Abstract
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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Antigens, Nuclear
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Cell Cycle Proteins
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Cell Polarity / physiology
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Cells, Cultured
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Chromosomes / drug effects
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Chromosomes / metabolism*
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Chromosomes / ultrastructure
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Eukaryotic Cells / metabolism*
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Eukaryotic Cells / ultrastructure
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Green Fluorescent Proteins
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Humans
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Kinesins / antagonists & inhibitors
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Kinesins / metabolism
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Kinetochores / drug effects
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Kinetochores / metabolism*
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Kinetochores / ultrastructure
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Luminescent Proteins
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Microscopy, Fluorescence
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Microtubules / drug effects
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Microtubules / metabolism*
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Microtubules / ultrastructure
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Mitosis / drug effects
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Mitosis / physiology*
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Nuclear Matrix-Associated Proteins
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Nuclear Proteins / antagonists & inhibitors
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Nuclear Proteins / metabolism
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Pyrimidines / pharmacology
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Recombinant Fusion Proteins
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Spindle Apparatus / drug effects
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Spindle Apparatus / metabolism*
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Spindle Apparatus / ultrastructure
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Thiones / pharmacology
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Tubulin
Substances
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Antigens, Nuclear
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Cell Cycle Proteins
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KIF11 protein, human
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Luminescent Proteins
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NUMA1 protein, human
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Nuclear Matrix-Associated Proteins
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Nuclear Proteins
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Pyrimidines
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Recombinant Fusion Proteins
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Thiones
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Tubulin
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Green Fluorescent Proteins
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monastrol
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Kinesins