Abstract
The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. ‘Spindle free’ nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability.
Highlights
Establishment of a bipolar spindle in mitotic cells is a critical aspect of maintaining genomic integrity [1,2]
We found that depletion of CENP-W induced a more rapid and profound disruption of mitosis than depletion of CENP-T and that this was associated with a pronounced multipolar spindle phenotype
HeLa cells revealed a high frequency of multipolar spindle formation, suggesting difficulties in either the formation or maintenance of bipolar spindles [30]
Summary
Establishment of a bipolar spindle in mitotic cells is a critical aspect of maintaining genomic integrity [1,2]. The spindle poles and the kinetochores of the chromosomes, regulate the structure and function of the spindle through dominant effects on microtubule stability and organization [3]. Maintenance of spindle pole integrity is essential as defects in spindle pole organization lead to multipolar mitoses and mitotic failure [7]. Aberrations in centrosome number are frequent in tumor cells and contribute to mechanisms of chromosome instability in cancerous cells [8,9]. Knowledge of the mechanisms of maintenance of spindle poles is of critical importance for understanding tumor cells and their adaptations and weaknesses
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