Spatial distribution and mobility of the Ran GTPase in live interphase cells.
The GTPase Ran is a key regulator of molecular transport through nuclear pore complex (NPC) channels. To analyze the role of Ran in its nuclear transport function, we used several quantitative fluorescence techniques to follow the distribution and dynamics of an enhanced yellow fluorescent protein (EYFP)-Ran in HeLa cells. The diffusion coefficient of the majority of EYFP-Ran molecules throughout the cells corresponded to an unbound state, revealing the remarkably dynamic Ran regulation. Although we observed no significant immobile Ran populations in cells, approximately 10% of the cytoplasmic EYFP-Ran and 30% of the nuclear EYFP-Ran exhibited low mobility indicative of molecular interactions. The high fraction of slow nuclear EYFP-Ran reflects the expected numerous interactions of nuclear RanGTP with nuclear transport receptors. However, it is not high enough to support retention mechanisms as the main cause for the observed nuclear accumulation of Ran. The highest cellular concentration of EYFP-Ran was detected at the nuclear envelope, corresponding to approximately 200 endogenous Ran molecules for each NPC, and exceeding the currently estimated NPC channel transport capacity. Together with the relatively long residence time of EYFP-Ran at the nuclear envelope (33 +/- 14 ms), these observations suggest that only a fraction of the Ran concentrated at NPCs transits through NPC channels.
Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, Canada.
Asmahan
A
Abu-Arish
Petr
P
Kalab
Josh
J
Ng-Kamstra
Karsten
K
Weis
Cécile
C
Fradin
D000465:Algorithms; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000502:physiology; D003593:Cytoplasm / Q000502:physiology; D004058:Diffusion; D005453:Fluorescence; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000502:physiology*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D009038:Motion; D009685:Nuclear Envelope / Q000502:physiology; D022022:Nuclear Pore / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013997:Time Factors; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C120587:RAN protein, human; C054562:yellow fluorescent protein, Bacteria; D020931:ran GTP-Binding Protein