Molecular Behavior in Living Mitotic Cells of Human Centromere Heterochromatin Protein HPLalpha Ectopically Expressed as a Fusion to Red Fluorescent Protein

Cell Struct Funct. 2001 Dec;26(6):705-18. doi: 10.1247/csf.26.705.

Abstract

We constructed stable mammalian cell lines in which human heterochromatin protein HP1alpha and kinetochore protein CENP-A were differentially expressed as fusions to red (RFP-HP1) and green fluorescent proteins (GFP-CENP-A). Heterochromatin localization of RFP-HP1 was clearly shown in mouse and Indian muntjac cells. By preparing mitotic chromosome spreads, the inner centromere localization of RFP-HP1 was observed in human and Indian muntjac cells. To characterize its molecular behavior in living mitotic cells, time-lapse images of RFP-HP1 were obtained by computer-assisted image analyzing system, mainly with mouse cells. In G2 phase, a significant portion of RFP-HP1 diffused homogeneously in the nucleus and further dispersed into the cytoplasm soon after the nuclear membrane breakdown, while some remained in the centromeric region. Simultaneous observations with GFP-CENP-A in human cells showed that RFP-HP1 was located just between the sister kinetochores and then aligned to the spindle midzone. With the onset of anaphase, once it was released from there, it moved to the centromeres of segregating chromosomes or returned to the spindle equator. As cytokinesis proceeded, HP1alpha was predominantly found in the newly formed daughter nuclei and again displayed a heterochromatin-like distribution. These results suggested that, although the majority of HP1alpha diffuses into the cytoplasm, some populations are retained in the centromeric region and involved in the association and segregation of sister kinetochores during mitosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoantigens*
  • Cells, Cultured
  • Centromere Protein A
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Chromosomes / genetics
  • Chromosomes / metabolism
  • Green Fluorescent Proteins
  • Humans
  • Indicators and Reagents / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Mice
  • Microscopy, Fluorescence
  • Microscopy, Video
  • Mitosis / physiology*
  • Muntjacs / physiology
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Time Factors
  • Transfection

Substances

  • Autoantigens
  • CENPA protein, human
  • Cenpa protein, mouse
  • Centromere Protein A
  • Chromosomal Proteins, Non-Histone
  • Indicators and Reagents
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • red fluorescent protein
  • heterochromatin-specific nonhistone chromosomal protein HP-1
  • Green Fluorescent Proteins