Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast
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 li...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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The Royal Society
2012-01-01
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| Series: | Open Biology |
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| Online Access: | https://royalsocietypublishing.org/doi/pdf/10.1098/rsob.120078 |
| _version_ | 1818522002016698368 |
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| author | 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 |
| author_facet | 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 |
| author_sort | David Lando |
| collection | DOAJ |
| description | 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-ACnp1 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-ACnp1 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. |
| first_indexed | 2024-12-11T01:58:54Z |
| format | Article |
| id | doaj.art-d4434d82101348dfb7371e24d1b3bdca |
| institution | Directory Open Access Journal |
| issn | 2046-2441 |
| language | English |
| last_indexed | 2024-12-11T01:58:54Z |
| publishDate | 2012-01-01 |
| publisher | The Royal Society |
| record_format | Article |
| series | Open Biology |
| spelling | doaj.art-d4434d82101348dfb7371e24d1b3bdca2022-12-22T01:24:32ZengThe Royal SocietyOpen Biology2046-24412012-01-012710.1098/rsob.120078120078Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeastDavid LandoUlrike EndesfelderHarald BergerLakxmi SubramanianPaul D. DunneJames McCollDavid KlenermanAntony M. CarrMarkus SauerRobin C. AllshireMike HeilemannErnest D. LaueThe 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-ACnp1 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-ACnp1 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.https://royalsocietypublishing.org/doi/pdf/10.1098/rsob.120078cenp-acentromeresingle-molecule microscopyfission yeast |
| spellingShingle | 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 Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast Open Biology cenp-a centromere single-molecule microscopy fission yeast |
| title | Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast |
| title_full | Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast |
| title_fullStr | Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast |
| title_full_unstemmed | Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast |
| title_short | Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast |
| title_sort | quantitative single molecule microscopy reveals that cenp acnp1 deposition occurs during g2 in fission yeast |
| topic | cenp-a centromere single-molecule microscopy fission yeast |
| url | https://royalsocietypublishing.org/doi/pdf/10.1098/rsob.120078 |
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