Analysis of detached human kinetochores.
A method has recently been established for inducing the physical detachment of kinetochores from chromosomes in human HeLa cells, and was used in the studies reported here to investigate the organization and function of dissociated HeLa kinetochores. Immunofluorescence labeling demonstrated that the detached HeLa kinetochores were relatively intact, with the number of detached kinetochores being only moderately more than the diploid number of chromosomes in HeLa cells. In addition, the detached kinetochores could be labeled with antibodies specific for the inner kinetochore plate, outer kinetochore, and subjacent centromeric heterochromatin. A functional assay demonstrated that detached kinetochores retained the capacity to activate the spindle checkpoint, leading to metaphase arrest. Analysis of kinetochore DNA indicated that it consisted primarily of DNA fragments of 130-160 kb in size, while the remainder of the chromosomes were sheared into much smaller fragments during the kinetochore detachment event. Further analysis of kinetochore DNA indicated that it was first cleaved into high molecular weight DNA (>200 kb) fragments during the initial stages of the kinetochore detachment process, and then underwent further maturation following nuclear envelope breakdown to give rise to the 130-160 kb fragment in detached kinetochores. Collectively, these data indicate that detached human kinetochores will be a useful system for investigating the organization, assembly, and function of human kinetochores.
Balczon
Ron
R
Department of Cell Biology and Neuroscience, The University of South Alabama, Mobile, AL 36688, USA. balczonr@sungcg.usouthal.edu
Wilson
Misti
M
Bhatnagar
Y M
YM
D000906:Antibodies / Q000378:metabolism; D015139:Blotting, Southern; D002110:Caffeine / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis; D002875:Chromosomes / Q000737:chemistry; D004247:DNA / Q000032:analysis; D016521:Electrophoresis, Gel, Pulsed-Field; D004591:Electrophoresis, Polyacrylamide Gel; D019085:Fluorescent Antibody Technique, Direct; D006367:HeLa Cells; D006570:Heterochromatin / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D018386:Kinetochores / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D015739:Nocodazole / Q000494:pharmacology; D000076988:Sulfate Transporters; D014162:Transfection
D000906:Antibodies; D002352:Carrier Proteins; D006570:Heterochromatin; D026901:Membrane Transport Proteins; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D002110:Caffeine; D004247:DNA; D015739:Nocodazole; D006918:Hydroxyurea