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Actin stringently accumulated in the specifically positioned, differentiating female gametangia of Allomyces
NGUYEN THI BICH, Nga, TURIAN, Gilbert
Abstract
Female gametangia of the normal bisexual Allomyces species are richer in fluorescently probed (FITC) actin, independent of their apical or subapical positioning during differentiation on the fertile hyphae. The anti-actin, cytochalasin D, can selectively suppress female differentiation in both species.
NGUYEN THI BICH, Nga, TURIAN, Gilbert. Actin stringently accumulated in the specifically positioned, differentiating female gametangia of Allomyces . Sexual Plant Reproduction , 1992, vol. 5, no. 4, p. 310-312
DOI : 10.1007/BF00197384
Available at:
http://archive-ouverte.unige.ch/unige:124610
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Sex Plant Reprod (1992) 5:310-312
Sexual Plant
Reproduction
9 Springer-Verlag 1992
Short communication
Actin stringently accumulated in the specifically positioned, differentiating female gametangia of Allomyces
B.N. Nguyen Thi and G. Turian
Laboratory of General Microbiology, Sciences III, Universit~ de Gen6ve, CH-1211 Geneva 4, Switzerland
Summary. Female gametangia o f the normal bisexual Allomyces species are richer in fluorescently probed (FITC) actin, independent o f their apical or subapical positioning during differentiation on the fertile hyphae.
The anti-actin, cytochalasin D, can selectively suppress male differentiation in both species.
Key words: Actin - Female G a m e t a n g i a - Differentia- tion - Allomyces
Introduction
The aquatic molds o f the genus Allomyces (Phycomy- ceres) present an experimentally attractive system of su- perposed gametangia specifically differentiated in an in- verse position, namely epigynous (male-female) in A.
macrogynus versus h y p o g y n o u s (female-male) in A. ar- buscula (Emerson and Wilson 1954). Their value in sex- ratio studies is enhanced by the easy recognition o f the male gametangia by their yellowish-orange, y-carotene pigmentation (Turian 1963; Ojha 1985).
By means o f fluorescent probing we have recently shown the predominant localization o f actin i n female gametangia apically differentiated on the fertile hyphae of h y p o g y n o u s A. arbuscula (Turian et al. 1992). How- ever, this selective accumulation o f actin m a y only have been the normal consequence o f apical localization, which has been observed in all fungal hyphae studied so far (see references in Heath 1990). To establish a link between actin and female gametangial differentiation in the Allomyces, it was therefore deemed necessary to fur- ther probe actin localization in the epigynous A. macro- gynus. The positive cytotopological link that was estab- lished led to the question of its stringency, which also could be positively answered by the selective prevention o f female gametangial differentiation by the most effec- tive anti-actin drug available, cytochalasin D (Tanen- Correspondence ." G. Turian
baum 1978; Bereiter-Hahn and Strohmeier 1987; Betina 1989).
Materials and methods
The gametophytic phases of Allomyces arbuscula Butl. (strain Stumm) and A. macrogynus Em. and W. (strain Burma) were grown on standard Difco YpSs agar medium (Turian and Ojha 1987), and meiospores were liberated into sterile water. They were then germinated, grown to small colonies in semi-synthetic medium PYG (peptone, yeast extract, glucose) (Turian 1963), and differen- tiated into gametangia following their transfer to DS solution (Machlis and Ossia 1953).
For single-labelling fluorescence microscopy, the preparations were incubated in fluorescein isothiocyanate (FITC)-phalloidin (Sigma Biochemicals, St. Louis, Mo.) at a concentration of l0 gg/ml for 1 h at room temperature (Turian et al. 1992). After a final rinse the ceils were mounted in p-phenylenediamine-glycerol (10% w/v) (Johnson and De Nogueira-Araujo 1981). A Leitz Orth- oplan epiillumination microscope (Ernst Leitz, Wetzlar, FRG) equipped with fluotar optics and a selective filter combination was used for viewing the FITC-phalloidin fluorescence patterns. Photo- graphs were taken on HP 5 Ilford black and white fiIm (Ilford, Basel, Switzerland).
For the inhibition experiments, Cytochalasin D or CD (Serva Feinbiochemica, Heidelberg, FRG) was used. Meiospores and ve- getative hyphae were grown on dialysis membranes (1.5 • 1.5 cm) placed on YpSs medium. After ~ 5 h of culture individual mem- brane-bound colonies were transferred either onto the same medi- um supplemented with the cytochalasin or into DS solution con- taining different concentrations of CD, dissolved in dimethyl sul- foxide (DMSO) to a final stock concentration of 1 rag/100 ml.
The concentrations tested varied from 10 ~tg to 100 ~tg/ml. Controls consisted of mycelia treated with DMSO at the working concentra- tions as well as untreated mycelia. An Olympus microscope coupled with an Olympus camera C-35 AD-4 was used, and micrographs were taken on Kodak Ektachrome 64T.
Results and discussion
Actin has now been found to be predominantly localized not only in the apical female gametangium o f the hypo- gynous A. arbuscula (Fig. I b) but also in the female ga- metangium o f the epigynous A. macrogynus (Fig. I a).
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Fig. I a, b. Predominant localization of actin fluorescently probed with fluorescein-isothiocyanate (FITC)-phalloidin in the female ga- metangia of Allomyces; localization is independent of the specific positioning of the female gametangial with respect to that of the males, a Epigynous in A. rnacrogynus, b hypogynous in A. arbuscu- la. Note that fluorescent actin dots are restricted to the female gametangia. Bar: 20 gm
In both o f the inversely positioned female gametangia, actin is visualized as fluorescent dots and as more diffuse fluorescent areas over the ribosomal nuclear caps o f the differentiating gametes. In contrast, males, whether api- cally or subapically positioned, show a weak fluores- cence during gametic differentiation (Fig. 1). These ob- servations indicate that a close relationship exists be- tween actin and female differentiation that is indepen- dent of their positioning on the fertile hyphae.
To further ascertain the stringency o f the topo-cyto- logical symmetry o f actin-female differentiation, we at- tempted to prevent the synthesis/accumulation o f actin in order to see which type o f differentiation would be more detrimentally affected - the female type or the male type. We then tested the effects o f the anti-actin cytochalasin D (CD), which is k n o w n to efficiently dis- rupt the fine, fibrillary actin meshwork (Bereiter-Hahn and Strohmeier 1987; Schliwa 1986).
Colonies o f both species o f Allornyces grown in the presence o f 20-40 gg/ml of CD could only differentiate a few bisexual couples o f gametangia. On 40 gg/ml, there were predominantly abnormal couples o f incompletely separated male gametangia - an effect previously ob- served with CE (Turian and Ojha 1987; Turian et al.
1992) - as well as a few single males. It is only with 60 gg/ml C D that female differentiation was fully pre- vented, leaving only single yellowish-orange male game- tangia on the tips o f the fertile hyphae, and this occurred independently o f their specific positioning in the control cultures (Fig. 2a-d).
Our observation o f the differential accumulation o f actin in the female gametangia presents the problem o f whether the differential control is expressed by the actin gene(s), as suggested (Nguyen Thi et al. 1991); this gene is still uncharacterized in the Allornyces. Preliminary re-
Fig. 2a-c. Selective prevention of female gametangial differentia- tion by cytochalasin D (CD) added at 60 gg/ml to the medium inducing sexual differentiation in both Allomyees species (6 h at 30 ~ C for controls and 10 h for CD-treated colonies). Bisexual ga- metangial couples and males are more chromogenic (7-carotene) in A. macrogynus (a) than A. arbuscula (c); CD-treated colonies with only male gametangia are differentiated and highly pigmented in both A. macrogynus (b) and A. arbuscula (d). Bar: 15 gm
sults obtained with a practically fully feminized (98 fe- males/2 males) hybrid strain (A. arbuscula x A . macro- gynus, Emerson and Wilson 1954) have shown that its sex-ratio drops to an average o f 45/55 (females/males) after having been grown in the presence o f 60 gg/ml CD. Inversely, the genetic male ('~ m u t a n t o f A.
arbuscula (Stumm 1958) is undisturbed by the same con- centration of CD (unpublished observation). Conversely to the situation in the female strain, male differentiation on the short hyphae o f the " m a s " m u t a n t thus supports a relaxed expression o f the actin gene(s).
The sex-linked difference in CD sensitivity confirms that the major localization o f actin in the female game- tangia is of functional significance for that type o f sexual orientation. Presumably, it could be related to its cyto- plasmic status - oxidatively competent mitochondria (Turian 1975; Olson 1984) - as regards the monoecious type o f A l l o m y c e s sexuality. There might therefore be a close interrelationship between the selective enrich- ment in ribosomal R N A that leads to the larger baso- philic nuclear caps o f the female gametes (Turian 1963) - as opposed to a deficiency in ribosomal genes in the male gametes (Ojha and Turian 1978) - and actin accu- mulation in the female zones. A subtle relationship could then occur between the high actin content o f female ga- metangia and their high ribosomal R N A content, as has also been suggested for m R N A in the yeast system (Singer 1992).
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The observation of a gradient of synthesis/accumula- tion of actin with its top either in the apical or the subap- ical female gametangium is relevant to the fundamental problem of the direction of the bipolar axis of sexual differentiation previously found to be controlled by an interspecifically transferable, positioning D N A (Ojha and Turian 1971).
Acknowledgements. We thank Drs. F. Barja and M. Ojha for their competent advises, Dr. R. Peck for critical reading of the manu- script typed by Mrs. F. Grange, and Mrs. A. Cattaneo for her technical help. B.N.N.T, is also grateful for financial support from the "Commision f6d6rale suisse des bourses pour 6tudiants 6trangers", Bern, Switzerland.
References
Bereiter-Hahn J, Strohmeier R (1987) Hydrostatic pressure in me- tazoan cells in culture: its involvement in locomotion and shape generation. In: Bereiter-Hahn J, Anderson OR, Reif WE (eds) Cytomechanics. The mechanical basis of cell form and struc- ture. Springer, Berlin Heidelberg New York, pp 261-272 Betina V (1989) Mycotoxins. Chemical, biological and environmen-
tal aspects. (Bioactive molecules, vol 9). Elsevier, Amsterdam, pp 317-321
Emerson R, Wilson CM (1954) Interspecific hybrids and the cyto- genetics and cytotaxonomy of Euallomyces. Mycologia 46:393- 434
Heath IB (1990) Tip growth in plant and fungal cells. Academic Press, New York London
Johnson GD, de Nogueira Araujo GM (1981) A simple method of reducing the fading of immunofluorescence during microsco- py. J Immunol Methods 43 : 349-350
Machlis L, Ossia E (1953) Maturation of the meiosporangia of Euallomyces. 1. The effect of cultural conditions. Am J Bot 40:358-365
Nguyen Thi BN, van Tuinen D, Turian G (1991) Actin in Allomyces arbuscula. Mycol Res 95:499 501
Ojha M (1985) Maleness in Allomyces arbuscula. J Gen Microbiol 131:693-695
Ojha M, Turian G (1971) Interspecific transformation and DNA characteristic in Allomyces. Mol Gen Genet 112:49-59 Ojha M, Turian G (1978) Loss of rRNA genes during a phenotypic,
masculinizing mutation in Allomyces arbuscula. Mol Gen Genet 166:225-227
Olson LW (1984) Allomyces: a different fungus. Opera Bot 73:1-96 Singer RH (1992) The cytoskeleton and mRNA localization. Curr
Opin Cell Biol 4:15-19
Schliwa M (1986) The cytoskeleton. Springer, Berlin Heidelberg New York
Stumm C (1958) Die Analyse von Genmutanten mit ge/inderten Fortpflanzungseigenschaften bei Allomyces arbuscula ButI. Z Vererbungsl 89 : 521-539
Tanenbaum J (1978) Approaches to the molecular biology of cyto- chalasin action. In: Tanenbaum SW (ed) Cytochalasin biochem- ical and cell biological aspects. North-Holland Sci Publ, Am- sterdam, pp 521 557
Turian G (1963) Synth6se diff6rentielle d'acide ribonucl6ique et diff6renciation sexuelle chez l'Allomyces. Dev Biol 6:61-72 Turian G (1975) Differentiation in Allomyces and Neurospora.
Trans Br Mycol Soc 64:367-380
Turian G, Ojha M (1987) Disruption of the bipolar gametangial segregation by anticytoskeletal chemicals in Allomyces. Bot Helv 97: 357-360
Turian G, Sch6nenberger I, Nguyen Thi BN (1992) Cytoskeletal involvement in bipolar disjunction of Allomyces gametangia.
Biomed Lett 47: 215~19
