Abstract
The small guanosine triphosphatase Ran is loaded with guanosine triphosphate (GTP) by the chromatin-bound guanine nucleotide exchange factor RCC1 and releases import cargoes in the nucleus during interphase. In mitosis, Ran-GTP promotes spindle assembly around chromosomes by locally discharging cargoes that regulate microtubule dynamics and organization. We used fluorescence resonance energy transfer-based biosensors to visualize gradients of Ran-GTP and liberated cargoes around chromosomes in mitotic Xenopus egg extracts. Both gradients were required to assemble and maintain spindle structure. During interphase, Ran-GTP was highly enriched in the nucleoplasm, and a steep concentration difference between nuclear and cytoplasmic Ran-GTP was established, providing evidence for a Ran-GTP gradient surrounding chromosomes throughout the cell cycle.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Active Transport, Cell Nucleus
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Animals
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Bacterial Proteins / metabolism
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Biosensing Techniques
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Carrier Proteins / chemistry
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Cell Nucleus / metabolism*
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Chromosomes / metabolism
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Cytoplasm / metabolism*
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Energy Transfer
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Fluorescence
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Fluorescent Dyes
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Green Fluorescent Proteins
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Guanosine Diphosphate / metabolism
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Guanosine Triphosphate / metabolism*
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Interphase*
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Luminescent Proteins / metabolism
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Male
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Microtubules / metabolism
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Mitosis*
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Ovum / metabolism
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / metabolism
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Spectrometry, Fluorescence
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Spindle Apparatus / metabolism
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Xenopus
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alpha Karyopherins / chemistry
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alpha Karyopherins / metabolism
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beta Karyopherins / chemistry
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beta Karyopherins / metabolism
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ran GTP-Binding Protein / metabolism*
Substances
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Bacterial Proteins
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Carrier Proteins
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Fluorescent Dyes
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Luminescent Proteins
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Recombinant Fusion Proteins
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alpha Karyopherins
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beta Karyopherins
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yellow fluorescent protein, Bacteria
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Guanosine Diphosphate
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Green Fluorescent Proteins
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Guanosine Triphosphate
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ran GTP-Binding Protein