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Monoclonal antibodies inhibiting mating reactivity exclusively of odd mating types in Paramecium caudatum
Europ. J. Protisto!' 32, Supp!. I: 4 - 9 (1996) October 31, 1996
European Journal of
PROTISTOLOGY
Monoclonal Antibodies Inhibiting Mating Reactivity Exclusively of Odd Mating Types in Paramecium caudatum Yoichiro Azuma, Emi Kaku, Fumiaki Maruo, and Mihoko Takahashi Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan
SUMMARY Paramecium caudatum has the bipolar system of mating types and thus each syngen consists of two complementary mating types, even and odd types. Mating types are distinguished by the agglutination reaction when complementary mating types are mixed, but the substances (called the mating type substance) involved in specific cell recognition between complementary mating types have not so far been identified as the molecules in Paramecium. To identify the mating type substances we obtained 5 monoclonal antibodies inhibiting the mating reactivity of odd mating type of syngen 3. Cilia detached from the highly reactive odd mating type, pretreated with salt alcohol, were injected into mice and 5 monoclonal ant ibodies (OmA, OmB, OmC, OmD and OmE) were obtained after screening. All of them inhibited mating reactivity of all odd strains of syngen 3 examined but of no even strain without inhibiting action on the ciliary movement. Among 7 other syngens than syngen 3, cross reactivity in inhibition of mating reactivity, against odd types was observed to some degree in syngen 1, 5, 13, but not in syngen 4, 6, 12 when the four antibodies except OmB were used. Thus, the inhibiting action has no syngen specificity. Localization of the antigen recognized by these antibodies was examined by indirect immunofluorescence. Immunofluorescence was observed on the cilia of the ventral surface, mostly in the anterior tip which is known to be reactive in mating clump formation, and looked like beads in a line on the surface of the cilia. The mating type specificity of the inhibiting action and the localization on the cell strongly suggest that these antibodies recognize the mating type substances .
Introduction When cells of complementary mating types in Paramecium caudatum are mixed together under appropriate physiological conditions, they undergo massive agglutination which eventually leads to conjugation. In this agglutination, the specific cell-to-cell recognition between complementary mating types takes place under the interactions between mating type substances [12] which are located on the cilia of the ventral surfaces of highly reactive complementary mating types in Paramecium [6]. No substances excreted from cells into the medium are involved [18]. Though indirect 0932-4739-96-0032-0004$3.50-0
evidences suggested the substances to be protein [8,11], none has so far succeeded in isolating the substances from the cilia. Immunological methods, if available, might be useful to identify and monitor the isolation of the mating type substance s. Antibodies blocking the mating reaction were first reported by Hiwatashi and Takahashi [9], but the antibody had no mating -type specificity. Similar antibodies were also reported in P. multimicronucleatum [1]. Mating-type specific antibodies have once been obtained by Sasaki et al. [15], but no further results are so far reported. To obtain antibodies against the mating type substances, removal of immobilization antigen [2, 14] might be © 1996 by Gu stav Fischer Verlag
Mating-Type Specific antibodies of Paramecium · 5
necessary, because the immobilization antigen is a major protein on the ciliary preparation and has a very high antigenic activity [17]. We succeeded in obtaining mating type specific monoclonal antibodies by injecting cilia which were pretreated with salt-alcohol to remove the immobilization antigen [14].
10,000 g for 30 min. Precipitated cilia were washed by the same centrifugation twice in K-DS. To remove the immobilization antigens, packed cilia were then treated with 30 ml of the solution containing 0.45% NaCl, 15% EtOH and 10 mM Tris-HCI (pH 7.2)[14] stirring for 6 h at 4 °C. After washing with 10 mM Tris-HCI (pH 7.2), the cilia were suspended in phosphate-buffered saline (PBS) and used as antigens .
Production of monoclonal antibodies
Material and Methods
Strains and culture The strain used for immunization was G3, an odd mating type belonging to syngen 3 (designated 0 3).To test the mating reactivity of 0 3, stra in C103s7 (a selfing progeny of C103, kindly supplied by T. Watanabe, Tohoku University) belonging to even mating type of syngen 3 (designated E3) was used as a tester. Other strains used of even mating type were N93 079 (EI), GTS05 (E2), Kyk402 (E3), YD-6 (E4 ) , N3024 (ES) , Yr-1 (E6),Y03-1 (EI2) and GT808 (EI3) . Strains of odd mating type used are listed in Table 2. Cells were cultured at 25 °C in 2.5% (w/v) fresh lettuce juice medium [7] diluted with modified Dryl's solution (called K-DS), prepared by substituting KH 2P04 for NaH 2P04 [3], and inoculated with Klebsiella pneumoniae one day before use.
Five hundred pi of the ciliary preparation (1- 2 mg/ml) were injected intra peritoneally into BALB/c mice 7 times in 1- to 2-week intervals. Three days after the last boost, the spleen cells were fused with PJ -X63-Ag8-653 mouse myeloma cells by polyethylene glycol (1500; Boehringer Mannheim) according to the method described by Galfer and Milstein [4]. Culture fluids from the hybridoma cells were subjected to screening. Fifty pi of the highly reactive cell suspension of G3 was incubated with the same volume of culture fluid of hybridomas for 20 min after adding 50 III of 50 mM Tris-HCI (pH 7.2), and then cells of a highly reactive complementary mating type (tester) were added. The hybridomas secreting the antibodies which inhibited mating reactivity of G3 were cloned by limiting dilution. Determination of IgG subclass of monoclonal antibodies were performed by using a mouse monoclonal antibody isotyping kit (Amersham).
Preparation of antigen Cilia detached from cells were obtained by the modified method of the previous repor t [18]. About 1.5 x 10 6 mating-reactive cells were washed by slow centrifugation (200 g, 2 min) in K-DS and then treated with ice-cold Triton X-100 solution which conta ined 0.005% (v/v) Triton X-100 (octylphenozypolyethoxyethanol), 10 mM of the tripotassium salt of EDTA (ethylenediaminetetraacetic acid), 20 mM KCI and 10 mM Mops buffer (pH 7.0 adjusted by NaOH) . After 15 min of this treatment, the cells were centr ifuged (200 g, 2 min) and transferred into ice-cold deciliation solution of 20 mM CaCI 2 in 10 mM Tris-HCI (pH 7.2). The deciliated cell bodies were precipitated by slow centrifugation (200 g, 2 min). Supernatant was then filtered through a sheet of filter paper (TOYO SA). The filtrat e was centrifuged at
Indirect immunofluorescence Cells in an eppendorf tube were fixed with 4% formalin in 2 mM phosphate buffer (pH 7.0) at room temperature for 20 min and washed 3 times with PBS containing 0.01 % Triton-Xl 00. The cells were incubated with culture supernatant of the hybridoma containing monoclonal antibodies (1:4 dilution in PBS) for 2 h at room temperature. After being washed 3 times with PBS, the cells were incubated with fluorescein-conjugated goat anti-mouse IgG (Kappel, 1:100 dilution) for 2 h at room tempe rature and then washed 3 times with PBS. The treated cells were mounted in 90% glycerol with 1% p-phenylene diamine and viewed with a Nikon microscope or a laser scanning confocal microscope (BioRad MRC-500) .
Table 1. Mating reactivity of the strain G3 (mating type 0 3) when treated with various antibodies. Cells of G3 were incubated for 20 min in diluted supernatant of the hybridoma containing the antibodies, and then tester cells of complementary mating type, E3, were added . a IgG subclass. - indicates no mating reaction ; ± weak mating reaction and +, strong mating reaction . The mark of - , ±, + in triplicate in every point indicate the results of three independent experiments . Monoclonal antibodies Dilution(lIx)
Myeloma OmA-3 (IgG I
8 16 32 64 128 256 512 no antibody
--± ±++ +++ +++ +++ +++
OmB-l IgG]
±++ +++ +++ +++ +++
OmC-1 IgG2b
- ±± +++ +++ +++
OmD -7 IgG I
--± ±±± +++ +++ +++
OmE-1 IgG I')
+++ +++
+++ +++ +++ +++ +++ +++ +++ +++
6 . Y. Azuma, E. Kaku, F. Maruo, and M . Takahashi
Determination of inhibition of mating reactivity Fifty ,Ill of the highly reactive cell suspension was incuba ted with the same volume of culture fluid of hybridomas diluted with K-OS containing 2 mM KCI for 20 min and then cells of the tester were added . Mating reactivity was observed 1015 min after adding the tester. The degree of inhibition of mat ing reactivity by antibodies was estimated by the size of the clumps or agglut inates compared with that of when the cells without treatment by antibo dies were mixed with the comp lementary mating type. Culture fluid of myeloma cells was used as the control to monoclonal antibodies .
higher than one fourth dilution, the mating reactivity was slightly weakened. Therefore, one eighth dilution of the culture fluid was used as the highest concentration of antibodies for testing living cells. The inhibition of mati ng reaction was observed in dilutions as high as 1/32 to 1/256 without ciliary immobilization . Whenever no mating reaction was observed, no pair formation was induced. Altho ugh all of them were derived from the same mouse, and all antibodies were identified as IgG class antibodies, OmC is different from the others, being the only one that belongs to IgG 2b subclass while the others belong to IgG t •
Results
Cross reaction to other syngens Inhibition of mating reactivity of the strain G3, mating type 0 3 After screening, 5 monoclonal antibodies, OmA, OmB, OmC, OmD and OrnE, inhibiting mating reactivity of 0 3were obtained. The inhibition profiles of the 5 antibodies are shown in Table 1. When cells of G3 were incubated for 20 min in the culture fluid of myeloma cells (control), the concentrations of which are
In the next experiment, syngen specificity of the inhibitory effects of antibodies was examined. To test the inhibition of mating reactivity, cells of odd mating types were incubated for 20 min in diluted supernatant of the hybridoma containing the antibodies and then tester cells of complementary even mating types were added. Since the highest effective dilutions were different depending on the hybridoma culture, inhibi-
Table 2. Mating-reaction inhibit ing effects of the antibodies on odd mating types of 8 syngens. + indicates that strong inhibition in the same dilution as those effective to G3 was observed . ± indicates that weak inhibition was observed. - indicates that the inhib ition was not observed in repeated experiments. NO indicates that the examination was not done . Antibodies Syngen
Strain
Myeloma OmA-3
1
N93 -035 N93-043 N216-5 RcFl -4 RcFl-5 RcFl -20
2
GT704 GT801
3
G3 Ym3 Kym
4
N93 -027
5
YO-1 N93 -018 N93 -020 N93 -021 N93-023
6
Houbenl BO-4
12
Yo16 N3 -011 dM2 -22 GTl05
13
Hoe6 AKl GT702
+ + +
OmB-l
OmC -l
OmO-7
OmE -1
±
+ +
+
+
+ ±
NO NO NO
+ + +
+ + +
± ± ±
+ + +
± ±
±
+ + +
± ± ±
+ ±
NO
±
± ± ± ±
± ±
±
+ + +
+ + +
±
±
+ + +
± ± ±
+
±
±
±
Mating-Type Specific antibodies of Paramecium· 7
Fig. 1. Localization of the antigen recognized by the antibody, OmA3. Cells of mating reactive G3 were fixed with 4% formalin and imrnunostained with the monoclonal antibody, OmA-3, followed by FITClabeled goat antimouse immunoglobulin. Laser scanning confocal microscopic image (A) and Nomarsky interference image (B) of the same cell are shown. The arrow indicates the region of oral apparatus (out of focus) . (C) A part of the cell in a higher magnification, immunofluorescence looks like beads in a line on the surface of the cilia. Bars = 50.um in (B) and l0.um in (C) .
HF29-1a.Tif
8 . Y. Azuma, E. Kaku, F. Maruo, and M . Takahashi
tion was determined always by comparison with the effect on the strain G3. Although 16 syngens have been known in P. caudatum [17], only 8 syngens, among which 4 have been checked with the 16 [5], are available for experiments in Japan. All five monoclonal antibodies were effective on all odd type strains of syngen 3 examined (Table 2). Among the five antibodies, OmB seems to be syngen 3 specific. Four other antibodies showed cross reactions in some degree to syngen 1, 5 and 13 but not to syngen 4, 6 and 12. Thus the syngen specificity of OmB is different from the other four.
Effects of antibodies on even mating types When the antibodies were added to a mating clump formed between cells of complementary mating types of syngen 3, dissociation of the clump was observed immediately after the addition of antibodies. This is due to the odd mating type specific inhibition of antibodies mentioned above. Therefore, to find the effects on even mating types the inhibitory effects of the antibodies on the tester odd type cells have to be avoided. To fulfill this requirement, some cells of even mating type, after 20 minutes of incubation in the antibodies, were transferred to a large volume of the tester odd type cell suspension. By this method, even if some odd type specific antibodies are introduced with the even type cells, they must be diluted out with a large volume of the tester cell suspension and give no effect on the mating reaction. No inhibitory effects on even mating types were observed in either strain of the eight syngens (see Material and Methods) tested.
bodies which specifically inhibit the mating reactivity of odd mating types. The following evidences strongly suggest that those monoclonal antibodies are recognizing the mating type substances. 1) Their inhibitory actions against mating reactivity are strictly mating type specific. 2) They show no inhibiting action on ciliary movement and thus exclude the possibility of side effects of ciliary immobilization [13]. 3) Experiments using fluorescent antibodies clearly show that the binding sites of the antibodies are limited to the surfaces of cilia at the anterior ventral side of the cell known to be mating reactive [6]. 4) The binding of the fluorescent antibodies are limited to cells in the stationary phase when the mating reactivity is expressed. As described in the results, the immunofluorescence appeared on the surface of the cilia as dots arranged like beads. This arrangement of the immunofluorescence was the same irrespective of the stage of antibody application, before or after fixation. It strongly suggests the actual distribution of the mating type substances on the ciliary surface. Cross reactions of inhibition observed in odd mating types of different syngens may reflect the similar nature of molecules of the odd mating type substances among syngens. Immunological approaches to the mating substances of Paramecium have been made by several investigators [1, 9, 10, 12]. None of those reports, however, fulfill the four requirements of specific antibodies against the mating type substances mentioned above . Thus, this work is the first visual presentation of the mating type substances since the discovery of the mating reaction by Sonneborn [16].
Localization of antigens recognized by the antibodies
If the inhibitory effects on mating reaction result from direct interactions between the antibodies and mating type substances, the antigens recognized by antibodies must be localized on the cilia of the ventral cell surface, because mating reactive cilia are localized on the ventral surfaces [6]. Figure 1 shows the immunofluorescence localization of the antigens recognized by the ant ibodies OmA-3. Under the laser scanning confocal microscope the immunofluorescence appeared on the ventral surface cilia, most strongly in the anterior tip (Fig.lA). In the higher magnifica tion, immunofluorescence looks like beads in a line on the surface of the cilia and clear dots appear at the base of the cilia (Fig. 1C). A similar localization was also observed in the other 4 antibodies, but immu nofluorescence was observed neither in cells of E3 nor in those of non mating-reactive 0 3 in the logarithmic growth pha se. Discussion Using a mating reactive cilia preparation from which immobilization antigens were removed by salt alcohol [14], we have succeeded in obtaining monoclonal anti-
Acknowledgements We thank Ms Uesaka for her correction of our English manuscript. This work was supported, in part, by Grantsin -Aid for Cooperative Research (A) No . 03304001 from the Ministry of Education, Science and Culture of Japan.
References 1 Barnet A. and Steers E. Jr. (1980): Antibodies blocking the mat ing reaction in Paramecium multimicronucleatum syngen 2. J. Protozool., 27, 103-107. 2 Beale G. H. (1954): The genetics of Paramecium aurelia. Cambridge Univ. Press, London. 3 Dryl S. (1959): Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool., 6, S. 25. 4 Galfer G. and Milstein C. (1981): Preparation of monoclonal antibodies: Strategies and procedures. Meth. Enzymol., 73, 3-46. 5 Gilman L. C. (1950): The position of Japanese varieties of Paramecium caudatum with respect to American varieties. BioI. Bull., 99, 348-349. 6 Hiwatashi K. (1961): Locality of mating reactivity on the surface of Paramecium caudatum. Sci. Rep. Tohoku Univ. Ser. 4, 27, 93-99.
Mating-Type Specific antibodies of Paramecium· 9 7 Hiwatashi K. (1968): Determination and inheritance of mating type in Paramecium caudatum. Genetics, 58, 373-386. 8 Hiwatashi K. and Kitamura A. (1985): Fertilization in Paramecium. In: Metz C. B. and Monroy A. (eds.): Biology of fertilization, pp. 57 - 85. Academic Press, Orlando Florida. 9 Hiwatashi K. and Takahashi M. (1967): Inhibition of mating reaction by antisera without ciliary immobilization in Paramecium . Sci. Rep. Tohoku Univ. Ser. 4, 33, 281-290. 10 Iwamoto M. and Fujishima M. (1995): Ventral surface cilia specific antigens of Paramecium caudatum. Zoo!' Sci., 12, s25. 11 Kitamura A. (1988): Mating-type substances . In: Gortz H. D. (ed.): Paramecium, pp. 85 -96. Springer-Verlag, Berlin, Heidelberg . 12 Metz C. B. (1954): Mating substances and the physiology of fertilization in ciliates. In: Wenrich D. H. (eds): Sex in micro-organisms, pp. 284-334 American Association for the Advancement of Science, Washington, D. C.
13 Metz C. B. and Fusco E. M. (1948): Inhibition of mating reaction in Paramecium aurelia with antiserum. Anat. Rec., 101, 654-655. 14 Preer J. R. Jr. and Preer L. B. (1959): Gel diffusion studies on the antigens of isolated cellular components of Paramecium. J. Protozoo!. 6, 88-100. 15 Sasaki S., Ito A., Kitamura A. and Hiwatashi K. (1972): Mating type specific antigen in Paramecium caudatum. Genetics, 71, S 55. 16 Sonneborn T. M. (1937): Sex, sex inheritance and sex determination in Paramecium aurelia. Proc. Natl . Acad. Sci. U. S. A. 23, 378-385. 17 Sonneborn T. M. (1957): Breeding systems, reproductive methods, and species problems in Protozoa. In: Mayr E. (ed.): The species problem, pp 155-324. American Association for the Advancement of Science, Washington, D. C.
18 Takahashi M., Takeuchi N. and Hiwatashi K. (1974): Mating agglutination of cilia detached from complementary mating types of Paramecium. Exp. Cell Res., 87, 415-417.
Key words: Paramecium - Mating type substances - Monoclonal antibodies - Mating type Mihako Takahashi, Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan
European Journal of
PROTISTOLOGY
Monoclonal Antibodies Inhibiting Mating Reactivity Exclusively of Odd Mating Types in Paramecium caudatum Yoichiro Azuma, Emi Kaku, Fumiaki Maruo, and Mihoko Takahashi Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan
SUMMARY Paramecium caudatum has the bipolar system of mating types and thus each syngen consists of two complementary mating types, even and odd types. Mating types are distinguished by the agglutination reaction when complementary mating types are mixed, but the substances (called the mating type substance) involved in specific cell recognition between complementary mating types have not so far been identified as the molecules in Paramecium. To identify the mating type substances we obtained 5 monoclonal antibodies inhibiting the mating reactivity of odd mating type of syngen 3. Cilia detached from the highly reactive odd mating type, pretreated with salt alcohol, were injected into mice and 5 monoclonal ant ibodies (OmA, OmB, OmC, OmD and OmE) were obtained after screening. All of them inhibited mating reactivity of all odd strains of syngen 3 examined but of no even strain without inhibiting action on the ciliary movement. Among 7 other syngens than syngen 3, cross reactivity in inhibition of mating reactivity, against odd types was observed to some degree in syngen 1, 5, 13, but not in syngen 4, 6, 12 when the four antibodies except OmB were used. Thus, the inhibiting action has no syngen specificity. Localization of the antigen recognized by these antibodies was examined by indirect immunofluorescence. Immunofluorescence was observed on the cilia of the ventral surface, mostly in the anterior tip which is known to be reactive in mating clump formation, and looked like beads in a line on the surface of the cilia. The mating type specificity of the inhibiting action and the localization on the cell strongly suggest that these antibodies recognize the mating type substances .
Introduction When cells of complementary mating types in Paramecium caudatum are mixed together under appropriate physiological conditions, they undergo massive agglutination which eventually leads to conjugation. In this agglutination, the specific cell-to-cell recognition between complementary mating types takes place under the interactions between mating type substances [12] which are located on the cilia of the ventral surfaces of highly reactive complementary mating types in Paramecium [6]. No substances excreted from cells into the medium are involved [18]. Though indirect 0932-4739-96-0032-0004$3.50-0
evidences suggested the substances to be protein [8,11], none has so far succeeded in isolating the substances from the cilia. Immunological methods, if available, might be useful to identify and monitor the isolation of the mating type substance s. Antibodies blocking the mating reaction were first reported by Hiwatashi and Takahashi [9], but the antibody had no mating -type specificity. Similar antibodies were also reported in P. multimicronucleatum [1]. Mating-type specific antibodies have once been obtained by Sasaki et al. [15], but no further results are so far reported. To obtain antibodies against the mating type substances, removal of immobilization antigen [2, 14] might be © 1996 by Gu stav Fischer Verlag
Mating-Type Specific antibodies of Paramecium · 5
necessary, because the immobilization antigen is a major protein on the ciliary preparation and has a very high antigenic activity [17]. We succeeded in obtaining mating type specific monoclonal antibodies by injecting cilia which were pretreated with salt-alcohol to remove the immobilization antigen [14].
10,000 g for 30 min. Precipitated cilia were washed by the same centrifugation twice in K-DS. To remove the immobilization antigens, packed cilia were then treated with 30 ml of the solution containing 0.45% NaCl, 15% EtOH and 10 mM Tris-HCI (pH 7.2)[14] stirring for 6 h at 4 °C. After washing with 10 mM Tris-HCI (pH 7.2), the cilia were suspended in phosphate-buffered saline (PBS) and used as antigens .
Production of monoclonal antibodies
Material and Methods
Strains and culture The strain used for immunization was G3, an odd mating type belonging to syngen 3 (designated 0 3).To test the mating reactivity of 0 3, stra in C103s7 (a selfing progeny of C103, kindly supplied by T. Watanabe, Tohoku University) belonging to even mating type of syngen 3 (designated E3) was used as a tester. Other strains used of even mating type were N93 079 (EI), GTS05 (E2), Kyk402 (E3), YD-6 (E4 ) , N3024 (ES) , Yr-1 (E6),Y03-1 (EI2) and GT808 (EI3) . Strains of odd mating type used are listed in Table 2. Cells were cultured at 25 °C in 2.5% (w/v) fresh lettuce juice medium [7] diluted with modified Dryl's solution (called K-DS), prepared by substituting KH 2P04 for NaH 2P04 [3], and inoculated with Klebsiella pneumoniae one day before use.
Five hundred pi of the ciliary preparation (1- 2 mg/ml) were injected intra peritoneally into BALB/c mice 7 times in 1- to 2-week intervals. Three days after the last boost, the spleen cells were fused with PJ -X63-Ag8-653 mouse myeloma cells by polyethylene glycol (1500; Boehringer Mannheim) according to the method described by Galfer and Milstein [4]. Culture fluids from the hybridoma cells were subjected to screening. Fifty pi of the highly reactive cell suspension of G3 was incubated with the same volume of culture fluid of hybridomas for 20 min after adding 50 III of 50 mM Tris-HCI (pH 7.2), and then cells of a highly reactive complementary mating type (tester) were added. The hybridomas secreting the antibodies which inhibited mating reactivity of G3 were cloned by limiting dilution. Determination of IgG subclass of monoclonal antibodies were performed by using a mouse monoclonal antibody isotyping kit (Amersham).
Preparation of antigen Cilia detached from cells were obtained by the modified method of the previous repor t [18]. About 1.5 x 10 6 mating-reactive cells were washed by slow centrifugation (200 g, 2 min) in K-DS and then treated with ice-cold Triton X-100 solution which conta ined 0.005% (v/v) Triton X-100 (octylphenozypolyethoxyethanol), 10 mM of the tripotassium salt of EDTA (ethylenediaminetetraacetic acid), 20 mM KCI and 10 mM Mops buffer (pH 7.0 adjusted by NaOH) . After 15 min of this treatment, the cells were centr ifuged (200 g, 2 min) and transferred into ice-cold deciliation solution of 20 mM CaCI 2 in 10 mM Tris-HCI (pH 7.2). The deciliated cell bodies were precipitated by slow centrifugation (200 g, 2 min). Supernatant was then filtered through a sheet of filter paper (TOYO SA). The filtrat e was centrifuged at
Indirect immunofluorescence Cells in an eppendorf tube were fixed with 4% formalin in 2 mM phosphate buffer (pH 7.0) at room temperature for 20 min and washed 3 times with PBS containing 0.01 % Triton-Xl 00. The cells were incubated with culture supernatant of the hybridoma containing monoclonal antibodies (1:4 dilution in PBS) for 2 h at room temperature. After being washed 3 times with PBS, the cells were incubated with fluorescein-conjugated goat anti-mouse IgG (Kappel, 1:100 dilution) for 2 h at room tempe rature and then washed 3 times with PBS. The treated cells were mounted in 90% glycerol with 1% p-phenylene diamine and viewed with a Nikon microscope or a laser scanning confocal microscope (BioRad MRC-500) .
Table 1. Mating reactivity of the strain G3 (mating type 0 3) when treated with various antibodies. Cells of G3 were incubated for 20 min in diluted supernatant of the hybridoma containing the antibodies, and then tester cells of complementary mating type, E3, were added . a IgG subclass. - indicates no mating reaction ; ± weak mating reaction and +, strong mating reaction . The mark of - , ±, + in triplicate in every point indicate the results of three independent experiments . Monoclonal antibodies Dilution(lIx)
Myeloma OmA-3 (IgG I
8 16 32 64 128 256 512 no antibody
--± ±++ +++ +++ +++ +++
OmB-l IgG]
±++ +++ +++ +++ +++
OmC-1 IgG2b
- ±± +++ +++ +++
OmD -7 IgG I
--± ±±± +++ +++ +++
OmE-1 IgG I')
+++ +++
+++ +++ +++ +++ +++ +++ +++ +++
6 . Y. Azuma, E. Kaku, F. Maruo, and M . Takahashi
Determination of inhibition of mating reactivity Fifty ,Ill of the highly reactive cell suspension was incuba ted with the same volume of culture fluid of hybridomas diluted with K-OS containing 2 mM KCI for 20 min and then cells of the tester were added . Mating reactivity was observed 1015 min after adding the tester. The degree of inhibition of mat ing reactivity by antibodies was estimated by the size of the clumps or agglut inates compared with that of when the cells without treatment by antibo dies were mixed with the comp lementary mating type. Culture fluid of myeloma cells was used as the control to monoclonal antibodies .
higher than one fourth dilution, the mating reactivity was slightly weakened. Therefore, one eighth dilution of the culture fluid was used as the highest concentration of antibodies for testing living cells. The inhibition of mati ng reaction was observed in dilutions as high as 1/32 to 1/256 without ciliary immobilization . Whenever no mating reaction was observed, no pair formation was induced. Altho ugh all of them were derived from the same mouse, and all antibodies were identified as IgG class antibodies, OmC is different from the others, being the only one that belongs to IgG 2b subclass while the others belong to IgG t •
Results
Cross reaction to other syngens Inhibition of mating reactivity of the strain G3, mating type 0 3 After screening, 5 monoclonal antibodies, OmA, OmB, OmC, OmD and OrnE, inhibiting mating reactivity of 0 3were obtained. The inhibition profiles of the 5 antibodies are shown in Table 1. When cells of G3 were incubated for 20 min in the culture fluid of myeloma cells (control), the concentrations of which are
In the next experiment, syngen specificity of the inhibitory effects of antibodies was examined. To test the inhibition of mating reactivity, cells of odd mating types were incubated for 20 min in diluted supernatant of the hybridoma containing the antibodies and then tester cells of complementary even mating types were added. Since the highest effective dilutions were different depending on the hybridoma culture, inhibi-
Table 2. Mating-reaction inhibit ing effects of the antibodies on odd mating types of 8 syngens. + indicates that strong inhibition in the same dilution as those effective to G3 was observed . ± indicates that weak inhibition was observed. - indicates that the inhib ition was not observed in repeated experiments. NO indicates that the examination was not done . Antibodies Syngen
Strain
Myeloma OmA-3
1
N93 -035 N93-043 N216-5 RcFl -4 RcFl-5 RcFl -20
2
GT704 GT801
3
G3 Ym3 Kym
4
N93 -027
5
YO-1 N93 -018 N93 -020 N93 -021 N93-023
6
Houbenl BO-4
12
Yo16 N3 -011 dM2 -22 GTl05
13
Hoe6 AKl GT702
+ + +
OmB-l
OmC -l
OmO-7
OmE -1
±
+ +
+
+
+ ±
NO NO NO
+ + +
+ + +
± ± ±
+ + +
± ±
±
+ + +
± ± ±
+ ±
NO
±
± ± ± ±
± ±
±
+ + +
+ + +
±
±
+ + +
± ± ±
+
±
±
±
Mating-Type Specific antibodies of Paramecium· 7
Fig. 1. Localization of the antigen recognized by the antibody, OmA3. Cells of mating reactive G3 were fixed with 4% formalin and imrnunostained with the monoclonal antibody, OmA-3, followed by FITClabeled goat antimouse immunoglobulin. Laser scanning confocal microscopic image (A) and Nomarsky interference image (B) of the same cell are shown. The arrow indicates the region of oral apparatus (out of focus) . (C) A part of the cell in a higher magnification, immunofluorescence looks like beads in a line on the surface of the cilia. Bars = 50.um in (B) and l0.um in (C) .
HF29-1a.Tif
8 . Y. Azuma, E. Kaku, F. Maruo, and M . Takahashi
tion was determined always by comparison with the effect on the strain G3. Although 16 syngens have been known in P. caudatum [17], only 8 syngens, among which 4 have been checked with the 16 [5], are available for experiments in Japan. All five monoclonal antibodies were effective on all odd type strains of syngen 3 examined (Table 2). Among the five antibodies, OmB seems to be syngen 3 specific. Four other antibodies showed cross reactions in some degree to syngen 1, 5 and 13 but not to syngen 4, 6 and 12. Thus the syngen specificity of OmB is different from the other four.
Effects of antibodies on even mating types When the antibodies were added to a mating clump formed between cells of complementary mating types of syngen 3, dissociation of the clump was observed immediately after the addition of antibodies. This is due to the odd mating type specific inhibition of antibodies mentioned above. Therefore, to find the effects on even mating types the inhibitory effects of the antibodies on the tester odd type cells have to be avoided. To fulfill this requirement, some cells of even mating type, after 20 minutes of incubation in the antibodies, were transferred to a large volume of the tester odd type cell suspension. By this method, even if some odd type specific antibodies are introduced with the even type cells, they must be diluted out with a large volume of the tester cell suspension and give no effect on the mating reaction. No inhibitory effects on even mating types were observed in either strain of the eight syngens (see Material and Methods) tested.
bodies which specifically inhibit the mating reactivity of odd mating types. The following evidences strongly suggest that those monoclonal antibodies are recognizing the mating type substances. 1) Their inhibitory actions against mating reactivity are strictly mating type specific. 2) They show no inhibiting action on ciliary movement and thus exclude the possibility of side effects of ciliary immobilization [13]. 3) Experiments using fluorescent antibodies clearly show that the binding sites of the antibodies are limited to the surfaces of cilia at the anterior ventral side of the cell known to be mating reactive [6]. 4) The binding of the fluorescent antibodies are limited to cells in the stationary phase when the mating reactivity is expressed. As described in the results, the immunofluorescence appeared on the surface of the cilia as dots arranged like beads. This arrangement of the immunofluorescence was the same irrespective of the stage of antibody application, before or after fixation. It strongly suggests the actual distribution of the mating type substances on the ciliary surface. Cross reactions of inhibition observed in odd mating types of different syngens may reflect the similar nature of molecules of the odd mating type substances among syngens. Immunological approaches to the mating substances of Paramecium have been made by several investigators [1, 9, 10, 12]. None of those reports, however, fulfill the four requirements of specific antibodies against the mating type substances mentioned above . Thus, this work is the first visual presentation of the mating type substances since the discovery of the mating reaction by Sonneborn [16].
Localization of antigens recognized by the antibodies
If the inhibitory effects on mating reaction result from direct interactions between the antibodies and mating type substances, the antigens recognized by antibodies must be localized on the cilia of the ventral cell surface, because mating reactive cilia are localized on the ventral surfaces [6]. Figure 1 shows the immunofluorescence localization of the antigens recognized by the ant ibodies OmA-3. Under the laser scanning confocal microscope the immunofluorescence appeared on the ventral surface cilia, most strongly in the anterior tip (Fig.lA). In the higher magnifica tion, immunofluorescence looks like beads in a line on the surface of the cilia and clear dots appear at the base of the cilia (Fig. 1C). A similar localization was also observed in the other 4 antibodies, but immu nofluorescence was observed neither in cells of E3 nor in those of non mating-reactive 0 3 in the logarithmic growth pha se. Discussion Using a mating reactive cilia preparation from which immobilization antigens were removed by salt alcohol [14], we have succeeded in obtaining monoclonal anti-
Acknowledgements We thank Ms Uesaka for her correction of our English manuscript. This work was supported, in part, by Grantsin -Aid for Cooperative Research (A) No . 03304001 from the Ministry of Education, Science and Culture of Japan.
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Key words: Paramecium - Mating type substances - Monoclonal antibodies - Mating type Mihako Takahashi, Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan