CULTURED-CELL EXTRACTS SUPPORT ORGANELLE MOVEMENT ON MICROTUBULES INVITRO
CULTURED-CELL EXTRACTS SUPPORT ORGANELLE MOVEMENT ON MICROTUBULES INVITRO
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DOI：
10.1002/cm.970100405
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发表时间：
1988-01-01
影响因子：
--
通讯作者：
SHEETZ, MP
中科院分区：
文献类型：
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作者：
DABORA, SL;SHEETZ, MP
在多种培养细胞中观察到细胞器的定向运动。为了研究胞内细胞器运动的调控及其分子基础，我们从培养的鸡胚成纤维细胞（CEF）中制备了支持膜细胞器沿微管运动的提取物。体外细胞器运动的速度、频率和特征与完整细胞内相似。细胞器和提取物包被的阴离子珠主要（80%）向中心体再生的微管的负端移动，对应于逆行易位。从其他培养细胞（非洲绿猴肾和3T3细胞）中提取的提取物中也观察到类似的微管依赖细胞器运动。细胞器的运动依赖于TP和微管。酸性（pH < 7）或碱性（pH < 8）溶液、高离子强度（[KCl] = 0.1 M）和游离镁离子的螯合作用均能抑制细胞器运动频率。用磷酸腺苷酰亚胺二磷酸（AMP-PNP, 7 mM）、正钒酸钠（vanadate; Na3VO4, 20 .mu）处理提取物。M)或n -乙基马来酰亚胺（NEM, 2 mM）阻断了所有细胞器的运动。在AMP-PNP或钒酸盐存在的情况下，观察到微管的细胞器装饰。细胞松弛素D(2。M)或cAMP （1mm）。Kinesin （Mr = 116000）是一种基于顺行微管的马达，在AMP-PNP存在下，通过微管亲和纯化从CEF提取物中部分纯化，并能够驱动玻璃盖上的微管运动。在钒酸盐存在下制成的类似制剂含有不同的蛋白质亚群，不支持运动性。这些结果表明，胞内细胞器运动可以在体外重现，并为研究细胞器运动复合物中单个分子组分的作用提供了基础。
Directed movements of organelles have been observed in a variety of cultured cells. To study the regulation and molecular basis of intracellular organelle motility, we have prepared extracts from cultured chick embryo fibroblasts (CEF cells) which support the movement of membranous organelles along microtubules. The velocity, frequency and characteristics of organelle movements in vitro were similar to those within intact cells. Organelles and extract-coated anionic beads moved predominantly (80%) toward the minus ends of microtubules that had been regrown from centrosomes, corresponding to retrograde translocation. Similar microtubule-dependent organelle movements were observed in extracts prepared from other cultured cells (African green monkey kidney and 3T3 cells). Organelle motility was TP and microtubule dependent. The frequency of organelle movement was inhibited by acidic (pH < 7) or alkaline (pH > 8) solutions, high ionic strength ([KCl] = 0.1 M), and the chelation of free magnesium ions. Treatment of the extracts with adenylyl imidodiphosphate (AMP-PNP, 7 mM), sodium orthovanadate (vanadate; Na3VO4, 20 .mu.M), or N-ethylmaleimide (NEM, 2 mM) blocked all organelle motility. The decoration of microtubules with organelles was observed in the presence of AMP-PNP or vanadate. Motility was not affected by cytochalasin D (2 .mu.M) or cAMP (1 mM). Kinesin (Mr = 116,000), an anterograde microtubule-based motor, was partially purified from the CEF extract by microtubule affinity purification in the presence of AMP-PNP, and was able to drive the movement of microtubule on glass coverslips. A similar preparation made in the presence of vanadate contained a different subset of proteins and did not support motility. These results demonstrate that intracellular organelle motility can be reproduced in vitro and provide the basis for investigating the roles of individual molecular components involved in the organelle motor complex.