Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts.
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.
Kalab
Petr
P
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
Weis
Karsten
K
Heald
Rebecca
R
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007399:Interphase; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D010063:Ovum / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D008941:Spindle Apparatus / Q000378:metabolism; D014981:Xenopus; D028901:alpha Karyopherins / Q000737:chemistry / Q000378:metabolism; D028961:beta Karyopherins / Q000737:chemistry / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C054562:yellow fluorescent protein, Bacteria; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein