A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle orientation
A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle orientation
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DOI：
10.1080/15384101.2014.1000693
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发表时间：
2015-03-15
期刊：
影响因子：
4.3
通讯作者：
Bastians, Holger
中科院分区：
文献类型：
--
作者：
Stolz, Ailine;Ertych, Norman;Bastians, Holger
在有丝分裂过程中，适当地调节微管动力学对于正确的染色体分离是至关重要的。事实上，我们最近发现，在人类癌细胞中经常观察到的微管和末端装配率增加是导致整个染色体错误分离和染色体不稳定(CIN)的重要机制。然而，导致癌细胞微管聚合率增加的基因改变在很大程度上仍不清楚。确定微管的动态参数通常涉及对活细胞的分析，这在技术上很难在大规模筛查环境中执行，这一事实阻碍了此类病变的识别。因此，我们试图寻找替代方案来系统地确定微管+末端聚合的调节因子。在这里，我们介绍了一种简单而可靠的表型筛选方法，它基于对单极有丝分裂纺锤体结构的分析，这种结构是由抑制有丝分裂运动EG5/KIF11诱导的。我们发现，在EG5/KIF11抑制的情况下，增加的微管聚合导致高度不对称的单单体，这种表型在活细胞和固定细胞中都可以可靠地评估。利用这一方法，我们进行了siRNA筛选，其中我们确定了几个微管+末端结合蛋白以及中心体和皮质相关蛋白作为微管+末端组装的重要调节蛋白。有趣的是，我们证明了这些调节子的一个亚群通过它们在抑制微管和末端聚合中的作用来调节纺锤体的取向。
Proper regulation of microtubule dynamics during mitosis is essential for faithful chromosome segregation. In fact, recently we discovered increased microtubule plus end assembly rates that are frequently observed in human cancer cells as an important mechanism leading to whole chromosome missegregation and chromosomal instability (CIN). However, the genetic alterations responsible for increased microtubule polymerization rates in cancer cells remain largely unknown. The identification of such lesions is hampered by the fact that determining dynamic parameters of microtubules usually involves analyses of living cells, which is technically difficult to perform in large-scale screening settings. Therefore, we sought to identify alternative options to systematically identify regulators of microtubule plus end polymerization. Here, we introduce a simple and robust phenotypic screening assay that is based on the analyses of monopolar mitotic spindle structures that are induced upon inhibition of the mitotic kinesin Eg5/KIF11. We show that increased microtubule polymerization causes highly asymmetric monoasters in the presence of Eg5/KIF11 inhibition and this phenotype can be reliably assessed in living as well as in fixed cells. Using this assay we performed a siRNA screen, in which we identify several microtubule plus end binding proteins as well as centrosomal and cortex associated proteins as important regulators of microtubule plus end assembly. Interestingly, we demonstrate that a subgroup of these regulators function in the regulation of spindle orientation through their role in dampening microtubule plus end polymerization.