|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
1) We performed double immunofluorecence staining of a hepatoma cell line, PLC/PRF/5, using antibodies specific to tubulin and calreticulin (CR) , which is a calcium-storing protein exclusively localized in endoplamic reticulum (ER). Cytoplasmic distribution of the ER is associated with that of microtubules (MT). After the treatment of MT-depolymerizing drug, the ER distribution is collapsed. Immunofluorescence of cytoplasmic dynein (CD) shows diffuse staining of the cytoplasm, correspondig to the ER distribution, and the detergent treatment is abolished the fluorescence. Western blotting of a crude microsomal fraction demonstrated that the CD is rich in the fraction. Complex of MT and microsomes by addition of taxol to low speed supernatant of the cell homogenate were formed. CD and microsomes were released from the complex by ATP treatment. These findings suggest that microtubles associated with CD are involved in the distribution of ER.
2) We examined the function of MAP-4 in the cyt
oskeleton in vivo. Intact MAP-4 was prepared from bovine adrenal cortex, and the truncated fragments of the N- and the C-terminal halves were expressed in E.coli and isolated. The injection of intact MAP-4 or C-terminal half into the ptK2 cells induced an increase in the number of cytoplasmic MT,as well as MT bundling. The N-terminal fragment did not affect the MT array. Injected MAP-4 and C-terminal half were associated with the increased MT.These results indicated that intact MAP-4 and the C-terminal fragment promoted MT assembly and stabilized MT,by binding to MT.
3) We demonstrate that the Golgi apparatus, an organelle positioned near the centrosome, is disorganized and dispersed by the incubation of the low pH medium. The Golgi apparatus was gradually dispersed in the acidic medium, and after 16 h, 61.5% of the cells showed the organelle dispersed throughout the cytoplasm. When the cells were transferred into the pH 7.4 medium after 16-h incubation in pH 6.6, the apparatus was immediately reorganized in the centrosomal region. Microtubules and endoplasmic reticulum were not changed by the acidification. Cytoplasmic dynein was localized in the Golgi membrane even when dispersed. Less