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A Stringent Role of F-Actin in the Ascogonial Differentiation of Neurospora crassa Fluffy Mutant
LIU, Jia Wei, TURIAN, Gilbert, BARJA, François
Abstract
Cytochalasin D, an inhibitor of actin polymerization, interferes with ascogonial differentiation in the fertile fluffy mutant of Neurospora crassa. As the total level of actin and its mRNA remain unchanged, this suggests that it is in its microfilamentous form (F-form) that actin is stringently required for female differentiation.
LIU, Jia Wei, TURIAN, Gilbert, BARJA, François. A Stringent Role of F-Actin in the Ascogonial Differentiation of Neurospora crassa Fluffy Mutant. Folia Microbiologica , 1995, vol. 40, no.
6, p. 665-668
DOI : 10.1007/BF02818527
Available at:
http://archive-ouverte.unige.ch/unige:122404
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Folia Microbiol. 40 (6), 665 -668 (1995)
A Stringent Role of F-Actin in the Ascogonial
Differentiation of Neurospora crassa Fluffy Mutant
J.W. LIU, G. TURIAN and F. BARJA*
Laboratory of General Microbiology, University of Geneva, Sciences 1II, CH-1211 Geneva 4, Switzerland
Received luly IL 1995
ABSTRACT. Cytochalasin D, an inhibitor of actin polymerization, interferes with ascogonial differentiation in the fertile fluffy mutant ofNeurospora crassa. As the total level of actin and its mRNA remain unchanged, this suggests that it is in its microfil- amentous form (F-form) that actin is stringently required for female differentiation.
It was recently found that actin in its polymerized form is stringently required for differentia- tion of the female gametangia of both Allomyces arbuscula and A. macrogynus as shown by their selec- tive inhibition by cytochalasin D (Nguyen Thi and Turian 1992), a specific inhibitor of the F-form (Tannenbaum 1978; Sampath and Pollard 1991). It was therefore of interest to confirm this female sen- sitivity with the acid of another fungal model of sexual differentiation, viz. Neurospora crassa, using its fluffy mutant, a fertile producer of female ascogonial coils (Turian and Bianchi 1972; Perkins et al.
1982) resembling N. tetrasperma previously studied for its heat sensitivity (Viswanath-Reddy and Turian 1975).
M A T E R I A L S A N D M E T H O D S
The macroconidialess mutant f/uffya (FGSC 809) was obtained from the Fungal Genetics Stock Center, University o f Kansas Medical Center, Kansas City (KS, USA). Mycelial discs of 2 mm in diame- ter were cut from colonies previously grown on solid perithecium sucrose (PS) medium for 2 d at 25 ~ These inocula were then incubated for 3 d at a shaking frequency of 2.5 Hz (25 ~ in the absence or presence of the anti-actin drug cytochalasin D (CD). CD was dissolved in dimethyl sulfoxide (MezSO) to a concentration of 10 g/L as stock solution, diluted to final concentrations from 50 to 200 lamol/L.
Control cultures with Me2SO at <2 % were shown not to have any detectable morphogenetic effects.
The presence or relative absence of ascogonial coils was recorded with a Leitz Orthoplan epiiUumina- tion microscope and photographed on 400 ASA HP5 black-and-white films (Ilford).
For chemical analysis, mycelia were harvested by filtration, pressed and resuspended in modified actin extraction buffer [5 mmol/L Tris-HC1, pH 7.9, 0.2 mmol/L CaCI2, 5 mmol/L disodium diphosphate, 1 mol/L ethylenediaminetetraacetic acid (EDTA), 0.2 mmoi/L KCI, 0.2 mmol/L ATP, 10 mmol/L 1,4-dithiothreitol (DTT), 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), 0.25 mg/L pepstatin and 10 mmol/L benzamidine] and disrupted in a mortar. The homogenate was centrifuged at 14 000 g for 10 min (4 ~ Concentration of total proteins in the supernatant was measured according to the method described by Spector (1978). Proteins (30 lag per slot) extracted from CD-treated and nontreated cultures were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophore- sis (SDS-PAGE) according to Laemmli (1970).
Proteins from unstained gel were transferred electrophoretically onto nitrocellulose discs strips (pore size 0.45 lam, Schleicher & Schuell, Dassel, Germany) using the transfer buffer described by Burnette (1981). The transferred proteins were stained with 500 ppm ( W / V ) Ponceau S in 3 % ( W / V ) trichloroacetic acid (TCA). The nonspecific binding sites were blocked with 5 % BSA in Tris-buffered saline (TBS) (150 mmol/L NaCI, 100 mmol/L Tris-HCl, pH 7.5) for 1 - 2 h at room temperature or overnight at 4 ~ For Western blot analysis, the nitrocellulose strip was then incubated for 4 h with commercial anti-actin monoclonal antibody (mAb) raised in mice against chicken ~ r : a r d actin (no.
350, Amersham International, Amersham, UK). The mAb was diluted to 1:7000 in TBS containing 1 % BSA. After four washes (10 min each) in TBS, incubation with a secondary antibody was done for 1 h at room temperature with peroxidase-labeled sheep anti-mouse IgM (Amersham). Following four
*Correspondence author.
6 6 6 J. L I U eta/. Vol. 41
washes, peroxidase activity was revealed by 0.5 g/L of 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Fluka) in 100 mmol/L Tris-HC! (pH 7.5) containing 300 ppm H202. When the bands became visible, the nitrocellulose strips were removed and washed with distilled water.
To compare mRNAs, total cellular RNA was purified from both control and CD-treated (125 lamol/L) cultures according to Chirgwin et al. (1979). Five lag RNA per lane were electrophoreti- cally separated and analyzed by Northern blots using formaldehyde-agarose gels and transferred to nitrocellulose membrane (Gene Screen Plus, New Research Products, Boston, MA, USA). Nonradioac- tive N. crassa actin eDNA probes (supplied by G. Wuethrich et al. 1994) were prepared by incorporat- ing a nucleotide analog (digoxigenin-ll-dUTP) according to the technique described in "The DIG Sys- tem User's Guide for Filter Hybridization" (Boehringer Mannheim, GmbH, Biochemica 1993). Hybridi- zation of the digoxigenin-labeled probe (25 ~tg/L) to immobilized RNA and detection of this probe based on digoxigenin-anti digoxigenin ELISA were performed according to the same technical guide.
R E S U L T S A N D DISCUSSION
The major effect of treatment by cytochalasin D on sexual differentiation of mycelia was a decrease in the number of ascogonia compared with control cultures incubated for the same time (Table I). The effect started at a concentration of 75 lamol/L which brought about a decrease of about 36 % in the number of coiled structures. Maximum inhibitory effect (mean decrease 82 %) was observed at a concentration of 200 lamol/L. The average concentration of 125 lamol/L which produced a 75 % ascogonial decrease within 3 d (Fig. 1) was chosen for further experiments.
Based on a previous Western-blot analysis (Barja et al.
1991) we detected a single band of M 43 kDa of equal in- tensity in extracts of both wild-type control and CD-treated
Table I. Effect of cytochalasin D on the f o r - cultures (Fig. 2). Therefore, CD did not seem to have in- mation of ascogonia in the flu.,~a mutant fluenced the total production of actin even at the concen- tration of 125 lamol/L which drastically prevented the for-
Cytochalasin D Number mation of ascogonia.
lamol/L of ascogonia a Results of a Northern-blot hybridization experiment are given in Fig. 3. An equal amount of total RNA from wild- 0 11.0 -+ 0.5 type N. crassa, fluffya and cytochalasin-treated fluffya were
50 11.0 +- 0.5
75 7.0 _+ 0.8 separated electrophoretically and hybridized with cDNA 100 5.0 -+ 0.4 probe for actin coding sequence. The probe hybridized to
125 b 3.0 -+ 0.4 two actin bands of a size similar to that of 17S rRNA which 150 2.6 _+ 0.3 corresponds approximately to that of 1.5-1.7 kb bands
175 2.6 -+ 0.6 (Fig. 3).
200 2.1 -+ 0.9 Comparative intensity of the mRNA bands extracted from control and CD-treated cultures (Fig. 3) confirms the
aPer cm 2 of culture, analytical results of total actin, i.e. the production of mRNA bMaximum inhibitory effect, coding for actin is not prevented by CD at the concentra- tion of 125 lamol/L which, in contrast, strongly restricted the number of differentiated ascogonia.
It can therefore be concluded that, as found in the phycomycetous Allomyces gametangia (Nguyen Thi and Turian 1993) it is in its polymerized F-form that actin plays an essential role in the differentiation of another female fungal organ, the ascomycetous ascogonium.
We thank Mrs M.-L. Chappuis for excellent technical help, Mrs E. Martinez and Mrs M. Vautravers for photo- graphic work and Dr. M. Ojha for reading the manuscript. Jiawei Liu is grateful for financial support from La Fondation Hans W'dsdorf des bourses pour ~tudiants ~trangers. We also express our gratitude to the Academic Society of Geneva.
1996 STRINGENT ROLE OF ACI'IN IN ASCOGONIAL DIFFERENTIATION 667
kDa 94 67
4 3
3 0 -
20.1 " d e f
9 Fig. 2. One-dimensional gel SDS-PAGE of total protein extract (30 gg per slot) probed with anti- actin mAbs. Staining with Ponceau S of total protein extracted (a) from mycelia of wild-type, (b) from non-treated fluffya, and (r from CD-treated (125 ~mol/L) fluffya. Visualization of actins in (el) wild type, (e) nontreated fluffYa, and (~ CD-treated fluffya. Standard low-molar mass markets (kDa):
phosphoryiase b (94), albumin (67), ovalbumin (45), carbonate dehydratase (30) (Pharmacia).
~1 Fig. 1. Microscopic view of the cytochalasin D effect; (a) nontreated fluffya, predominance of melanizing protoperithecia developed after 3 d at 25 ~ from many early ascogonial coils (arrows at two delayed ones); bar represents 10 IJm; (b) fluffya treated with CD (125 lamol/L) for 3 d at 25 ~ only one asco- gonial coil uncovered at higher magnification; bar represents 10 ~m.
26S- D
1 7 S -
a b c
F'gg. 3. Methylene blue staining and Northern-blot hybridization of total RNA (5 I~g per lane) with a cDNA probe derived from an actin-coding sequence. RNAs were from (a and d) wild-type IV. crassa; (b and e) nontreated fluffya; (0 and f) CD- treated fluffya. Note the constant methylene blue pattern of 26S and 17S rRNAs.
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