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The tubulin cytoskeleton and its sites of nucleation in hyphal tips ofAllomyces macrogynus

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Summary

The tubulin cytoskeleton in hyphal tip cells ofAllomyces macrogynus was detected with an α-tubulin monoclonal antibody and analyzed with microscopic and immunoblot techniques. The α-tubulin antibody identified a 52 kilodalton polypeptide band on immunoblots. Immunfluorescence data were collected from formaldehyde-and cryofixed hyphae. Both methods provided similar images of tubulin localization. However, cryofixation yielded more consistent labeling and did not require detergent extraction or cell-wall lytic treatments. Tubulin was primarily localized as microtubules observed in the peripheral and central cytoplasmic regions and in mitotic spindles. Cytoplasmic microtubules were oriented parallel to the cells' longitudinal axis, with central microtubules more often varied in their alignment, and emanated from a region in the hyphal apex resulting in an apical zone of bright fluorescence. A thin layer of microtubules appearing as bands of fluorescence encircled many nuclei. Discrete spots of fluorescence were also associated with nuclei. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins that may be involved in the regulation of microtubule nucleation, stained centrosomes but not apical regions of hyphae. Nocodazole was used to depolymerize the microtubule network and reveal its regions of origin. A hocodazole concentration of 0.01 μg/ ml (3.3× 10−8M) provided a 70 to 75% inhibition of hyphal tip growth and was used throughout this study. The number of cells having an apical zone of fluorescence declined by 15 min of exposure. This zone was present in only a few cells after 60 min. After 30 min, the central cytoplasm consisted of small microtubule fragments and nuclear-associated spots. A small number of peripheral microtubules and nuclear-associated spots persisted throughout nocodazole treatments. Spindle microtubules were restored by 30 min after removal of nocodazole. This was followed by the reappearance of the apical zone of fluorescence and then by central and peripheral cytoplasmic microtubules. Apical fluorescence coincided with the presence of a Spitzenkörper. The results suggest that the Spitzenkörper and centrosome function as centers of microtubule nucleation and organization during hyphal tip growth in this fungus.

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Abbreviations

BSA:

bovine serum albumin

DAPI:

4′,6-diamidino-2-phenylindole

DMSO:

dimethylsulfoxide

FITC:

fluorescein isothiocyanate

IB:

incubation buffer

LN2 :

liquid nitrogen

LSCM:

laser scanning confocal microscopy

MTOCs:

microtubule-organizing centers

PBS:

phosphate buffered saline

PIPES:

1,4-piperazinedietha-nesulfonic acid

PFB:

PIPES fixation buffer

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SPB:

spindle pole body

TEM:

transmission electron microscopy

YpSs:

yeast extract-inorganic phosphate-soluble starch

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  1. M. M. Vargas

    Present address: Department of Biology, University of Puerto Rico, Mayaguez, Puerto Rico

Authors and Affiliations

  1. Department of Botany, Arizona State University, P.O. Box 871601, 85287-1601, Tempe, AZ, USA

    R. W. Roberson & M. M. Vargas

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  2. M. M. Vargas
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Roberson, R.W., Vargas, M.M. The tubulin cytoskeleton and its sites of nucleation in hyphal tips ofAllomyces macrogynus . Protoplasma 182, 19–31 (1994). https://doi.org/10.1007/BF01403685

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  • DOI: https://doi.org/10.1007/BF01403685

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