Tao Li, Fasulo Barbara, Warecki Brandt, Sullivan William
Department of Biology, University of Hawaii at Hilo, 200 West Kawili Street, Hilo, Hawaii 96720, USA.
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Cruz, California 95064, USA.
Nat Commun. 2016 Apr 19;7:11182. doi: 10.1038/ncomms11182.
Centralspindlin is essential for central spindle and cleavage furrow formation. Drosophila centralspindlin consists of a kinesin-6 motor (Pav/kinesin-6) and a GTPase-activating protein (Tum/RacGAP). Centralspindlin localization to the central spindle is mediated by Pav/kinesin-6. While Tum/RacGAP has well-documented scaffolding functions, whether it influences Pav/kinesin-6 function is less well-explored. Here we demonstrate that both Pav/kinesin-6 and the centralspindlin complex (co-expressed Pav/Tum) have strong microtubule bundling activity. Centralspindlin also has robust plus-end-directed motility. In contrast, Pav/kinesin-6 alone cannot move microtubules. However, the addition of Tum/RacGAP or a 65 amino acid Tum/RacGAP fragment to Pav/kinesin-6 restores microtubule motility. Further, ATPase assays reveal that microtubule-stimulated ATPase activity of centralspindlin is seven times higher than that of Pav/kinesin-6. These findings are supported by in vivo studies demonstrating that in Tum/RacGAP-depleted S2 Drosophila cells, Pav/kinesin-6 exhibits severely reduced localization to the central spindle and an abnormal concentration at the centrosomes.
中心纺锤体微管蛋白复合体对于中心纺锤体和分裂沟的形成至关重要。果蝇的中心纺锤体微管蛋白复合体由一种驱动蛋白-6（Pav/驱动蛋白-6）和一种GTP酶激活蛋白（Tum/RacGAP）组成。Pav/驱动蛋白-6介导中心纺锤体微管蛋白复合体定位于中心纺锤体。虽然Tum/RacGAP具有充分记录的支架功能，但其是否影响Pav/驱动蛋白-6的功能却较少被研究。在此我们证明，Pav/驱动蛋白-6和中心纺锤体微管蛋白复合体（共表达的Pav/Tum）都具有很强的微管成束活性。中心纺锤体微管蛋白复合体也具有强大的正端定向运动性。相比之下，单独的Pav/驱动蛋白-6无法移动微管。然而，向Pav/驱动蛋白-6中添加Tum/RacGAP或一个65个氨基酸的Tum/RacGAP片段可恢复微管的运动性。此外，ATP酶分析表明，中心纺锤体微管蛋白复合体的微管刺激ATP酶活性比Pav/驱动蛋白-6高7倍。体内研究支持了这些发现，这些研究表明，在缺失Tum/RacGAP的果蝇S2细胞中，Pav/驱动蛋白-6在中心纺锤体上的定位严重减少，并且在中心体处异常聚集。
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