Regulation of mitotic cytoskeleton dynamics and cytokinesis by integrin-linked kinase in retinoblastoma cells

William K A Sikkema; Arend Strikwerda; Manju Sharma; Kiran Assi; Baljinder Salh; Michael E Cox; Julia Mills

doi:10.1371/journal.pone.0098838

Regulation of mitotic cytoskeleton dynamics and cytokinesis by integrin-linked kinase in retinoblastoma cells

PLoS One. 2014 Jun 9;9(6):e98838. doi: 10.1371/journal.pone.0098838. eCollection 2014.

Authors

William K A Sikkema¹, Arend Strikwerda¹, Manju Sharma², Kiran Assi³, Baljinder Salh³, Michael E Cox², Julia Mills⁴

Affiliations

¹ Department of Biology, Trinity Western University, Langley, British Columbia, Canada.
² Vancouver Prostate Centre, Vancouver Coastal Health Research Institute, Vancouver, British Columbia, Canada.
³ Division of Gastroenterology, University of British Columbia, Vancouver, British Columbia, Canada.
⁴ Department of Biology, Trinity Western University, Langley, British Columbia, Canada; Adjunct, Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada; External Associate Member, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Abstract

During cell division integrin-linked kinase (ILK) has been shown to regulate microtubule dynamics and centrosome clustering, processes involved in cell cycle progression, and malignant transformation. In this study, we examine the effects of downregulating ILK on mitotic function in human retinoblastoma cell lines. These retinal cancer cells, caused by the loss of function of two gene alleles (Rb1) that encode the retinoblastoma tumour suppressor, have elevated expression of ILK. Here we show that inhibition of ILK activity results in a concentration-dependent increase in nuclear area and multinucleated cells. Moreover, inhibition of ILK activity and expression increased the accumulation of multinucleated cells over time. In these cells, aberrant cytokinesis and karyokinesis correlate with altered mitotic spindle organization, decreased levels of cortical F-actin and centrosome de-clustering. Centrosome de-clustering, induced by ILK siRNA, was rescued in FLAG-ILK expressing Y79 cells as compared to those expressing FLAG-tag alone. Inhibition of ILK increased the proportion of cells exhibiting mitotic spindles and caused a significant G2/M arrest as early as 24 hours after exposure to QLT-0267. Live cell analysis indicate ILK downregulation causes an increase in multipolar anaphases and failed cytokinesis (bipolar and multipolar) of viable cells. These studies extend those indicating a critical function for ILK in mitotic cytoskeletal organization and describe a novel role for ILK in cytokinesis of Rb deficient cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Actins / metabolism
Cell Line, Tumor
Cell Nucleus / drug effects
Cell Nucleus / pathology
Cell Nucleus Size / drug effects
Cell Nucleus Size / genetics
Cell Proliferation / drug effects
Cell Proliferation / genetics
Cytokinesis* / drug effects
Cytokinesis* / genetics
Cytoskeleton* / drug effects
Cytoskeleton* / genetics
Down-Regulation / drug effects
Down-Regulation / genetics
Gene Knockdown Techniques
Humans
Mitosis* / drug effects
Mitosis* / genetics
Protein Kinase Inhibitors / pharmacology
Protein Serine-Threonine Kinases / antagonists & inhibitors
Protein Serine-Threonine Kinases / deficiency
Protein Serine-Threonine Kinases / genetics
Protein Serine-Threonine Kinases / metabolism*
RNA, Small Interfering / genetics
Retinoblastoma / pathology*
Spindle Apparatus / drug effects
Spindle Apparatus / genetics

Substances

Actins
Protein Kinase Inhibitors
RNA, Small Interfering
integrin-linked kinase
Protein Serine-Threonine Kinases

Grants and funding

The authors gratefully acknowledge the award from the Murdock College Research Program for Life Sciences that made this research possible. A Murdock General Science Grant as a match to the CFI Leaders Opportunity Fund Grant enabled the purchase of the Olympus DSU spinning disk confocal used in this study. The Molecular and Cell Biology laboratory and Cell Culture laboratory in which these studies took place were made possible by a grant from Murdock Trust as a match to the Industry Canada Knowledge Infrastructure Program Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.