Live Imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control
Live Imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control
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DOI：
10.1091/mbc.e05-04-0338
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发表时间：
2005-11-01
影响因子：
3.3
通讯作者：
Doe, CQ
中科院分区：
文献类型：
--
作者：
Siller, KH;Serr, M;Doe, CQ
Lis1是真菌细胞核迁移、哺乳动物细胞周期进程和人类折叠大脑皮层形成所必需的。Lis1结合动力蛋白和动力蛋白运动复合体，但Lis1在许多动力蛋白/动力蛋白依赖性过程中的作用尚不清楚。在这里，我们产生和/或表征果蝇的Lis1和dynactin亚基，胶合剂的突变体，调查Lis1/dynactin在有丝分裂检查点功能的作用。此外，我们开发了一种改进的延时视频显微镜技术，允许实时成像的GFP-Lis1，GFP-杆检查点蛋白，绿色荧光蛋白（GFP）标记的染色体，或GFP标记的有丝分裂纺锤体动力学在整个幼虫脑外植体内的成神经细胞。我们的突变体分析表明，Lis1/dynactin有至少两个独立的功能，在有丝分裂：首先促进中心体分离和双极纺锤体组装在前期/前中期，随后产生interkinetochore张力和转运检查点蛋白的着丝粒在中期，从而促进后期的及时发病。此外，我们发现，Lis1/dynactin/dynein物理关联和共定位在中心体，纺锤体MT，和着丝粒，和调节Lis1/dynactin着丝粒定位在果蝇不同的秀丽隐杆线虫和哺乳动物。我们的结论是，Lis1/dynactin共同作用，以调节多个，独立的功能，在有丝分裂细胞，包括纺锤体的形成和细胞周期检查点的释放。
Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.