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

高等植物细胞中肌球蛋白XI对端粒网的易位机制

基本信息

批准号：
17570043
负责人：
YOKOTA Etsuo
金额：
$ 2.3万
依托单位：
University of Hyogo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570043/
关键词：
myosin XI 175-kDa myosin endonlasmic reticulum Network structure in vitro motility assay GTP actin tobacco cultured cell, BY-2 原形質流動運動再構成系小胞体チューブ構造ミオシン細胞周期フラグモプラスト

项目摘要

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.

在烟草培养的细胞中，肌球蛋白X，175 kDa肌球蛋白的同工型由175 kDa重链组成，被认为与内质网（ER）的易位有关。为了进一步检查该肌球蛋白与ER易位的关系，通过使用表达GFP标记的ER（GFP-ER）的BY-2 TM细胞进行细胞分馏过程。通过离心机制备的微粒体和胞质分数检测到175 kDa肌球蛋白和GFP-ER。当这些馏分被蔗糖密度离心进一步分馏时，将175 kDa肌球蛋白的一部分与GFP-ER共取分，表明该肌球蛋白与ER紧密相关并负责其易位。然而，在体外运动测定中，即使在ATP存在的情况下，与175 kDa肌球蛋白相关的ER也无法沿着Chara节节间细胞中的肌动蛋白束移动。有趣的是，加入GTP时，由GFP-ER的囊泡形成管状结构。这些结构被认为对应于活细胞中皮质区域中观察到的ER的管状结构。但是，添加其他核苷酸（例如GDP或ATP）而不是GTP，无法组装ER的管状结构。已经在爪蟾卵中制备的ER分数中记录了相同的现象。然而，在BY-2细胞的GFP-ER的情况下，需要诸如流的力来诱导ER体外的肾小管结构的伸长。肌动蛋白细胞骨架的抑制剂可显着抑制活体by-2细胞中ER管的伸长和收缩。因此，建议通过肌动蛋白细胞骨架，尤其是分子运动肌球蛋白，肌球蛋白，肌动蛋白的肌骨架产生ER管伸长所需的力。