Stolz Ailine, Ertych Norman, Bastians Holger
a Georg-August University Göttingen; Göttingen Center for Molecular Biosciences (GZMB) and University Medical Center Göttingen (UMG) ; Institute of Molecular Oncology; Section for Cellular Oncology ; Göttingen , Germany.
Cell Cycle. 2015;14(6):827-37. doi: 10.1080/15384101.2014.1000693.
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.
有丝分裂期间微管动力学的适当调节对于准确的染色体分离至关重要。事实上，最近我们发现人类癌细胞中经常观察到微管正端组装速率增加，这是导致全染色体错分离和染色体不稳定性（CIN）的重要机制。然而，导致癌细胞中微管聚合速率增加的基因改变在很大程度上仍然未知。确定微管的动态参数通常涉及对活细胞的分析，而这在大规模筛选环境中技术上难以实施，这一事实阻碍了此类病变的识别。因此，我们寻求确定替代方法来系统地识别微管正端聚合的调节因子。在这里，我们介绍一种简单而稳健的表型筛选测定法，该方法基于对有丝分裂驱动蛋白Eg5/KIF11抑制后诱导的单极有丝分裂纺锤体结构的分析。我们表明，在存在Eg5/KIF11抑制的情况下，微管聚合增加会导致高度不对称的单星体，并且这种表型可以在活细胞和固定细胞中可靠地评估。使用该测定法我们进行了一项siRNA筛选，其中我们鉴定了几种微管正端结合蛋白以及中心体和皮质相关蛋白作为微管正端组装的重要调节因子。有趣的是，我们证明这些调节因子的一个亚组通过其在抑制微管正端聚合中的作用来调节纺锤体取向。
