Mechanism of the translocation of endcpinsmic reticulum by myosin XI in higher plant cells

在烟草培养细胞中，BY-Z是肌球蛋白X的一种异构体，由175-kDa重链组成，被认为参与内质网（ER）的移位。为了进一步研究这种肌球蛋白与ER易位的关系，使用表达GFP标记ER（GFP-ER）的BY-2 TM细胞进行细胞分级分离程序。175-kDa肌球蛋白和GFP-ER，检测到微粒体和细胞溶质组分通过离心制备。当这些组分通过蔗糖密度梯度离心进一步分级时，一部分175-kDa肌球蛋白与GFP-ER共分级，表明该肌球蛋白与ER紧密相关并负责其易位。然而，在体外运动试验中，这种ER与175-kDa肌球蛋白也不能移动沿着肌动蛋白束轮节间细胞，即使在ATP的存在下。有趣的是，当加入GTP时，GFP-ER的囊泡形成管状结构。这些结构被认为对应于在活细胞中的皮质区域中观察到的ER的管状结构。然而，添加其他核苷酸如GDP或ATP代替GTP，不能组装ER的管状结构。同样的现象已经在从非洲爪蟾卵制备的ER组分中记录。然而，在来自BY-2细胞的GFP-ER的情况下，诸如流的力是体外诱导ER管状结构伸长所必需的。肌动蛋白细胞骨架抑制剂能明显抑制BY-2细胞内质网管的伸长和收缩，但对微管细胞骨架抑制剂无明显作用。因此，ER管在体内伸长所需的力被认为是由肌动蛋白细胞骨架，特别是分子马达肌球蛋白产生的。

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.