Silencing Mitosin Induces Misaligned Chromosomes, Premature Chromosome Decondensation before Anaphase Onset, and Mitotic Cell Death

Abstract

Mitosin (also named CENP-F) is a large human nuclear protein transiently associated with the outer kinetochore plate in M phase. Using RNA interference and fluorescence microscopy, we showed that mitosin depletion attenuated chromosome congression and led to metaphase arrest with misaligned polar chromosomes whose kinetochores showed few cold-stable microtubules. Kinetochores of fully aligned chromosomes often failed to show orientation in the direction of the spindle long axis. Moreover, tension across their sister kinetochores was decreased by 53% on average. These phenotypes collectively imply defects in motor functions in mitosin-depleted cells and are similar to those of CENP-E depletion. Consistently, the intensities of CENP-E and cytoplasmic dynein and dynactin, which are motors controlling microtubule attachment and chromosome movement, were reduced at the kinetochore in a microtubule-dependent manner. In addition, after being arrested in pseudometaphase for approximately 2 h, mitosin-depleted cells died before anaphase initiation through apoptosis. The dying cells exhibited progressive chromosome arm decondensation, while the centromeres were still associated with spindles. Mitosin is therefore essential for full chromosome alignment, possibly by promoting proper kinetochore attachments through modulating CENP-E and dynein functions. Its depletion also prematurely triggers chromosome decondensation, a process that normally occurs from telophase for the nucleus reassembly, thus resulting in apoptosis.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org.

ACKNOWLEDGMENTS

We thank Qiongping Huang, Lirong Liu, and Wei Bian for technical assistance. We are also in debt to T. J. Yen (Fox Chase Cancer Center, Philadelphia, PA) for antibodies to CENP-E and BubR1, B. J. Howell and E. D. Salmon (University of North Carolina at Chapel Hill, Chapel Hill, North Carolina) for anti-Mad2 antibody, J. Kilmartin (MRC Laboratory of Molecular Biology, Cambridge, United Kingdom) for anti-hNuf2 antibody, S. S. Taylor (University of Manchester, Manchester, United Kingdom) for antibodies to Bub1 and Aurora B, W. R. Brinkley (Baylor College of Medicine, Houston, TX) for human anticentromere antibody, G. M. Wahl (Salk Institute for Biological Studies, La Jolla, California) for the HeLa stable cell line expressing histone 2B-GFP, and K. Liao of our institute for the pBS/U6 plasmid for silencing murine caveolin-1.

This work was supported by grants 30025021, 30330330, and 30421005 from the National Science Foundation of China, KSCX2-2-02 from the Chinese Academy of Sciences, and 2002CB713802 (the national key basic research and development program) from the Ministry of Science and Technology of China.