Han G, Liu B, Zhang J, Zuo W, Morris N R, Xiang X
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Curr Biol. 2001 May 1;11(9):719-24. doi: 10.1016/s0960-9822(01)00200-7.
Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.
胞质动力蛋白是一种多亚基的、向微管负端移动的微管马达蛋白，它利用动力蛋白激活蛋白作为辅助复合体来执行各种体内功能，包括囊泡运输、纺锤体组装和核分布[1]。我们之前发现，在丝状真菌构巢曲霉中，绿色荧光蛋白标记的胞质动力蛋白重链（NUDA）形成彗星状结构，该结构表现出依赖微管的向菌丝顶端移动和从菌丝顶端返回的运动[2]。在此，我们证明在NUDA途径中的另一种蛋白NUDF，它与参与大脑发育的人类LIS1蛋白同源[3,4]，也表现出这种动态行为。NUDA和NUDF都位于微管末端，这一观察结果表明，所观察到的动态行为是由于它们与动态的微管末端相关联。为了研究NUDA和NUDF在体内是否在调节微管动力学中发挥作用，我们构建了一个绿色荧光蛋白标记的α-微管蛋白菌株，并用它来比较野生型构巢曲霉与nudA和nudF温度敏感功能缺失突变体在体内的微管动力学。突变体显示微管灾变频率较低，灾变期间收缩速率较低，以及拯救频率较低。突变体细胞中的微管在菌丝顶端停留的时间也比野生型微管更长。这些结果表明，胞质动力蛋白和LIS1同源物NUDF在体内影响微管动力学。
