Relative contributions of chromatin and kinetochores to mitotic spindle assembly.
During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly.
O'Connell
Christopher B
CB
Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA. oconnell@wadsworth.org
Loncarek
Jadranka
J
Kaláb
Petr
P
Khodjakov
Alexey
A
D002843:Chromatin / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004247:DNA / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism
D002843:Chromatin; D003598:Cytoskeletal Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D014404:Tubulin; D004247:DNA; D020931:ran GTP-Binding Protein