Immunocharacterization of actin and its immunofluorescent localization during the developmental gametophytic stages of <i>Allomyces arbuscula</i>

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Immunocharacterization of actin and its immunofluorescent localization during the developmental gametophytic stages of

Allomyces arbuscula

NGUYEN THI BICH, Nga, TURIAN, Gilbert

Abstract

Highly specific polyclonal antibodies against actin from Allomyces arbuscula were produced in rabbits, immunopurified by immunoblotting and specified with actin isolated from Neurospora crassa and mouse skeletal muscle. Used as immunofluorescence probes, they allowed localization of actin in the sequential gametophytic stages of the mould.

NGUYEN THI BICH, Nga, TURIAN, Gilbert. Immunocharacterization of actin and its

immunofluorescent localization during the developmental gametophytic stages of Allomyces arbuscula . FEMS Microbiology Letters , 1992, vol. 94, no. 3, p. 241-246

DOI : 10.1111/j.1574-6968.1992.tb05325.x

Available at:

http://archive-ouverte.unige.ch/unige:124609

Disclaimer: layout of this document may differ from the published version.

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FEMSLE 04956

Immunocharacterization of actin and its immunofluorescent localization during the developmental gametophytic stages

of Allomyces arbuscula

Bich-Nga Nguyen Thi and Gilbert Turian

Laboratory of General Microbiology, University of Geneua, Sciences III, Gene va, Switzerland

Received 10 April 1992 Accepted 4 May 1992

Key words: Allomyces arbuscula; Actin; Gametophyte; Immunofluorescence

1. SUMMARY

Highly specific polyclonal antibodies against actin from Allomyces arbuscula were produced in rabbits, immunopurified by immunoblotting and specified with actin isolated from Neurospora crassa and mouse skeletal muscle. Used as im- munofluorescence probes, they allowed localization of actin in the sequential gametophytic stages of the mould.

2. INTRODUCTION

We have recently isolated a 42-kDa protein from Allomyces arbuscula, purified by affinity chromatography on DNAse 1-Sepharose and identified as actin by immunoblots with a monoclonal antibody against chicken gizzard actin [l].

Using this purification procedure, a highly spe-

Correspondence to: G. Turian, Lab. Microbiologie générale, Université de Genève, Sciences III, 30, Quai Ernest-Anser- met, 1211 Genève 4, Switzerland.

cific polyclonal antibody against actin from A.

arbuscula has been prepared, and its specificity ascertained by comparison with actin isolated from Neurospora crassa [2] and mouse skeletal muscle. This paper reports the use of those anti- actin antibodies as immunofluorescence probes to localize actin along the sequential, gametophytic stages in this hypogynous species of Al- lomyces, namely meiospores, germ tubes, vegeta- tive and sexually differentiating hyphae.

3. MATERIALS AND METHODS 3.1. Organisms and growth conditions

The experimental Stumm strain of Allomyces arbuscula Butl. was grown and maintained on the standard YpSs agar medium. Meiospores liber- ated from resistant sporangia were germinated and their vegetative hyphae grown in the GCY liquid medium as described elsewhere [3]. Sexual differentiation of the gametangia was then in- duced by transfer of the vegetative hyphae into DS solution [4].

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The wild-type strain, St. Lawrence 74A of Neurospora crassa (FGSC 262) was obtained

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Fungal Genetics Stock Center, University of Kansas, Medical Center, Kansas City, KS. It was grown in the standard manner according to the method of Barja et al. [2].

3.2. Purification of anti-actin polyclonal antibodies Actin was purified as described previously [l].

Antisera against actin were produced by injecting 2 New Zealand white rabbits subcutaneously with 300 µ,g of electophoretically pure actin from A.

arbuscula emulsified with either complete (initial injection) or incomplete (subsequent injection) Freund's adjuvant. After a total of four injec- tions, done at three-week intervals, the rabbits were bled and the serum collected.

Affinity purified antibody was prepared according to the method of Cox et al. [5]. Purified actin was then electrophoresed under denaturing conditions and transferred to nitrocellulose by electroblotting [6]. Proteins were visualized by staining with 0.05% Ponceau S in 3% TCA. The 42-kDa subunit was excised and the nitrocellulose strip was destained in water. After incubation for 1 h in 3% gelatin in Tris-buffered saline (TBS) (50 mM Tris, pH 7.5, 500 mM NaCI) at 25°C, the strips were incubated with 4 ml of antisera (25%

sera in TBS) for 4 h at room temperature. The strips were subsequently washed twice with TBS containing 0.05% Tween-20 and once with TBS.

Bound antibody was eluted in 1.0 ml of 0.5 M glycine, pH 2.9, for 2 min in the presence of 100 µ,g of BSA. The eluted affinity-purified antibody was immediately neutralized with Tris base and stored in TBS containing 0.5% NaN₃at 4°C until use.

The specificity of the immunopurified anti- actin IG was tested by immunoblotting and immunofluorescence. Proteins eluted from DNase 1-Sepharose column (70% pure) were then elec- trotransferred to nitrocellulose membrane.

3.3. Immunoblotting

Proteins from unstained gels were elec- trophoretically transferred onto nitrocellulose paper BA 85 (pore size 0.45 µ,m, Schleicher and

Schuell, Dassel, FRG) using the transfer buffer described by Burnette [7]. The transferred proteins were stained with 0.05% (w /v) Ponceau Sin 3% (w /v) trichloracetic acid (TCA). The non- specific binding sites were blocked with 2% BSA in Tris-buffered Saline (TBS) (100 mM Tris· HCI pH 7.5, 500 mM NaCI) for 2 h at room

tempera~

ture. The nitrocellulose was then incubated for 4 h with anti-actin polyclonal antibodies diluted 1: 10 in TBS supplemented with 0.5% (v /v) Tween-20, 0.5% (w /v) BSA. After three washes in TBS, an incubation with secondary antibodies was performed for 1 h at room temperature with peroxidase-labeled sheep-anti-rabbit lgG (Amer- sham, UK) diluted 1 : 1000 in TBS. Following four washes, the peroxidase activity was revealed with 0.5 mg/ml of 3,3 diaminobenzidine tetrahydro- chloride (DAB) (F!uka Chemie, Buchs, Switzer- land) in 100 mM Tris· HCI, pH 7.5, containing 0.03% H₂0_{2 .}The reaction was stopped with dis- tilled water. The concentration of proteins was determined by Coomassie blue staining using BSA (Bovine serum albumin) as standard.

3.4. Fixation, wall digestion and membrane perme- abilization

The cells were fixed with 3.7% formaldehyde, 2.5% glutaraldehyde in the microfilaments-stabi- lizing buffer (MF-buffer) 10 mM imidazole, pH 7.0, containing 2 mM MgCl_{2 ,} 2 mM EGTA, 2 mM ATP, 1 mM PMSF, 20 µ,M leupeptin and 2% PEG-400 for 30 min at room temperature.

They were then treated with a 0.1 % sodium boro- hydride in phosphate-buffered saline solution (PBS), pH 7.3, for 2 h to quench the autofluores- cence of glutaraldehyde. The solution of borohy- dride was prepared immediately before use and changed twice to fresh medium during the treat- ment [8]. For the partial digestion of the wall, the cells were incubated for 45 min at 37°C with a lytic enzyme preparation in Sürensen's buffer (66.6 mM), pH 6.0. The enzyme mixture consisted of 2 mg/ml chitinase (Calbiochem, Lucerne, Switzerland), 5 mg/ml cellulase (Sigma, St. Louis, MO), and 55 µ,!/ml f3-glucuronidase (Sigma).

Digestion was stopped by rinsing three times in Sêirensen's buffer. The cell membrane of digested cells was finely permeabilized with 1 % Triton

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1 2 3

Fig. !. lmmunochemical characterization of actin: proteins were revealed by serum original (lane !). Recognition by the affinity-purified antibodies of the 42-kDa protein eluted from the column DNase 1-Sepharose (lane 2); contrai identification of the 42-kDa protein as actin by an anti-actin monoclonal

antibody raised against chicken gizzards (lane 3).

X-100 in Sorensen's buffer for 20 min at room temperature.

3.5. Immunofluorescence microscopy

Following a rinse with MF-buffer for 15 min and with TBS for 10 min, the preparations were incubated overnight at room temperature with the primary antibodies (anti-actin) at a dilution of 1 : 10 in TBS. Following a rinse in TBS, the preparations were incubated with Fluorescein- isothiocyanate (FITC) goat-anti-rabbit immu- noglobulin (Nordic Biogenzia Lemania, Lau- sanne, Switzerland) of 1 : 1000 dilution for 1 h at 37°C. A Leitz Orthoplan epiillumination micro- scope (Ernst Leitz, Wetzlar, FRG) equipped with fluotar optics and selective filter combination was used for viewing the FITC-fluorescence patterns.

Photographs were taken on HP 5 Ilford black and white film (Ilford, Base!, Switzerland).

4. RESULTS AND DISCUSSION

In the first phase of the immunochemical work, a few bands of proteins were revealed in the total serum anti-actin (Fig. 1, Jane 1). After their im- munopurification, the antibodies recognized a single 42-kDa protein (Fig. 1, Jane 2) identified as actin when tested with anti-actin monoclonal chicken gizzards (Fig. 1, Jane 3) as used previously [l].

The anti-actin specificity of our polyclonal antibody was confirmed by immunostaining colo- ration of actin isolated from both N. crassa ac- cording to Barja et al. [2] (Fig. 2, Jane 1) and mouse skeletal muscle as described previously by Mooseker [9] (Fig. 2, Jane 2).

In the second, cyto-immunochemical phase, the specific actin-antibody was used to detect the intracellular distribution of actin by immunofluorescence. The preservation of actin microfilaments and penetration of the antibodies through the chitinous thick wall of hyphae and gametan-

1 2

...

Fig. 2. Further detection of Allomyces anti-actin with actin purified from N. crassa by column DNase I-Sepharose, 70%

pure (lane 1) and from mouse skeletal muscle (lane 2).

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gia were ensured by the use of the enzymatic mixture described above.

In the meiospores (Fig. 3a), as in their elongating hyphal germ tube (Fig. 3b), immunofluorescence was found as a set of dots or patches. The same pattern has also been observed in Saproleg- nia ferax [10] and N. crassa hyphae [2]. lt was not possible to detect clear filamentous structures in these first developmental stages.

Immunofluorescence was generalized but with a subapical concentration in the vegetative hypha (Fig. 3c). This fluorescence moved into the apical

cytoplasm of sexually differentiating hyphae in which it appeared as a set of cytoplasmic dots among microfilaments (Fig. 3d). At the first stage of gametangial differentiation, a concentration of fluorescent dots appeared in the widening, club- like apical zone of sexually differentiating hyphae (Fig. 3e). This fluorescence probed with FITC- phalloidin was apparent as much more distinct and discrete regions in the female gametangia (Fig. 3f).

Our findings confirm previous works empha- sizing the apical localization of actin in hyphae

Fig. 3. Immunofluorescence with affinity-purified anti-actin antibodies of sequential stages of sexual differentiation in A. arbuscula:

meiospore (a); 5-h outgrown hyphal germ tube (two planes of focus, left one with arrows on dots) (b); 15-h-old elongating (directional arrow) vegetative hypha (c); 'club-like' stage of a sexually differentiating hypha (d); gametangial differentiation (arrow on the early intergametangial septum) (e); FITC-phalloidin probe with predominant, fluorescent dotted actin network in the apical

female gametangium (f). Bar= 5 µ,m.

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(see Gooday and Gow, ref. 11) and extend it to the initial differentiation stage of Allomyces ga- metangia.

ACKNOWLEDGEMENTS

We thank Dr. F. Barja and Dr. M. Ojha for their competent scientific advice, Dr. J. Fahrni (Department of Animal Biology) for his gift of actin standard mouse skeletal muscle, Mrs. A.

Cattaneo, Mr. R. Hachadourian and Mrs. E.

Martinez, Mrs. A. Fehr and F. Grange for their respective technical, photographie, and dactylo- graphie help. B.N.N.T. is grateful for her finan- cial support from La Commision fédérale suisse des bourses pour étudiants étrangers.

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[3] Turian, G., Schonenberger, I. and Nguyen Thi, B.N.

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[IO] Heath, LB. (1987) J. Cell Bio!. 44, 10-16.

[11] Gooday, G.W. and Gow, N.A.R. (1990) In: Tip growth in plant and fungal cells. (Heath, LB .. Ed.), pp. 31-58.

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