Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.
Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.
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DOI：
10.1083/jcb.139.2.469
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发表时间：
1997-10-20
期刊：
影响因子：
--
通讯作者：
Vallee RB
中科院分区：
文献类型：
--
作者：
Burkhardt JK;Echeverri CJ;Nilsson T;Vallee RB
动力蛋白是一种多亚基复合物，在细胞质动力蛋白功能中起辅助作用。一种动力蛋白亚基动力蛋白在哺乳动物细胞中的过表达破坏了复合物，导致细胞质动力蛋白从前中期动粒中解离，从而干扰有丝分裂（Echeverri，C. J.，B.M. Paschal，K.T. Vaughan和R.B.瓦利1996. 132：617-634）。基于这些结果，动力蛋白被认为在连接细胞质动力蛋白与动粒，以及潜在的膜细胞器中发挥作用。目前的研究报告dynamitin间期表型。在动力蛋白过表达细胞中，早期内体（用抗转铁蛋白受体标记）以及晚期内体和溶酶体（用抗溶酶体相关膜蛋白-1 [LAMP-1]标记）重新分布到细胞外周。这种再分配被诺考达唑破坏，暗示了一种潜在的加末端定向微管运动活性。使用唾液酸转移酶、半乳糖基转移酶和N-乙酰葡糖胺转移酶I监测的高尔基体堆叠被显著破坏成与中间室的组分（ERGIC-53和ERD-2）共定位的分散结构。被破坏的高尔基体元件被EM显示为代表短堆栈类似于微管解聚剂形成的那些。高尔基体到内质网交通的堆栈标记布雷菲德菌素A诱导的动力素过表达不受抑制。从布雷菲德菌素A治疗中恢复的dynamitin过表达细胞的延时观察显示，分散的高尔基体元件不像在对照细胞中所见的那样经历基于微管的运输，而是在其ER出口位点处或附近保持静止。这些结果表明，dynactin是专门需要进行向心运动的内吞细胞器和组件的中间室。通过显微注射抗动力蛋白中间链抗体获得类似于动力蛋白过度表达的结果，这与动力蛋白在介导细胞质动力蛋白与特定膜细胞器相互作用中的作用一致。这些结果表明，dynamitin起着关键作用，在调节细胞器运动的水平上的电机货物绑定。
Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617–634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti–lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end–directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor–cargo binding.