title: Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast.
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 super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A(Cnp1) 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(Cnp1) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.
authors.affiliation: Department of Biochemistry, University of Cambridge , Cambridge CB2 1EW, UK.
authors.forename: David
authors.initials: D
authors.lastname: Lando
authors.forename: Ulrike
authors.initials: U
authors.lastname: Endesfelder
authors.forename: Harald
authors.initials: H
authors.lastname: Berger
authors.forename: Lakxmi
authors.initials: L
authors.lastname: Subramanian
authors.forename: Paul D
authors.initials: PD
authors.lastname: Dunne
authors.forename: James
authors.initials: J
authors.lastname: McColl
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.forename: Antony M
authors.initials: AM
authors.lastname: Carr
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
authors.forename: Robin C
authors.initials: RC
authors.lastname: Allshire
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Ernest D
authors.initials: ED
authors.lastname: Laue
mesh_terms: D002503:Centromere / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis* / Q000235:genetics; D044127:Epigenesis, Genetic / Q000502:physiology; D016195:G2 Phase / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C410349:Cnp1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins