Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon, United States of America.
PLoS One. 2012;7(1):e29611. doi: 10.1371/journal.pone.0029611. Epub 2012 Jan 10.
During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud).
在不对称细胞分裂过程中，有丝分裂纺锤体与细胞极性轴的对准确保了分裂沟将命运决定因素分离到不同的子细胞中。蛋白 Inscuteable（Insc）被认为通过结合极性蛋白 Bazooka（Baz；又名 Par-3）和纺锤体定向蛋白 Partner of Inscuteable（Pins；哺乳动物中的 mPins 或 LGN）在各种系统中连接细胞极性和纺锤体定位。在这里，我们研究了 Insc-Pins 复合物的纺锤体定向机制。之前，我们定义了两个 Pins 纺锤体定向途径：需要与蘑菇体缺陷（Mud；哺乳动物中的 NuMA）形成复合物才能发挥完全活性，而与 Discs large（Dlg）结合足以发挥部分活性。在当前的研究中，我们研究了 Inscuteable 在介导下游 Pins 介导的纺锤体定向途径中的作用。我们发现，Insc-Pins 复合物需要 Gαi 才能发挥部分活性，并且该复合物特异性招募 Dlg 但不招募 Mud。体外竞争实验表明，Insc 和 Mud 竞争与 Pins TPR 基序结合，而 Dlg 可以与 Insc-Pins 形成三元复合物。我们的结果表明，Insc 不会被动地将极性和纺锤体定向联系起来，而是优先抑制 Mud 途径，同时允许 Dlg 途径保持活性。Insc 调节的复合物组装可能确保纺锤体在激活由 Dynein/Dynactin（通过 Mud）产生的纺锤体拉力之前附着到质膜（通过 Dlg）。
