Assembly of the inner kinetochore proteins CENP-A and CENP-B in living human cells

Chembiochem. 2008 Jan 4;9(1):77-92. doi: 10.1002/cbic.200700358.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoantigens / metabolism*
  • Cell Survival
  • Centromere Protein A
  • Centromere Protein B / metabolism*
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes
  • Hepatocytes / cytology*
  • Hepatocytes / metabolism*
  • Histones / metabolism
  • Humans
  • Kinetochores / chemistry*
  • Kinetochores / metabolism*


  • Autoantigens
  • CENPA protein, human
  • CENPB protein, human
  • Centromere Protein A
  • Centromere Protein B
  • Chromosomal Proteins, Non-Histone
  • Fluorescent Dyes
  • Histones