Significant proportions of nuclear transport proteins with reduced intracellular mobilities resolved by fluorescence correlation spectroscopy.
Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin alpha, importin beta, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.
Paradise
Allison
A
Department of Molecular Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Levin
Mikhail K
MK
Korza
George
G
Carson
John H
JH
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009685:Nuclear Envelope / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D011257:Pregnancy Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D014407:Tumor Cells, Cultured; D028901:alpha Karyopherins / Q000737:chemistry / Q000378:metabolism; D028961:beta Karyopherins / Q000737:chemistry / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism
C038965:NUTF2 protein, human; D029543:Nucleocytoplasmic Transport Proteins; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein