Assembly of the inner kinetochore proteins CENP-A and CENP-B in living human cells.
DNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. A subkinetochore complex, the "inner kinetochore", is present at the centromere during the entire cell cycle. Its location seems to be defined by the settlement of CENP-A (CENH3), which replaces histone H3 in centromeric nucleosomes. This suggests that CENP-A can recruit further inner kinetochore proteins by direct binding. Surprisingly, intense in vitro studies could not identify an interaction of CENP-A with any other inner kinetochore protein. Instead, centromere identity seems to be maintained by a unique nucleosome, which might have a modified structure or epigenetic state that serves to distinguish the centromere from the rest of the chromosome. We investigated the association of CENP-A and CENP-B by fluorescence intensity and lifetime-based FRET measurements in living human HEp-2 cells. We observed Förster resonance energy transfer (FRET) between CENP-A and CENP-B at centromere locations; this indicates that these proteins are in the molecular vicinity (<10 nm) of each other. In addition, we analysed protein-protein interactions within the centromeric nucleosome. We could detect energy transfer between CENP-A and histone H4 as well as between CENP-A molecules themselves. On the other hand, no FRET was detected between CENP-A and H2A.1 or H3.1. Our data support the view that two CENP-A molecules are packed with H4, but not with H3, in a single centromeric nucleosome.
Orthaus
Sandra
S
Department of Molecular Biology, Leibniz Institute for Age Research-Fritz Lipmann Institute, Beutenbergstrasse 11, 07745 Jena, Germany.
Biskup
Christoph
C
Hoffmann
Birgit
B
Hoischen
Christian
C
Ohndorf
Sabine
S
Benndorf
Klaus
K
Diekmann
Stephan
S
D001324:Autoantigens / Q000378:metabolism*; D002470:Cell Survival; D000076247:Centromere Protein A; D051936:Centromere Protein B / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D018386:Kinetochores / Q000737:chemistry* / Q000378:metabolism*
D001324:Autoantigens; C000619700:CENPA protein, human; C497926:CENPB protein, human; D000076247:Centromere Protein A; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D005456:Fluorescent Dyes; D006657:Histones