|Budget Amount *help
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥5,300,000 (Direct Cost: ¥5,300,000)
In mesophyll cells of the aquatic angiosperm Vallisneria gigantea, bundles of microfilaments(MFs)serve as the tracks for rotational streaming of the cytoplasm, which occurs along the two longer side walls and the two shorter end walls. The stationary organization of these bundles has been shown to depend on the association of the bundles with the cell wall through the plasma membrane at the end walls(Masuda et al., 1991). To confirm further the sites of such association and to get further insights on the mechanism of the stationary organization of the MF bundles, the effects of cytochalasin B(CB)on the configuration of the bundles of MFs were first examined. In the side walls, MFs were completely disrupted after treatment with CB at 100mug/ml for 24 h. By contrast, in the end walls, a number of partially disrupted MFs remained even after 48 h of treatment. After removal of CB,a completely normal arrangement of bundles of MFs was once again evident within 24 h after a rather complicated
process of reassembly. When reassembly had been completed, the direction of cytoplasmic streaming was reversed only in a small fraction of the treated cells, suggesting that bundles of MFs are anchored and stabilized at the end walls of each cell and that the polarity of reorganized bundles and, therefore, the direction of the cytoplasmic streaming is determined in a manner that depends on the original polarity of MFs that remained in spite of the disruptive action of CB.By contrast, the direction of reinitiated cytoplasmic streaming was reversed in 50% of cells in which the bundles of MFs had been completely disrupted by exogenously applied trypsin prior treatment with CB.The results confirm that protease-sensitive anchoring of MF bundles at the end walls is crucial for maintenance of the stationary organization of the bundles of MFs.
A synthetic hexapeptide, GRGDSP is known to inhibit in animals as well as in plants certain developmental events such as cell-matrix adheision or cellular migration in vivo. RYD motif is known to be conserved in the surface glycoprotein of Leishmania and in plant vitronection-like protein and to mimic functionally and antigenically RGD motif in extracelluar matrix protein, fibronection of animal cells. Taking these facts into considerations, we applied exogenously a synthetic peptide, GRGDSP or ARYDEI that contains RYD motif to mesophyll cells of Vallisneria. Both peptides induced extremely abnormal patterns of cytoplasmic streaming concomitantly with striking disorganization of arrangement of bundles of MFs at concenteations of 1-15 mM within 24 hrs. GRGESP or ARYEEI peptide used used for the control had no detectable effects. Immunofluoresence microscopy revealed that polyclonal antibodies against ARYDEI peptide heavily stained the cell wall of mesophyll cells while preimmune serum did not. Western blotting analysis demonstrated that the antibodies recognized a protein of 54 kD in total proteins extracted from leaf cells of Vallisneria. The results suggest that protein of 54 kD which contains RGD or RYD motif in the cell wall is implicated in anchoring mechanism of MFs. Less