title: A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle orientation.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ailine
authors.initials: A
authors.lastname: Stolz
authors.forename: Norman
authors.initials: N
authors.lastname: Ertych
authors.forename: Holger
authors.initials: H
authors.lastname: Bastians
mesh_terms: D043171:Chromosomal Instability; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D016547:Kinesins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis; D010641:Phenotype; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: C526596:Cep192 protein, human; D002868:Chromosomal Proteins, Non-Histone; C470243:KIF11 protein, human; D016547:Kinesins
keywords: aneuploidy; chromosomal instability; chromosome segregation; microtubule assembly; microtubule dynamics; mitosis; monastrol