Transcription and Translation involved in Pellicle Formation in the Chlamydomonas reinhardtii Zygote

Transcription

1 1997 The Japan Mendel Society Cytologia 62 : , 1997 Transcription and Translation involved in Pellicle Formation in the Chlamydomonas reinhardtii Zygote Lena Suzuki 1, 2, *, Yasuhito Yuasa1 and Tsuneyoshi Kuroiwa2 Department of Hygiene and Oncology, Tokyo Medical and Dental University School of Medicine, Yushima, Tokyo 113, Japan 2 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113, Japan Accepted November 12, 1997 Multicellular organisms are constructed in two ways. In higher plants and animals, there is adhesion of cells during embryogenesis following fertilization, while cell aggregation occurs in animal cell cultures, in pseudoplasmodium in Dictyosterium discoideum and in pellicle formation in green algae (Harris 1989). There are many reports on the molecular mechanisms for the adhesion of animal cells (Alberts et al. 1995). However, no studies of the molecular mechanisms of adhesion or aggregation consistently explain the relationship between the transcription of a specific gene and the formation of multicellular organisms from its products. The pellicle the green alga Chlamydomonas reinhardtii is formed by the adhesion of zygotic cells soon after mating between mt+ and mt- gametes. It is a kind of multi-celled sheet. It confers physical strength and resistance to desiccation to the organism (Harris 1989). Pellicle formation in C. reinhardtii occurs soon after mating, making this system of pellicle formation very suitable for studies of the molecular mechanism of inter-cellular adhesion. The pellicle is formed by the zygote-specific wall (Cavalier-Smith 1976), which is constructed to replace the vegetative cell wall shed before mating (Claes 1971). The zygotic cell wall is composed of a fibrous material, which contains hydroxyproline as a major amino acid, glucose as the most abundant sugar residue, and a (1-3)ƒÀ-D-glucan as the major structural polysaccharide (Grief et al. 1987). At least six zygote specific peptides with molecular masses of 76 kda, 120 kda, 150 kda and 200 kda (A, B and C) have been identified as components of zygotic walls (Minami and Goodenough 1978). Zygote-specific cdna clones, which encoded the extensin-like proteins ZSP-1 and ZSP-2 (Woessner and Goodenough 1989), have also been obtained in previous studies (Ferris and Goodenough 1987). However, no gene was characterized that played a role in cell adhesion for pellicle formation, in Chlamydomonas. To elucidate the relationships between the pellicle and these peptides and genes, a detailed time schedule of gene expression in pellicle formation needs to be constructed. The aims of this study are to clarify when the pellicle is formed, and to estimate when mrna and proteins involved in pellicle formation are synthesized, by using transcription and translation inhibitors administered at various times after mating. Material and methods Strain and culture conditions The wild-type strains of C. reinhardtii, 137c mt+ and mt-, were used. Cells of the two mating types were grown on Medium I plates (Sager and Granick 1954) containing 1.2% agar. Five days Corresponding author: Lena Suzuki. Tel: , ext Fax: lena@biol.s.utokyo.ac.jp.

2 422 Lena Suzuki, Yasuhito Yuasa and Tsuneyoshi Kuroiwa Cytologia 62 after plating, the cells were collected with a glass spreader, suspended in mating buffer (0.6 mm MgC12, 1.2 mm HEPES (ph 6.8)) at a density of approximate 2-5 ~ 107 cell per ml, induced to form gametes, and then mixed to allow matings described in a previous report (Nakamura et al. 1986). Aliquots of the cell suspension at appropriate stages were used for the analysis described below. Assessment of pellicle formation Complete pellicle formation was defined as when 100% of the cells adhered to each other. The rate of pellicle formation was calculated by using the formula: % Pellicle formation= [ 100 -(X/N)] Where X is the number of non-aggregated zygotic cells and N is the total number of zygotic cells. Numbers of non-aggregated cells were counted at different times, using hemacytometer after fixation with 1% glutaraldehyde and vigorously vortexing aliquots of the cell suspension. Treatment with inhibitors Actinomycin D (Sigma) and rifampicin (Wako, Osaka, Japan) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/ml. Cycloheximide (Sigma) and chloramphenicol (Wako, Osaka, Japan) were dissolved in ethanol at concentrations of 10 mg/ml and 34 mg/ml, respectively. Stock solutions of these inhibitors were stored at-20 C until use, and then added to the cell suspensions in mating buffer to give the final concentrations used in previous studies (Kuroiwa et al. 1983a, b) (actinomycin D, 20ƒÊg/ml; cycloheximide, 10ƒÊg/ml; rifampicin, 80 ƒêg/ml, and chloramphenicol, 80ƒÊg/ml). Each inhibitor was added to the mating medium at eight different stages: 30 min before the gametes were mixed, and 5 min, 10 min, 20 min, 40 min, 1 hr, 2 hr and 3 hr after mating. Pellicle formation was assessed 8 hr after mating for each inhibition series. Results Pellicle formation was observed with phase contrast microscopy as growing clusters of zygotic cells. Solitary zygotic cells swam actively until up to 2 hr after gamete mixing (Fig. 1A). At 2.5 hr, small aggregations of zygotic cells were observed for the first time (Fig. 1B). These aggregations continued growing until 3 hr after gamete mixing (Fig. 1C). This was quantified by subtracting the number of non-aggregated cells from the total number of zygotic cells per ml medium. The results of these observations determined that pellicles were formed synchronously between 2.5 and 3.5 hr after mating (Fig. 1D). Inhibitor treatment with four antibiotics was perfomred, to clarify whether the transcription/ translation necessary for pellicle formation occurred in the cytoplasm or organelles (i.e. plastids and mitochondria). Inhibitors of organelle transcription (rifampicin) and translation (chloramphenicol) given at the gametic stage had no effect on pellicle formation. On the other hand, inhibitors of cytoplasm transcription (actinomycin D) and translation (cycloheximide) completely inhibited pellicle formation (Table 1). This indicates that pellicle formation requires de novo transcription and translation in the cytoplasm after gamete mixing. To estimate when this transcription and translation occurs, the inhibitors were given at various times after mating. Actinomycin D completely inhibited pellicle formation when it was given 5 min after mating, but partial pellicle formation occurred when this inhibitor was given 10 min after mating. Actinomycin D had no effect on pellicle formation when it was given 20 min or more after mating (Fig. 2). This suggests that the minimum transcription period for pellicle formation is from 10 to 20 min after gamete mixing. Cycloheximide administered up to 1 hr after mixing resulted in complete inhibition. However,

3 1997 Zygote Specific Gene Expressions (1) 423 A B c D Fig. 1. Phase contrast images of pellicle formation. (A), (B) and (C) show zygotic cells, before and during pellicle formation, at 2, 2.5 and 3.5 hr after mating, respectively. The cluster that formed from adhesion of zygotic cells grew as time passed. The bar indicates 100 Đm. (D) Quantitative assessment of pellicle formation. The rate of zygotic cell adhesion, expressed as % pellicle formation, was calculated from the formula: % pellicle formation= [10-X/N)] (X, number of non-aggregated zygotic cells; N, total number of zygotic cells, per ml medium). Table 1. Inhibition of transcription/translation in the cytoplasm and the organelles on pellicle formation Inhibitors were given to gametes 30 min before mating, and observed at 8 hr after mating. Relative percentages against the control values are shown. * The same concentrations given with chloramphenicol and rifampicin as a solvent of the stock solutions, respectiely.

4 424 Lena Suzuki, Yasuhito Yuasa and Tsuneyoshi Kuroiwa Cytologia 62 Fig. 2. The effects of antibiotics on pellicle formation. Either 20ƒÊg/ml actinomycin D or 10ƒÊg/ml cycloheximide was given to gametes and zygotes at various stages. Pellicle formation was evaluated 8 hr after mating. the addition of cycloheximide 2 hr or more after mixing had no effect on pellicle formation. These results suggest that translation occurs between 1 and 2 hr after mating. Discussion The results of this study reveal the periods required for pellicle formation and for mrna and protein synthesis. They imply that pellicle-inducing protein(s) is (are) encoded by a zygote-specific gene, which initiates transcription within 10 min of mating. The Pellicle Formation Inducing gene might be one of the early zygote-specific genes identified in previous studies (Ferris and Goodenough 1987, Uchida et al. 1993). Possible candidates are zsp-1 /class IV and zsp-2/class IV, which encode extensin-like zygote wall proteins (Woessner and Goodenough 1989). On the other hand, the zygote specific class V gene, which is suspected of encoding cysteine-rich zygotic wall protein, can be removed from the list of candidates because this gene is expressed 90 min after mating (Matters and Goodenough 1992). However, no specific gene was identified to cause adhesion of zygotic cells, and proteins unrelated to adhesion are probably found in the zygote wall. The characteristics of pellicle-inducing factor, as distinguish from non-pellicle wall proteins, are well illustrated by this and previous studies of the zygote cell wall and pellicle. Electron microscopic observations (Cavalier-Smith 1976, Grief et al. 1987, Minami and Goodenough 1978) suggest that adhesion of zygotic cells results from interaction between the pellicle material layer, the outermost layer of the zygote-specific cell wall, and the zygotic extracellular matrix, which consists of a complex of 4-8 layers formed successively from the outermost layer in. Hence, the product(s) of the Pellicle Formation Inducing gene is (are) expected to be localized to the outermost layer of the extracellular matrix protein(s) during the pellicle formation period hr after mating, although the pellicle inducing protein is synthesized before the pellicle formation period, between 1 and 2 hr after mating. It is suspected that the pellicle inducing protein is either not localized in the pellicle material layer, or is localized in an adhesioninactivated from, before the pellicle formation period. Further searches for the pellicle inducing gene are now in progress.

5 1997 Zygote Specific Gene Expressions (1) 425 Summary The pellicle, filmy aggregate of zygotic cells formed in the green alga Chlamydomonas reinhardtii, was characterized as a model of inter-cellular adhesion. Pellicle formation was observed following treatment with inhibitors of transcription and translation in the gamate and at various times after mating. The pellicle forms between 2.5 and 3.5 hr after gamete mixing and is sensitive to cytoplasmic inhibitors of transcription and translation, when they are mixed with gametes or zygotes soon after mating. This indicates that the pellicle formation related gene(s) is (are) one of the early zygote-specific genes, and that transcription occurs within the first 20 min after gamete mixing. The protein(s) required for pellicle formation is (are) synthesized predominantly between 1 and 2 hr after mating, just before pellicle formation. Acknowledgments The authors would like to thank Dr. H. Takano and Dr. A. Sakai for their helpful discussions and advice. This work was supported by a Grant for Special Promoted Research (project no ) to T. K. from the Ministry of Education, Science, Sports and Culture of Japan. References Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J. D. (eds.) Molecular biology of the cell (3rd Ed.). Garland Publishing, Inc., New York, London. Cavalier-Smith, T Electron microscopy of zygospore formation in Chlamydomonas reinhardii. Protoplasma 87: Claes, H Autolyse der zell wand bei den gameten von Chamydomonas reinhardtii. Arch. Mikrobiol. 78: Ferris, P. J. and Goodenough, U. W Transcription of novel genes, including a gene linked to the mating-type locus, induced by Chlamydomonas fertilization. Mol. Cell. Biol. 7: Grief, C., O'Neill, M. A. and Shaw, P. J The zygote cell wall of Chlamydomonas reinhardii: a structural, chemical and immunological approach. Planta 170: Harris, E. H The Chlamydomonas source book. San Diego: Academic. Kuroiwa, T., Kawano, S. and Sato, C. 1983a. Mechanisms of maternal inheritance. I. Protein synthesis involved in preferential destruction of chloroplast DNA of male origin. Proc. Japan Acad. 59: \, \and \1983b. Mechanisms of maternal inheritance. II. RNA synthesis involved in preferential destruction of chloroplast DNA of male origin. Proc. Japan Acad. 59: Matters, G. L. and Goodenough, U. W A gene/pseudogene tandem duplication encodes a cysteine-rich protein expressed during zygote development in Chlamydomonas reinhardtii. Mol. Gen. Genet. 232: Minami, S. A. and Goodenough, U. W Novel glycopeptide synthesis induced by gametic cell fusion in Chlamydomonas reinhardtii. J. Cell Biol. 77: Nakamura, S., Itoh, S. and Kuroiwa, T Behavior of chloroplast nucleus during chloroplast development and degeneration in Chlamydomonas reinhardtii. Plant Cell Physiol. 27: Sager, R. and Granick, S Nutritional control of sexuality in Chlamydomonas reinhardi. J. Gen. Physiol. 3: Uchida, H., Kawano, S., Sato, N. and Kuroiwa, T Isolation and characterization of novel genes which are expressed during the very early stage of zygote formation in Chlamydomonas reinhardtii. Curr. Genet. 24: Woessner, J. P. and Goodenough, U. W Molecular characterization of a zygote wall protein: An extensin-like molecule in Chlamydomonas reinhardtii. The Plant Cell. 1: