Visualization of the chromosome scaffold and intermediates of loop domain compaction in extracted mitotic cells.
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 IIalpha, in extracted metaphase and anaphase cells. An extraction using 2M 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 IIalpha. 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 IIalpha 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.
Sheval
Eugene V
EV
Department of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, GSP-2, Moscow 119992, Russia. sheval_e@genebee.msu.su
Polyakov
Vladimir Y
VY
D000818:Animals; D000951:Antigens, Neoplasm / Q000648:ultrastructure*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002875:Chromosomes / Q000378:metabolism* / Q000648:ultrastructure*; D004250:DNA Topoisomerases, Type II / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000648:ultrastructure*; D005347:Fibroblasts / Q000166:cytology; D045170:Matrix Attachment Regions; D051379:Mice; D008938:Mitosis; D013194:Staining and Labeling / Q000379:methods
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D004250:DNA Topoisomerases, Type II