Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis.
Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.
Khodjakov
Alexey
A
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Copenagle
Lily
L
Gordon
Michael B
MB
Compton
Duane A
DA
Kapoor
Tarun M
TM
D000818:Animals; D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D002875:Chromosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005057:Eukaryotic Cells / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013871:Thiones / Q000494:pharmacology; D014404:Tubulin
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C470243:KIF11 protein, human; D008164:Luminescent Proteins; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D013871:Thiones; D014404:Tubulin; D049452:Green Fluorescent Proteins; C400223:monastrol; D016547:Kinesins