Visualization of the chromosome scaffold and intermediates of loop domain compaction in extracted mitotic cells

Authors

  • Eugene V. Sheval,

    Corresponding author
    1. Department of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, GSP-2, Moscow 119992, Russia
    2. Laboratory of Cell Biology, Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Timiryazevskaya 42, Moscow 127550, Russia
    Search for more papers by this author
  • Vladimir Y. Polyakov

    1. Department of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, GSP-2, Moscow 119992, Russia
    Search for more papers by this author

Corresponding author. Department of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, GSP-2, Moscow 119992, Russia. Tel.: +7 495 939 5528; fax: +7 495 939 3181. sheval_e@genebee.msu.su

Abstract

A novel extraction protocol for cells cultured on coverslips is described. Observations of the extraction process in a perfusion chamber reveal that cells of all mitotic stages are not detached from coverslips during extraction, and all stages can be recognized using phase contrast images. We studied the extracted cell morphology and distribution of a major scaffold component—topoisomerase IIα, in extracted metaphase and anaphase cells. An extraction using 2 M NaCl leads to destruction of chromosomes at the light microscope level. Immunogold studies demonstrate that the only residual structure observed is an axial chromosome scaffold that contains topoisomerase IIα. In contrast, mitotic chromosomes are swelled only partially after an extraction using dextran sulphate and heparin, and it appears that this treatment does not lead to total destruction of loop domains. In this case, the chromosome scaffold and numerous structures resembling small rosettes are revealed inside extracted cells. The rosettes observed condense after addition of Mg2+-ions and do not contain topoisomerase IIα suggesting that these structures correspond to intermediates of loop domain compaction. We propose a model of chromosome structure in which the loop domains are condensed into highly regular structures with rosette organization.

Ancillary