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Assembly of the Inner Kinetochore Proteins CENP‐A and CENP‐B in Living Human Cells
Author(s) -
Orthaus Sandra,
Biskup Christoph,
Hoffmann Birgit,
Hoischen Christian,
Ohndorf Sabine,
Benndorf Klaus,
Diekmann Stephan
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700358
Subject(s) - kinetochore , centromere , förster resonance energy transfer , nucleosome , microbiology and biotechnology , histone h3 , biology , histone , mitosis , chromatin , chromosome segregation , biophysics , chromosome , genetics , dna , fluorescence , gene , physics , quantum mechanics
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.