Functional analysis after rapid degradation of condensins and 3D-EM reveals chromatin volume is uncoupled from chromosome architecture in mitosis

Kumiko Samejima, Daniel G Booth, Hiromi Ogawa, James R. Paulson, Linfeng Xie, Cara A Watson, Melpomeni Platani, Masato T Kanemaki, William C Earnshaw

Research output: Contribution to journalArticlepeer-review

Abstract

The requirement for condensin in chromosome formation in somatic cells remains unclear, as imperfectly condensed chromosomes do form in cells depleted of condensin by conventional methodologies. In order to dissect the roles of condensin at different stages of vertebrate mitosis, we have established a versatile cellular system that combines auxin-mediated rapid degradation with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells in the presence and absence of condensin. We analyzed the outcome by live- and fixed-cell microscopy methods, including serial block face scanning electron microscopy with digital reconstruction. Following rapid depletion of condensin, chromosomal defects were much more obvious than those seen after a slow depletion of condensin. The total mitotic chromatin volume was similar to that in control cells, but a single mass of mitotic chromosomes was clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments where the auxin concentration was titrated showed that different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.

Original languageEnglish
Number of pages17
JournalJournal of Cell Science
Volume131
Issue number4
Early online date19 Jan 2018
DOIs
Publication statusPublished - 22 Feb 2018

Keywords

  • Auxin inducible degron system
  • CDK1
  • Chromosome
  • Condensin
  • DT40
  • Mitosis

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