Archives of
Hicrobiology
Arch. Microbiol. 107, 113-114 (1976)
9 by Springer-Verlag 1976
Microfilament Bundles in Antheridial Nuclei of Achlya ambisexualis E 87 JOANNE ELLZEY, ELAINE HUIZAR, and DAVID YANEZ Department of Biological Sciences, University of Texas at E1 Paso, El Paso, Texas 79968, U.S.A.
Abstract. This is the first report of intranuclear microfilaments within gametangial nuclei of o6mycetous fungi. Longitudinal sections of four to six microfilaments were frequently observed in meiotic antheridial nuclei of Achlya ambisexualis. Each microfilament measured approximately 7 - 1 0 nm in diameter. Spindle tubules (25 nm in diameter) were also observed within some of the nuclei possessing microfilaments. Key words ."Achlya - Electron microscopy - Nuclear microfilaments - Antheridia - Mycology.
Wessels et al. (1971) reviewed the distribution of cytoplasmic filaments within plants and animals, as well as the ability of cytochalasin B to disrupt microfilaments ( 5 - 7 nm in diameter). They mentioned that karyokinesis is one of the processes not affected by cytochalasin B. Patton and Marchant (1975) recently reviewed the few papers reporting cytoplasmic microfilaments in fungi and effects of cytochalasin B on hyphal morphology. Observations of nuclear microfilaments in fungi have been very rare (Beck et al., 1970; Heath et al., 1970; Gleason, 1973; Allen et al., 1974). Gleason (1973) reported observing 1 0 - 1 5 nm tubules in Saprolegnia nuclei of "actively growing hyphal tips". Heath etal. (1970) described 8 n m diameter nuclear fibrils in colchicine treated zoospores of SaproIegnia ferax. Hemmes and Bartnicki-Garcia (1975) have shown "undulating rods approximately 20 nm in diameter and of undetermined length" within the oosphere nucleus of Phytophthora capsici. We have found a bundle of nuclear filaments similar in size and appearance to those of Heath et al. (1970), Gleason (1973), and Allen et al. (1974).
MATERIALS AND METHODS Achlya ambisexualis E 87 was obtained from Dr. Alma W. Barksdale, grown in liquid Barksdale Medium no. 5 and induced to produce antheridia by the additions of 1000 units/ml of antheridiol, according to Barksdale (1968). Such cultures were fixed for electron microscopy 141/2h after the addition of the hormone. Fungal colonies were transferred into 6~ glutaraldehyde-6~ acrolein, diluted 1:1 with 0.2 M sodium cacodylate buffer, pH 7.2 (Hess, 1966). After 11/2h (first 5 min in vacuo) at room temperature the fungus was washed four times at 15-min intervals in 0.1 M Na cacodylate buffer and left overnight in the buffer. Postfixing with Na cacodylate buffered 1~ osmium tetroxide proceeded for 4 h at 4~C. After washing the antheridial cultures were transferred to 0.5 ~ aqueous uranyl acetate overnight at 4~C, washed, dehydrated in a standard ethanol-acetone series and embedded in a plastic mixture of AralditeM and Epon812 (Plastic II; Mollenhauer, 1964). Ultrathin sections were obtained with glass knives on a Porter-Blum MT-1Ultramicrotome,poststained with 0.5 ~ aqueous uranyl acetate for 10 min and lead citrate 5 min (Reynolds, 1963). Specimenswerephotographedin a Zeiss EM-10electronmicroscope operating at 60 kV.
RESULTS AND DISCUSSION During our observations of meiotic nuclei within
Achlya ambisexualis antheridia we frequently observed bundles of four to six filaments sectioned longitudinally. Each filament had a diameter of approximately 7 nm. In Figure I the bundle of filaments contains six microfilaments aligned perpendicularly to the 25 nm diam. microtubules which compose the spindle fibers. We have also observed these bundles ofmicrofilaments extending almost the entire length of some nuclei running parallel to the spindle fibers in others, and in close association with the nucleolus. The function of these nuclear microfilaments is not known. In Figure 2 five parallel microfilaments are visible. Some of these microfilaments appear to extend obliquely across 2/3 of the nucleus. Figure 3 shows a cross section of approximately fourteen nuclear microfila-
114
J. Ellzey et al.
Fig. 1. Longitudinal section through a meiotic antheridial nucleus of Achlya ambisexualis. The microtubules which compose the spindle fibers (SF) are greater than twice the size of the individual microfilaments (MF). x 57 750 Fig. 2.
A higher magnification of nuclear microfilaments (MF) sectioned lengthwise, x 61000
Fig. 3.
A cross section of nuclear microfilaments (MF). x 83000
ments which appear to be consistent with the diameter and subunit structure of Nem'ospora crassa microfilaments (Allen et al., 1974). Acknowledgements'. The authors wish to thank Mr. Benjamin Rosario for technical assistance. This work was supported in part by Grant RR-08012-04 from the General Research Support Branch, Division of Research Resources, National Institutes of Health.
REFERENCES Allen, E. D., Lowry, R. J., Sussman, A. S. : Accumulation of microfsIaments in a colonial mutant of Neurospora crassa. J. Ultrastruct. Res. 48, 455-464 (1974) Barksdale, A. W. : Meiosis in the antheridium ofAchlya ambisexualis E 87. J. Elisha Mitch. Sci. Soc. 84, 187-194 (1968) Beck, D. P., Decker, G. L., Greenawalt, J. W.: Ultrastructure of striated inclusions in Neurospora. J. Ultrastruct. Res. 33, 245-251 (1970) Gleason, F. H. : Nuclear tubules in Saprolegnia. Cytobios 8, 185187 (1973)
Heath, I. B., Greenwood, A. D., Griffiths, H. B.: The origin of flimmer in Saprolegnia, Dietyuchus, Synura and Cryptomonas. J. Cell Sci. 7, 445-461 (1970) Heroines, D. E., Bartnicki-Garcia, S. : Electron microscopy of gametangial interaction and oospore development in Phytophthora eapsici. Arch. Microbiol. 103, 9 1 - 1 1 2 (1975) Hess, W. M.: Fixation and staining of fungus hyphae and host plant root tissues for electron microscopy. Stain Technol. 41, 2 7 - 35 (1966) Mollenhauer, H. H. : Plastic mabedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114 (1964) Patton, A. M., Marchant, R. : The effect of cytochalasin B on hyphal morphogenesis in Polyporus biennis. J. gen. Microbiol. 86, 301-310 (1975) Reynolds, E. S. : The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Wessells, N. K., $pooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M.A., Taylor, E.L., Wrenn, J.T., Yamada, K. M.: Microfilaments in cellular and developmental processes. Science 171, 135-143 (1971)
Received October 27, 1975
Hicrobiology
Arch. Microbiol. 107, 113-114 (1976)
9 by Springer-Verlag 1976
Microfilament Bundles in Antheridial Nuclei of Achlya ambisexualis E 87 JOANNE ELLZEY, ELAINE HUIZAR, and DAVID YANEZ Department of Biological Sciences, University of Texas at E1 Paso, El Paso, Texas 79968, U.S.A.
Abstract. This is the first report of intranuclear microfilaments within gametangial nuclei of o6mycetous fungi. Longitudinal sections of four to six microfilaments were frequently observed in meiotic antheridial nuclei of Achlya ambisexualis. Each microfilament measured approximately 7 - 1 0 nm in diameter. Spindle tubules (25 nm in diameter) were also observed within some of the nuclei possessing microfilaments. Key words ."Achlya - Electron microscopy - Nuclear microfilaments - Antheridia - Mycology.
Wessels et al. (1971) reviewed the distribution of cytoplasmic filaments within plants and animals, as well as the ability of cytochalasin B to disrupt microfilaments ( 5 - 7 nm in diameter). They mentioned that karyokinesis is one of the processes not affected by cytochalasin B. Patton and Marchant (1975) recently reviewed the few papers reporting cytoplasmic microfilaments in fungi and effects of cytochalasin B on hyphal morphology. Observations of nuclear microfilaments in fungi have been very rare (Beck et al., 1970; Heath et al., 1970; Gleason, 1973; Allen et al., 1974). Gleason (1973) reported observing 1 0 - 1 5 nm tubules in Saprolegnia nuclei of "actively growing hyphal tips". Heath etal. (1970) described 8 n m diameter nuclear fibrils in colchicine treated zoospores of SaproIegnia ferax. Hemmes and Bartnicki-Garcia (1975) have shown "undulating rods approximately 20 nm in diameter and of undetermined length" within the oosphere nucleus of Phytophthora capsici. We have found a bundle of nuclear filaments similar in size and appearance to those of Heath et al. (1970), Gleason (1973), and Allen et al. (1974).
MATERIALS AND METHODS Achlya ambisexualis E 87 was obtained from Dr. Alma W. Barksdale, grown in liquid Barksdale Medium no. 5 and induced to produce antheridia by the additions of 1000 units/ml of antheridiol, according to Barksdale (1968). Such cultures were fixed for electron microscopy 141/2h after the addition of the hormone. Fungal colonies were transferred into 6~ glutaraldehyde-6~ acrolein, diluted 1:1 with 0.2 M sodium cacodylate buffer, pH 7.2 (Hess, 1966). After 11/2h (first 5 min in vacuo) at room temperature the fungus was washed four times at 15-min intervals in 0.1 M Na cacodylate buffer and left overnight in the buffer. Postfixing with Na cacodylate buffered 1~ osmium tetroxide proceeded for 4 h at 4~C. After washing the antheridial cultures were transferred to 0.5 ~ aqueous uranyl acetate overnight at 4~C, washed, dehydrated in a standard ethanol-acetone series and embedded in a plastic mixture of AralditeM and Epon812 (Plastic II; Mollenhauer, 1964). Ultrathin sections were obtained with glass knives on a Porter-Blum MT-1Ultramicrotome,poststained with 0.5 ~ aqueous uranyl acetate for 10 min and lead citrate 5 min (Reynolds, 1963). Specimenswerephotographedin a Zeiss EM-10electronmicroscope operating at 60 kV.
RESULTS AND DISCUSSION During our observations of meiotic nuclei within
Achlya ambisexualis antheridia we frequently observed bundles of four to six filaments sectioned longitudinally. Each filament had a diameter of approximately 7 nm. In Figure I the bundle of filaments contains six microfilaments aligned perpendicularly to the 25 nm diam. microtubules which compose the spindle fibers. We have also observed these bundles ofmicrofilaments extending almost the entire length of some nuclei running parallel to the spindle fibers in others, and in close association with the nucleolus. The function of these nuclear microfilaments is not known. In Figure 2 five parallel microfilaments are visible. Some of these microfilaments appear to extend obliquely across 2/3 of the nucleus. Figure 3 shows a cross section of approximately fourteen nuclear microfila-
114
J. Ellzey et al.
Fig. 1. Longitudinal section through a meiotic antheridial nucleus of Achlya ambisexualis. The microtubules which compose the spindle fibers (SF) are greater than twice the size of the individual microfilaments (MF). x 57 750 Fig. 2.
A higher magnification of nuclear microfilaments (MF) sectioned lengthwise, x 61000
Fig. 3.
A cross section of nuclear microfilaments (MF). x 83000
ments which appear to be consistent with the diameter and subunit structure of Nem'ospora crassa microfilaments (Allen et al., 1974). Acknowledgements'. The authors wish to thank Mr. Benjamin Rosario for technical assistance. This work was supported in part by Grant RR-08012-04 from the General Research Support Branch, Division of Research Resources, National Institutes of Health.
REFERENCES Allen, E. D., Lowry, R. J., Sussman, A. S. : Accumulation of microfsIaments in a colonial mutant of Neurospora crassa. J. Ultrastruct. Res. 48, 455-464 (1974) Barksdale, A. W. : Meiosis in the antheridium ofAchlya ambisexualis E 87. J. Elisha Mitch. Sci. Soc. 84, 187-194 (1968) Beck, D. P., Decker, G. L., Greenawalt, J. W.: Ultrastructure of striated inclusions in Neurospora. J. Ultrastruct. Res. 33, 245-251 (1970) Gleason, F. H. : Nuclear tubules in Saprolegnia. Cytobios 8, 185187 (1973)
Heath, I. B., Greenwood, A. D., Griffiths, H. B.: The origin of flimmer in Saprolegnia, Dietyuchus, Synura and Cryptomonas. J. Cell Sci. 7, 445-461 (1970) Heroines, D. E., Bartnicki-Garcia, S. : Electron microscopy of gametangial interaction and oospore development in Phytophthora eapsici. Arch. Microbiol. 103, 9 1 - 1 1 2 (1975) Hess, W. M.: Fixation and staining of fungus hyphae and host plant root tissues for electron microscopy. Stain Technol. 41, 2 7 - 35 (1966) Mollenhauer, H. H. : Plastic mabedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114 (1964) Patton, A. M., Marchant, R. : The effect of cytochalasin B on hyphal morphogenesis in Polyporus biennis. J. gen. Microbiol. 86, 301-310 (1975) Reynolds, E. S. : The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Wessells, N. K., $pooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M.A., Taylor, E.L., Wrenn, J.T., Yamada, K. M.: Microfilaments in cellular and developmental processes. Science 171, 135-143 (1971)
Received October 27, 1975
