| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
In tobacco cultured cells, BY-Z an isoform of myosin X, 175-kDa myosin, which is composed of 175-kDa heavy chain, is believed to be involved in the translocation of endoplasmic reticulum (ER). To further examine the relation of this myosin with ER translocation, cell fractionation procedure was carried out by using BY-2 TM cells expressing GFP-labeled ER (GFP-ER). Both 175-kDa myosin and GFP-ER, were detected in microsomal and cytosolic fractions prepared by the centrifugation. When these fractions were further fractioned by the sucrose-densityOgradient centrifugation, one portion of 175-kDa myosin was co-fractionated with GFP-ER, indicating that this myosin was tightly associated with ER and responsible for its translocation. In in vitro motility assay however, such ER associated with 175-kDa myosin was nor able to move along actin bundles in Chara internodal cells even in the presence of ATP. Interestingly, tubular structures were formed from vesicles of GFP-ER when GTP was added. These structures were considered to correspond to the tubular structures of ER observed in the cortical regions in living cells. However, the addition of other nucleotides such as GDP or ATP, instead of GTP, were not able to assemble the tubular structures of ER. Same phenomenon has been documented in the ER fraction prepared from Xenopus eggs. However, in the case of GFP-ER from BY-2 cells, the force such as stream was necessary to induce the elongation of tubular structures of ER in vitro. The elongation and shrinking of ER tubes in living BY-2 cells was significantly suppressed by inhibitors for actin cytoskeleton, but not for microtuble cytoskeleton. Hence, the force needed for the ER-tube elongation in vivo was suggested to be generated by actin cytoskeleton, especially molecular motor, myosin.
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