I. M. 2001; MTOC formation during mitotic exit in fission yeast. J Cell Sci 114:4521–4532
    [Google Scholar]
  25. Hepler P. K., Vidali L., Cheung A. Y. 2001; Polarized cell growth in higher plants. Annu Rev Cell Dev Biol 17:159–187 [CrossRef]
    [Google Scholar]
  26. Herr F. B., Heath M. C. 1982; The effects of antimicrotubule agents on organelle positioning in the cowpea rust fungus, Uromyces phaseoli var. vignae. Exp Mycol 6:15–24 [CrossRef]
    [Google Scholar]
  27. Heuser J. 2003; Whatever happened to the ‘microtrabecular concept’?. Biol Cell 94:561–596
    [Google Scholar]
  28. Hoch H. C., Staples R. C. 1985; The microtubule cytoskeleton in hyphae of Uromyces phaseoli germlings: its relationship to the region of nucleation and to the F-actin cytoskeleton. Protoplasma 124:112–122 [CrossRef]
    [Google Scholar]
  29. Horio T., Oakley B. R. 2005; The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans. Mol Biol Cell 16:918–926
    [Google Scholar]
  30. Howard R. J., Aist J. R. 1977; Effects of MBC on hyphal tip organization, growth, and mitosis of Fusarium acuminatum, and their antagonism by D2O. Protoplasma 92:195–210 [CrossRef]
    [Google Scholar]
  31. Howard R. J., Aist J. R. 1980; Cytoplasmic microtubules and fungal morphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze-substitution of hyphal tip cells. J Cell Biol 87:55–64 [CrossRef]
    [Google Scholar]
  32. Hyde G. J., Heath I. B. 1997; Ca2+ gradients in hyphae and branches of Saprolegnia ferax. Fungal Genet Biol 21:238–247 [CrossRef]
    [Google Scholar]
  33. Hyde G. J., Davies D., Perasso L., Cole L., Ashford A. E. 1999; Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius. Cell Motil Cytoskeleton 42:114–124 [CrossRef]
    [Google Scholar]
  34. Jackson S. L., Heath I. B. 1993; Roles of calcium ions in hyphal tip growth. Microbiol Rev 57:367–382
    [Google Scholar]
  35. Jochova J., Rupes I., Peberdy J. 1993; Effect of the microtubule inhibitor benomyl on protein secretion in Aspergillus nidulans. Mycol Res 97:22–27
    [Google Scholar]
  36. Kaminskyj S. G. W., Heath I. B. 1996; Studies on Saprolegnia ferax suggest the general importance of the cytoplasm in determining hyphal morphology. Mycologia 88:20–37 [CrossRef]
    [Google Scholar]
  37. Kaminskyj S. G. W., Yoon K. S., Heath I. B. 1989; Cytoskeletal interactions with post-mitotic migrating nuclei in the oyster mushroom fungus, Pleurotus ostreatus: evidence against a force-generating role for astral microtubules. J Cell Sci 94:663–674
    [Google Scholar]
  38. Keating T. J., Peloquin J. G., Rodionov V. I., Momcilovic D., Borisy G. G. 1997; Microtubule release from the centrosome. Proc Natl Acad Sci U S A 94:5078–5083 [CrossRef]
    [Google Scholar]
  39. Ketelaar T., de Ruijter N. C., Emons A. M. 2003; Unstable F-actin specifies the area and microtubules direction of cell expansion in Arabidopsis root hairs. Plant Cell 15:285–292 [CrossRef]
    [Google Scholar]
  40. Konzack S., Rischitor P. E., Enke C., Fischer R. 2005; The role of the kinesin motor KipA in microtubule organization and polarized growth of Aspergillus nidulans. Mol Biol Cell 16:497–506
    [Google Scholar]
  41. Lehmler C., Steinberg G., Snetselaar K. M., Schliwa M., Kahmann R., Bolker M. 1997; Identification of a motor protein required for filamentous growth in Ustilago maydis. EMBO J 16:3464–3473 [CrossRef]
    [Google Scholar]
  42. Luby-Phelps K. 1993; Effect of cytoarchitecture on the transport and localization of protein synthetic machinery. J Cell Biochem 52:140–147 [CrossRef]
    [Google Scholar]
  43. Martin M. A., Osmani S. A., Oakley B. R. 1997; The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans. J Cell Sci 110:623–633
    [Google Scholar]
  44. Mata J., Nurse P. 1998; Discovering the poles in yeast. Trends Cell Biol 8:163–167 [CrossRef]
    [Google Scholar]
  45. McDaniel D. P., Roberson R. W. 1998; γ-Tubulin is a component of the Spitzenkörper and centrosomes in hyphal-tip cells ofAllomyces macrogynus . Protoplasma 203:118–123 [CrossRef]
    [Google Scholar]
  46. McKerracher L. J., Heath I. B. 1986; Fungal nuclear behaviour analysed by ultraviolet microbeam irradiation. Cell Motil Cytoskeleton 6:35–47 [CrossRef]
    [Google Scholar]
  47. McNiven M. A. 2003; The solid state cell. Biol Cell 94:555–556
    [Google Scholar]
  48. Meyer S. L. F., Kaminskyj S. G. W., Heath I. B. 1988; Nuclear migration in a Nud mutant of Aspergillus nidulans is inhibited in the presence of a quantitatively normal population of cytoplasmic microtubules. J Cell Biol 106:773–778 [CrossRef]
    [Google Scholar]
  49. Minke P. F., Lee I. H., Tinsley J. H., Bruno K. S., Plamann M. 1999; Neurospora crassa ro-10 and ro-11 genes encode novel proteins required for nuclear distribution. Mol Microbiol 32:1065–1076 [CrossRef]
    [Google Scholar]
  50. Murphy S. M., Stearns T. 1996; Cytoskeleton: microtubule nucleation takes shape. Curr Biol 6:642–644 [CrossRef]
    [Google Scholar]
  51. Niini S., Raudskoski M. 1993; Response of ectomycorrhizal fungi to benomyl and nocodazole – growth-inhibition and microtubule depolymerization. Mycorrhiza 3:83–91 [CrossRef]
    [Google Scholar]
  52. Oakley B. R. 2000; γ-Tubulin. Curr Top Dev Biol 49:27–54
    [Google Scholar]
  53. Oakley B. R., Morris N. R. 1980; Nuclear movement is β-tubulin-dependent in Aspergillus nidulans . Cell 19:255–262 [CrossRef]
    [Google Scholar]
  54. Oakley B. R., Rinehart J. E. 1985; Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans. J Cell Biol 101:2392–2397 [CrossRef]
    [Google Scholar]
  55. Oakley B. R., Oakley C. E., Yoon Y., Jung M. K. 1990; Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans . Cell 61:1289–1301 [CrossRef]
    [Google Scholar]
  56. Ovechkina Y., Maddox P., Oakley C. E., Xiang X., Osmani S. A., Salmon E. D., Oakley B. R. 2003; Spindle formation in Aspergillus is coupled to tubulin movement into the nucleus. Mol Biol Cell 14:2192–2200 [CrossRef]
    [Google Scholar]
  57. Pedregosa A., Rios S., Monistrol I., Laborda F. 1995; Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on protein secretion and microtubule distribution in Cladosporium cucumerinum. Mycol Res 99:43–48 [CrossRef]
    [Google Scholar]
  58. Peterbauer C. K., Heidenreich E., Baker R. T., Kubicek C. P. 1992; Effect of benomyl and benomyl resistance on cellulase formation by Trichoderma reesi and Trichoderma harzianum . Can J Microbiol 38:1292–1297 [CrossRef]
    [Google Scholar]
  59. Raudaskoski M., Mao W. Z., Yli-Mattila T. 1994; Microtubule cytoskeleton in hyphal growth. Response to nocodazole in a sensitive and a tolerant strain of the homobasidiomycete Schizophyllum commune. Eur J Cell Biol 64:131–141
    [Google Scholar]
  60. Requena N., Alberti-Segui C., Winzenburg E., Horn C., Schliwa M., Philippsen P., Liese R., Fischer R. 2001; Genetic evidence for a microtubule-destabilizing effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution in Aspergillus nidulans. Mol Microbiol 42:121–132
    [Google Scholar]
  61. Rieder C. L., Davison E. A., Jensen L. C., Cassimeris L., Salmon E. D. 1986; Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J Cell Biol 103:581–591 [CrossRef]
    [Google Scholar]
  62. Rischitor P. E., Konzack S., Fischer R. 2004; The Kip3-like kinesin KipB moves along microtubules and determines spindle position during synchronized mitoses in Aspergillus nidulans hyphae. Eukaryot Cell 3:632–645 [CrossRef]
    [Google Scholar]
  63. Roberson R. W., Fuller M. S. 1988; Ultrastructural aspects of the hyphal tip of Sclerotium rolfsii preserved by freeze substitution. Protoplasma 146:143–149 [CrossRef]
    [Google Scholar]
  64. Rupes I., Mao W., Astrom H., Raudaskoski M. 1995; Effect of nocodazole and brefeldin-A on microtuble cytoskeleton and membrane organization in the homobasidiomycete Schizophyllum commune. Protoplasma 185:212–221 [CrossRef]
    [Google Scholar]
  65. Sawin K. E., Nurse P. 1998; Regulation of cell polarity by microtubules in fission yeast. J Cell Biol 142:457–471 [CrossRef]
    [Google Scholar]
  66. Seiler S., Nargang F. E., Steinberg G., Schliwa M. 1997; Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa. EMBO J 16:3025–3034 [CrossRef]
    [Google Scholar]
  67. Shepherd V., Orlovich D., Ashford A. 1993; A dynamic continuum of pleiomorphic tubules and vacuoles in growing hyphae of a fungus. J Cell Sci 104:495–507
    [Google Scholar]
  68. Srinivasan S., Vargas M., Roberson R. 1996; Functional, organizational, and biochemical analysis of actin in hyphal tip cells of Allomyces macrogynus. Mycologia 88:57–70 [CrossRef]
    [Google Scholar]
  69. Steinberg G., Schliwa M. 1993; Organelle movements in the wild type and wall-less fz; sg; os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules. J Cell Sci 106:555–564
    [Google Scholar]
  70. Steinberg G., Schliwa M. 1995; The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties. Mol Biol Cell 6:1605–1618 [CrossRef]
    [Google Scholar]
  71. Steinberg G., Schliwa M., Lehmler C., Bolker M., Kahmann R., McIntosh J. R. 1998; Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration. J Cell Sci 111:2235–2246
    [Google Scholar]
  72. Straube A., Brill M., Oakley B. R., Horio T., Steinberg G. 2003; Microtubule organization requires cell cycle-dependent nucleation at dispersed cytoplasmic sites: polar and perinuclear microtubule organizing centers in the plant pathogen Ustilago maydis. Mol Biol Cell 14:642–657 [CrossRef]
    [Google Scholar]
  73. Temperli E., Roos U.-P., Hohl H. 1991; Germ tube growth and the microtubule cytoskeleton in Phytophthora infestans. Effects of antagonists of hyphal growth, microtubule inhibitors, and ionophores. Mycol Res 95:611–617 [CrossRef]
    [Google Scholar]
  74. That T. C., Rossier C., Barja F., Turian G., Roos U.-P. 1988; Induction of multiple germ tubes in Neurospora crassa by antitubulin agents. Eur J Cell Biol 46:68–79
    [Google Scholar]
  75. Torralba S., Heath I. B. 2001; Cytoskeletal and Ca2+ regulation of hyphal tip growth and initiation. Curr Top Dev Biol 51:135–187
    [Google Scholar]
  76. Torralba S., Pedregosa A., Lucas J. D., Dias M., Monistrol I., Laborda F. 1996; Effects of the microtubule inhibitor methyl benzimidazole-2-yl carbamate (MBC) on production and secretion of enzymes in Aspergillus nidulans. Mycol Res 100:1375–1382 [CrossRef]
    [Google Scholar]
  77. Torralba S., Raudaskoski M., Pedregosa A. 1998; Effects of methyl benzimidazole-2-yl carbamate on microtubule and actin cytoskeleton in Aspergillus nidulans. Protoplasma 202:54–64 [CrossRef]
    [Google Scholar]
  78. Trinci A. P. J., Collinge A. J. 1973; Occlusion of septal pores of damaged hyphae of Neurospora crassa by hexagonal crystals. Protoplasma 80:57–67
    [Google Scholar]
  79. Vidali L., Hepler P. 2001; Actin and pollen tube growth. Protoplasma 215:64–76 [CrossRef]
    [Google Scholar]
  80. Wedlich-Soldner R., Bolker M., Kahmann R., Steinberg G. 2000; A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis. EMBO J 19:1974–1986 [CrossRef]
    [Google Scholar]
  81. Wedlich-Soldner R., Schulz I., Straube A., Steinberg G. 2002; Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis. Mol Biol Cell 13:965–977 [CrossRef]
    [Google Scholar]
  82. Wu Q., Sandrock T. M., Turgeon B. G., Yoder O. C., Wirsel S. G., Aist J. R. 1998; A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis. Mol Biol Cell 9:89–101 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27750-0
Loading
/content/journal/micro/10.1099/mic.0.27750-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error