NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF CELL PROLIFERATION

(Adjunct)

Professor:
Masayuki Yamamoto
Research Associate:
Masuo Goto
Institute Research Fellow:
Takashi Kuromori 1
Visiting Fellow:
Akira Yamashita
Satsuki Okamoto


The major goal of this Division is to elucidate regulatory mechanisms of meiosis. Meiosis is a crucial step in gamete formation and is essential for sexual reproduction. Meiotic steps are highly conserved among eukaryotic species. We have been screening for genes relevant to the regulation of meiosis in animals and plants, by isolating genes that either complement Schizosaccharomyces pombe meiotic mutants or affect the meiotic behavior of this fission yeast. We have isolated a number of animal and plant genes in this manner, and analysis of the cloned genes is in progress. In addition, efforts have been also paid to elucidate the regulatory mechanisms of meiosis in fission yeast in more detail, so that the above strategy may be reinforced. Some examples of the analysis are presented below.


I. Microtubule-binding protein which functions specifically during meiosis.

In fission yeast, an RNA species named meiRNA is specifically required for the promotion of the first meiotic division. To dissect the function of this RNA and its partner RNA-binding protein Mei2p, we screened for high-copy-number suppressors of the arrest prior to the first meiotic division caused by loss of meiRNA. Analysis of one of the suppressors thus isolated, named ssm4, suggested that it encodes a coiled-coil protein carrying a microtubule-binding motif at its N-terminus. Expression of ssm4 was restricted to cells undergoing meiosis. Disruption of ssm4 affected neither vegetative growth nor conjugation, but resulted in frequent generation of asci carrying less than four spores. Tagged Ssm4p localized at spindles during both the first and the second meiotic division. The microtubule-binding motif was essential for the association of Ssm4p with microtubules and for its function during meiosis, but not for the suppression of loss of meiRNA. Ssm4p appeared to possess a potential to migrate to the nucleus. Thus, Ssm4p is a microtubule-colocalizing protein that plays a role specifically in meiosis. Ssm4p appears to modify the structure or the function of nuclear microtubules in order to promote the meiotic nuclear division.


II. Arabidopsis 14-3-3 isoform.

14-3-3 proteins constitute a highly conserved family among eukaryotes, and diverse biochemical activities have been ascribed to them. We isolated a variety of Arabidopsis 14-3-3 genes in a screening for cDNAs that could block ectopic meiosis driven by the pat1 mutation in fission yeast. In addition to suppressing the pat1 mutation, these cDNAs could cure the deformed morphology and raised UV sensitivity of fission yeast cells defective in rad24, which encodes a 14-3-3 protein. This indicates that most Arabidopsis 14-3-3 proteins can execute the function of the fission yeast 14-3-3 protein encoded by rad24, at least partially. We analyzed one of the Arabidopsis 14-3-3 clones in more detail, as it was not fully characterized in literature. This 14-3-3 gene turned out to be expressed in all plant organs examined.

Fig. 1
Cellular localization of S. pombe type II myosin heavy chain Myo2p fused to the jellyfish green fluorescent protein (GFP) and that of S. pombe actin. Cells grown to the mid-log phase were fixed, immediately stained with Phalloidin-TRITC, and observed under the microscope as rapidly as possible. Micrographs show GFP fluorescence of Myo2p (A) and phalloidin-TRITC-stained actin (B).


III. Identification of fission yeast type II myosin heavy chain.

We cloned the myo2 gene of Schizosaccharomyces pombe, which encodes a type II myosin heavy chain, by virtue of its ability to promote meiosis in certain mutants. The myo2 gene encoded 1526 amino acids, and deduced Myo2p showed homology to the head domains and the coiled-coil tail of the conventional type II myosin heavy chain, and carried putative binding sites for ATP and actin. Disruption of myo2 inhibited cell proliferation. myo2-defective cells showed normal punctate distribution of interphase actin, but they produced irregular actin rings and septa, and were impaired in cell separation. Overproduction of Myo2p was also lethal, apparently blocking actin relocation. Nuclear division proceeded without actin ring formation and cytokinesis in cells overexpressing Myo2p, giving rise to multinucleated cells with dumbbell morphology. Analysis using tagged Myo2p revealed that Myo2p colocalizes with actin in the contractile ring, suggesting that Myo2p is a component of the ring and responsible for its contraction. Furthermore, genetic evidence suggested that the acto-myosin system may interact with the Ras pathway, which regulates mating and the maintenance of cell morphology in S. pombe.


Publication List:
Hirayama, T., Ishida, C., Kuromori, T., Obata, S., Shimoda, C., Yamamoto, M., Shinozaki, K., and Ohto, C. (1997). Functional cloning of a cDNA encoding Mei2-like protein from Arabidopsis thaliana using a fission yeast pheromone receptor deficient mutant. FEBS Lett. 413, 16-20.
Imai, Y., Davey, J., Kawagishi-Kobayashi, M., and Yamamoto, M. (1997). Genes encoding farnesyl cysteine carboxyl methyltransferase in Schizosaccharomyces pombe and Xenopus laevis. Mol. Cell. Biol. 17, 1543-1551.
Kitayama, C., Sugimoto, A., and Yamamoto, M. (1997). Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe. J. Cell Biol. 137, 1309-1329.
Yamashita, A., Watanabe, Y., and Yamamoto, M. (1997). Microtubule-associated coiled-coil protein Ssm4 is involved in the meiotic development in fission yeast. Genes Cells 2, 155-166.

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