Analysis of a RanGTP-regulated gradient in mitotic somatic cells.
The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran-importin-beta-cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
Petr
P
Kaláb
Arnd
A
Pralle
Ehud Y
EY
Isacoff
Rebecca
R
Heald
Karsten
K
Weis
D000818:Animals; D015374:Biosensing Techniques; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D018386:Kinetochores / Q000378:metabolism; D008540:Meiosis; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D014981:Xenopus; D028961:beta Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C062521:RCC1 protein, human; D028961:beta Karyopherins; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein