Vaisberg E A, Koonce M P, McIntosh J R
Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder 80309-0347.
J Cell Biol. 1993 Nov;123(4):849-58. doi: 10.1083/jcb.123.4.849.
The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.
有丝分裂纺锤体的形成和功能不仅取决于微管的组装/拆卸，还取决于运动酶的作用。细胞质动力蛋白已定位到纺锤体，但它在有丝分裂过程中是否发挥作用或如何发挥作用尚不清楚。我们已经克隆并表达了编码来自HeLa细胞和盘基网柄菌细胞质动力蛋白重链假定ATP水解位点的DNA片段。针对所得多肽制备了单特异性抗体，这些抗体在体外抑制动力蛋白的运动活性。将它们注射到有丝分裂的哺乳动物细胞中会在前期或有丝分裂后期从诺考达唑处理恢复过程中阻止纺锤体的形成。细胞因中心体未分离而停滞，并形成单极纺锤体。注射的抗体对染色体附着于双极纺锤体或后期运动没有可检测到的影响。这些数据表明，细胞质动力蛋白在双极纺锤体形成的初始事件中发挥独特而重要的作用，而它可能发挥的任何后期作用都是多余的。讨论了动力蛋白参与有丝分裂的可能机制。
