Quantitative single-molecule microscopy reveals that CENP-A deposition occurs during G2 in fission yeast

David Lando, Ulrike Endesfelder, Harald Berger, Lakxmi Subramanian, Paul D Dunne, James McColl, David Klenerman, Antony M Carr, Markus Sauer, Robin C Allshire, Mike Heilemann, Ernest D Laue

Research output: Contribution to journalArticlepeer-review

Abstract

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called superresolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.
Original languageEnglish
Pages (from-to)1-11
JournalOpen Biology
Volume2
Issue number7
DOIs
Publication statusPublished - Jul 2012

Keywords

  • CENP-A
  • centromere
  • single-molecule microscopy
  • fission yeast

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