CONSTRUCTION OF THE ENDOPLASMIC-RETICULUM
CONSTRUCTION OF THE ENDOPLASMIC-RETICULUM
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DOI：
10.1083/jcb.109.5.2045
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发表时间：
1989-11-01
影响因子：
7.8
通讯作者：
CHEN, LB
中科院分区：
文献类型：
--
作者：
LEE, C;FERGUSON, M;CHEN, LB
为了研究 ER 的构建，我们使用微管破坏药物诺考达唑诱导活细胞中 ER 结构的完全破坏，然后在无药物培养基中恢复，从而在 15 分钟内再生 ER 网络。使用荧光染料3,3''-二己基氧杂碳氰碘化物来可视化内质网，我们直接观察到了活细胞中的网络构建过程。在这些实验中，ER 网络是通过由先前描述为小管分支的 ER 运动驱动的内质网小管的延伸、分支和交叉的迭代过程构建的。我们已经测试了该过程的细胞骨架要求。我们发现新形成的内质网小管与单个微管对齐，但与波形蛋白中间丝的肌动蛋白纤维不对齐。微管聚合先于内质网小管的延伸，并且在使用各种不同药物的实验中，微管聚合似乎是内质网网络形成的必要条件。此外，微管特异性药物对微管聚合模式的扰动引起了 ER 构建模式的精确相关扰动。用20μM紫杉醇诱导异常短的、不相交的微管阻止了ER网络的形成； ER 小管仅沿着与聚集的 ER 膜接触的少数微管延伸。这种对相交微管的连续网络的要求表明内质网网络的形成是通过内质网膜沿着微管的分支和运动而发生的。尽管鬼笔环肽染色的肌动蛋白纤维明显完全破坏，但细胞松弛素 B 在恢复期间对 ER 网络的构建没有明显影响。用放线菌酮预处理阻断蛋白质合成和解体中间丝也未能扰乱 ER 构建。
To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 min. Using the fluorescent dye 3,3''-dihexyloxacarbocxyanine ioidide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, an intersection of ne ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubles but not actin fiber of vimentin intermediate filaments. Microtubule polymerization preceded the extenison of ER tubules and, in experiments with a variety of different drugs, appeared to be necessary condition for the ER network formation. Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 .mu.M taxol prevented the ER network formation; ER tubules only entended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalsain B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.