CENP-W plays a role in maintaining bipolar spindle structure

PLoS One. 2014 Oct 15;9(10):e106464. doi: 10.1371/journal.pone.0106464. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bipolar Disorder / genetics*
  • Bipolar Disorder / pathology
  • Cell Cycle Proteins / biosynthesis
  • Cell Cycle Proteins / genetics*
  • Centrosome
  • Chromosomal Proteins, Non-Histone / biosynthesis
  • Chromosomal Proteins, Non-Histone / genetics*
  • Chromosome Segregation / genetics
  • Gene Expression Regulation
  • HeLa Cells
  • Humans
  • Microtubule-Associated Proteins / biosynthesis
  • Microtubule-Associated Proteins / genetics*
  • Microtubule-Associated Proteins / metabolism
  • Microtubules / genetics
  • Mitosis / genetics*
  • Nuclear Proteins / biosynthesis
  • Nuclear Proteins / genetics*
  • RNA, Small Interfering
  • Spindle Apparatus / genetics*
  • Spindle Apparatus / metabolism

Substances

  • CENPW protein, human
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • Microtubule-Associated Proteins
  • Nuclear Proteins
  • RNA, Small Interfering
  • TPX2 protein, human

Grants and funding

This work was funded by a grant from Science Foundation Ireland (SFI 05/RP1/B793). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.