Article
Reference
Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora
crassa
BARJA, François, CHAPPUIS, Marie-Louise, TURIAN, Gilbert
Abstract
Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa. Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia.
BARJA, François, CHAPPUIS, Marie-Louise, TURIAN, Gilbert. Differential effects of
anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa . FEMS Microbiology Letters , 1993, vol. 107, no. 2-3, p.
261-266
DOI : 10.1111/j.1574-6968.1993.tb06040.x
Available at:
http://archive-ouverte.unige.ch/unige:119368
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1 / 1
© 1993 Federation of European Microbiological Societies 0378-1097 /93/$06.00 Published by Elsevier
FEMSLE 05311
Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa
Francisco Barja, Marie-Louise Chappuis and Gilbert Turian
Laboratory of General Microbiology, University of Geneva, Geneva, Switzerland
(Received 10 December 1992; accepted 17 December 1992)
Abstract: Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa. Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia.
Key words: Actin; Cytoskeletal inhibitors; Multiple germ tubes; Neurospora crassa
Introduction
During germination, macroconidia of Neuro- spora crassa are genetically programmed to out- grow a single germ tube from an unpredictable site on their plasma membrane [1]. This mono- polar axiation [2] is controlled by microtubules, as shown by its deviation to multipolar outgrowth following germination of conidia in the presence of the anti-microtubule agent benomyl [3,4]. In contrast, the localized initiation of the germ
Correspondence to: F. Barja, Laboratory of General Microbio- logy, University of Geneva, Sciences III, 30, Quai Ernest- Ansermet, CH-1211 Geneva 4, Switzerland.
tube(s) could be prevented by the anti-actin drug cytochalasin B [5]. Those preliminary results which suggest a differential role of the two main components of the cytoskeleton in the initial mor- phogenetic events of conidial germination, have now been confirmed and extended.
Conidia of N. crassa were germinated in the separate or combined presence of the specific anticytoskeletal drugs, benomyl against micro- tubules [6] and cytochalasins A or B against actin [7-10]. Changes induced in the cytotopology of actin were also studied in parallel, by antibody and phalloidin fluorescence probing microscopy.
The conidial amounts of total actin and of its recently characterized actin isoforms have been chemically compared [11].
262
Materials and Methods
Organism and culture conditions
Wild-type Neurospora crassa (FGSC 262, strain St. Lawrence 74A) was obtained from the Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS.
In order to produce large numbers of macro- conidia, the fungus was first grown on solid Vo- gel's minimal medium [12] for 3 days at 33°C in the dark and then at 25°C in artificial light for 4 days. Conidia were germinated at an inoculum size of 5 X 106 conidia/ml in liquid Vogel's medium containing 1.5% sucrose, at 37°C, on a rotary shaker (150 rpm).
Anticytoskeletal drug treatments
Cytochalasins A (CA) and B (CB) (Sigma Chemical Co., St. Louis, MO) and benomyl (ana- lytical grade, Du Pont de Nemours Co., Wilming- ton, DE) were dissolved respectively in dimethyl sulfoxide (DMSO) (1 mg/ml stock solution) and absolute ethanol (1 mM stock solution). These solutions were added to the media after autoclav- ing. Conidia were germinated in the presence of 1.0 µ,M benomyl, 125 µ,M CA or 167 µ,M CB.
DMSO and ethanol were tested separately as controls. One percent DMSO or 0.8% ethanol had no detectable effects on the germination of conidia.
Fixation, wall digestion and membrane permeabi- lization
The conidia were fixed with 3.7% para-for- maldehyde in 10 mM imidazole buffer, pH 7.0 for 30 min at room temperature. Cells were incu- bated with 5 mg/ml Novozym 234 (Sigma) in imidazole buffer for 15-30 min at 40°C for partial wall digestion. The degree of digestion was moni- tored periodically by light microscopy. Digestion was stopped by rinsing three times in imidazole buffer. The cell membranes were then permeabi- lized with 0.5% Triton X-100 in Tris-buffered saline (TBS) (500 mM NaCl, 100 mM Tris · HCl, pH 7.5) for 10 min at room temperature. Triton was removed by washing five times for 10-min periods in TBS and cells settled on poly-L-lysine coated slides.
Immunofluorescence of actin microfilaments The distribution of actin in formaldehyde-fixed germlings was examined using either FITC-con- jugated phalloidin [11] or anti-actin antibodies.
After rinsing with TBS the slides were incubated for 2 h at room temperature or overnight at 4°C with the primary anti-actin antibody (Amersham N.350, Amersham International plc., Amersham, UK) at a dilution of 1 : 100 in TBS. Following a rinse in TBS, the material was incubated with fluorescein-isothiocyanate (FITC)-goat-anti- mouse IgM (Sigma) at a dilution of 1 : 40 in TBS for 1 h at 37°C. After a final rinse in TBS, the cells were mounted in p-phenylenediamine- glycerol (10% w /v). An Orthoplan epiillumina- tion microscope (Ernst Leitz, Wetzlar Germany) equipped with Fluotar optics and selective filter combinations was used for viewing the FITC-flu- orescence patterns. Photographs were taken on 400 ASA HP5 black and white films (Ilford, Basel Switzerland).
Protein extraction and determination
Germlings were harvested by filtration through Millipore filters (pore size 0.5-1 ,um; Millipore Corp., Bedford, MA, USA), washed three times with distilled water, frozen in liquid nitrogen and ground in a mortar. Frozen powder was sus- pended in 1 ml of extraction buffer (5 mM Tris · HCl, 0.2 mM CaC12 , 5 mM pyrophosphate, 1 mM EDTA, 0.2 mM KCl, 10 mM 1,4 dithio-DL-threi- tol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.25 µ,g/ml pepstatin, 10 mM benza- midin, 0.2 mM ATP) and the homogenate cen- trifuged at 15000 rpm for 15 min at 4°C. Protein concentration in the supernatants was deter- mined using Coomassie blue (Serva, Heidelberg, Germany) [13] with bovine serum albumin (BSA) as a standard.
One- and two-dimensional gel electrophoresis Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (10% polyacrylamide) using the method of Laemmli [14]. Total extracted proteins (30-50 µ,g/slot) were electrophoresed along with the following standard molecular mass markers:
phosphorylase b, 94000; albumin, 67000; ovalbu-
min, 43000; carbonic anhydrase, 30000; trypsine inhibitor, 20100 (all from Pharmacia, Uppsala, Sweden).
Two-dimensional SDS-PAGE separation of actin isoforms was achieved by isoelectric fo- cussing [ 15] using ampholytes (1/5 pH 3-10 and 4/5 pH 5-7; Bio-Rad Laboratories, Richmond,
CA) followed by migration on SDS-PAGE (10%
polyacrylamide ).
Immunoblotting
Proteins from unstained gels were elec- trophoretically transferred to nitrocellulose paper BA85 (pore size 0.45 ,um; Schleicher and Schull
Fig. 1. Effects of 125 µM CA, 160 µM CB and 1 µM benomyl on the distribution of actin during germination of N. crassa. The actin was visualized with the anti-actin monoclonal antibody N.350. Conidia were germinated for 4-6 h in the presence of the inhibitors. (a) Untreated conidia from the isometrically swollen to the outgrowing germ tub'e stages (emergence-elongation). (b, c) Cytochalasin-treated conidia with 125 µM CA and 160 µM CB, respectively. (d) Multiple germ tubes induced by 1 µM benomyl.
(e) Fully prevented germ tube outgrowth from conidia doubly treated with 1 µM benomyl and 125 µMCA. Bar= 10 µm.
264
GmbH, Dassel, FRG) using the transfer buffer described by Burnette [16]. The transferred pro- teins were stained with 0.05% (w /v) Ponceau Sin 3% (w /v) trichloroacetic acid (TCA). The non- specific binding sites were blocked with 5% BSA in TBS for 4 h or overnight at room temperature.
The nitrocellulose paper was then incubated for 4 h with commercial anti-actin monoclonal antibod- ies (mAbs) raised in mouse against actin purified from chicken gizzards (N.350). The mAbs were diluted 1: 5000 in TBS containing 0.5% BSA After 4 washes in TBS, an incubation with the second antibody was performed for 1 h at room temperature with peroxidase-labelled sheep anti- mouse IgM (Amersham). Following four washes in TBS, the peroxidase activity was revealed with 0.5 mg/ml of 3,3'-diaminobenzidine tetrahydro- chloride (DAB) (Fluka Chemie AG, Buchs, Switzerland) in 100 mM Tris · HCl, pH 7.5 con- taining 0.03% H202 •
Results and Discussion
Inhibitory effects and cytotopology of actin
The fluorescence patterns of actin in germ tubes revealed with antibodies or FITC-phalloi- din were found to be virtually identical. The immunofluorescence control with the second antibody alone revealed only a slight background, as did samples incubated with unlabelled phal- loidin prior to addition of FITC-phalloidin (data not shown).
CA fully prevented outgrowth of the germ tubes at a concentration of 125 ,uM (Fig. lb). In contrast to untreated, non-germinated conidia, in which the actin stained either with FITC-phal- loidin or anti-actin antibodies which appeared as homogeneously distributed diffuse patches (Fig.
la), the cytoplasm of conidia inhibited with CA exhibited only diffuse actin fluorescence, mainly restricted to a few more fluorescent spots, pre- sumably of residual actin (Fig. lb).
CB (167 ,uM) only reduced the rate of germi- nation and produced abnormal germlings with a highly fluorescent, short, widening germ tube (Fig.
le) previously shown by electron microcopy to be surrounded by a thickened wall [17]. On similar
ultrathin sections of conidia, nucleolar dense spots have recently been noticed, which suggests a role for actin in the preribosomal nuclear export [18], presumably to meet the increased need for ribo- somes at spore germination [19].
The inhibitory effects of cytochalasins might thus be due to the disorganization of actin [20,21], suggesting that this protein should be intact for initiation and linear growth of the germ tube in N. crassa. Recently Yokoyama et al. [22] came to the same conclusion with the dimorphic yeast Candida albicans following their experiments of differential disruption of microfilaments and mi- crotubules. This conclusion is now strengthened by our observation that the benomyl-induced multipolar outgrowth (Fig. ld) could also be fully inhibited by CA (Fig. le). In the cytopasm of such inhibited, isometrically swollen conidia, actin re- mained unpolarized.
Apparently, the cytoskeletal inhibitors used af- fect only the polymerization-depolymerization cy- cle of actin without preventing the synthesis of the monomers [23-25]. The mechanism of the inhibition of polymerization at the pointed ends of actin filaments has not yet been solved.
After 5 h of germination in the presence of 1 ,uM benomyl which induces multiple germ tubes [3,4], actin stained with monoclonal antibodies was found, in contrast with its concentration in the tips of the normally single outgrowing germ tube (Fig. la), to be concentrated in each of the 3-5 tiny outgrowing germ tubes (Fig. ld).
Thus, germ tube initiation can still occur in benomyl-treated conidia devoid of intact micro- tubules. These tubular structures thus appear to be stringently required for the monopolar axia- tion of N. crassa conidia rather than for the initiation of germ tube growth. In contrast, actin microfilaments would be essential for such out- growth as much for further linear growth of the normally unique germ tube as for drug-induced multipolar germination. This interpretation is supported by immunofluorescent microscopic ob- servations which constantly showed the presence of actin patches at the tip of each germ tube of benomyl-treated conidia (Fig. ld). At present, we can only suggest that in the multitubes outgrow- ing in the absence or at the most in the presence
of fragmented residual microtubules [3], the main actin capping network would be fragmented into a few actin patches functioning as elicitors of the multipolar outgrowing germ tubes.
Chemical patterns of actin and its isoforms in treated germlings
Immunoblots of one-dimensional SDS-poly- acrylamide gels of total proteins extracted from N crassa (4-6 h germination) revealed a single actin band of Mr 43 kDa when tested with anti- actin mAbs (Fig. 2). Benomyl and cytochalasin treatments did not induce any significant qualita- tive or quantitative changes in actin (Fig. 2). The same observation applied for tubulin after a benomyl treatment [26]. Moreover, preliminary data show that both drug treatments did not affect the levels of actin mRNAs.
MW kDa
94
._. - 67- 43
,..._ 30
- - 20,1
a b ; c d e
Fig. 2. One-dimensional SDS-PAGE (10% acrylamide) of total proteins (50 µ,g/slot) extracted from germlings of N crassa. Visualization of actin performed by immunoblotting with the monoclonal antibody N.350. (a) Total proteins stained with Ponceau S. (b) No treatment. (c) Treatment with 125 µ,M CA. (d) Treatment with 160 µ,M CB. (e) Treatment with 1 µ,M benomyl. MW: Standard low molecular mass markers.
I~··~·
IEFMr / \ \
l~-
Mr / \ \
'Y (3 a
b
'Y (3 aa
I·~·
IEF Mr / [ \c
'Y (3 aFig. 3. Two-dimensional gel electrophoresis (10% acrylamide) showing the three actin isoforms after treatment of conidia with the various cytoskeletal inhibitors. Experimental condi- tions as in Fig. 2. (a) No treatment. (b) Treatment with 125
µ,MCA. (c) Treatment with 1 µ,M benomyl.
It is known that CA or CB prevent polymeriza- tion of G- to F-actin [8]. The fact that the total actin content of conidia was similar when out- growth was inhibited by treatment with either cytochalasin A or B, suggests that it is that funda- mental filamentization which is required for po- larized outgrowth during normal germination of conidia.
Immunoblotting of two-dimensional gel elec- trophoresis of the same extract revealed three actin isoforms (a, f3 and y) with a predominance of {3-actin (p/ = 6.4) (Fig. 3). Such predominance of the f3 isoform is also known in the smooth muscle cells during proliferation [27]. Similarly, chemical treatments did not induce any qualita- tive or quantitative change in the proportions of the isoforms (Fig. 3). Unfortunately, an explana- tion of this individualized role of each of the actin isoforms is still elusive. It would be interest- ing to know whether, among the three isoforms, one polymerizes quicker than the others and thereby would have more significance for the process of polarized growth.
266
Acknowledgements
We are grateful to Dr. C. Rossier for critical reading of the manuscript. We thank Mrs. E.
Martinez and Mr. R. Hachadourian for photo- graphic work and Mrs. A. Fehr for typing the manuscript.
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