Significant Proportions of Nuclear Transport Proteins with Reduced Intracellular Mobilities Resolved by Fluorescence Correlation Spectroscopy

Department of Molecular, Microbial and Structural Biology, University of Connecticut, Storrs, Connecticut, United States
Journal of Molecular Biology (Impact Factor: 4.33). 02/2007; 365(1):50-65. DOI: 10.1016/j.jmb.2006.09.089
Source: PubMed


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.

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    • "But in general, these results indicate strong binding of NTRs to the NPC and high capacity of binding sites at single NPCs. As the cytoplasmic concentrations of NTRs, like mammalian Kap␤1 and yeast Kap95p, have been found to be in the micromolar range (Paradise et al., 2007; Pyhtila and Rexach, 2003), i.e. orders of magnitude above the effective k D values of FG–NTR complexes (Ben-Efraim and Gerace, 2001; Pyhtila and Rexach, 2003), it is likely that under physiological conditions the NPC is saturated with NTRs. In such an arrangement NTRs cannot be regarded as soluble interaction partners but instead, constituent parts of the NPC. "
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    ABSTRACT: It is generally accepted that transport through the nuclear pore complex (NPC) involves an abundance of phenylalanine-glycine rich protein domains (FG-domains) that serve as docking sites for soluble nuclear transport receptors (NTRs) and their cargo complexes. But the precise mechanism of translocation through the NPC allowing for high speed and selectivity is still vividly debated. To ultimately decipher the underlying gating mechanism it is indispensable to shed more light on the molecular arrangement of FG-domains and the distribution of NTR-binding sites within the central channel of the NPC. In this review we revisit current transport models, summarize recent results regarding translocation through the NPC obtained by super-resolution microscopy and finally discuss the status and potential of optical methods in the analysis of the NPC.
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    • "Brightness analysis showed that the karyopherins were monomeric at all observed concentrations—from 100 to 1000 nM—and thus the slow diffusion was not due to receptor aggregation. Using FCS, Paradise et al. (2007) also noted the reduced mobility of nuclear transport proteins, both in the cytoplasm and in the nucleus, and also used photobleaching methods to ascertain the contribution from an immobile fraction. Dross et al. (2009) studied the diffusion of EGFP in cell interiors and also present a useful discussion of the FCS-specific artifacts typically encountered in live cell studies as well as strategies for minimizing them. "
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    • "The main function of NTF2 is to facilitate transport of certain proteins into the nucleus via interaction with nucleoporin FxFG [12,13]. NTF2 also works as a GDP-dissociation inhibitor to mediate the GDP-Ran gradient, which is also involved nucleocytoplasmic transport [14-16]. The importance of NTF2 is illustrated by the findings that deletion of this gene results in lethality [17]. "
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    ABSTRACT: We performed human, animal, and in vitro studies to examine the potential role of nuclear transport factor 2 (NTF2) in conferring resistance to diabetic retinopathy (DR). Blood NTF2 levels were assessed in two groups of patients with type 2 diabetes mellitus. Group P patients had a history of proliferative DR (PDR), while group N patients did not. The retinal vasculature was examined in diabetic rats three months after they received an intravitreal injection of a recombinant adeno-associated virus (rAAV) vector overexpressing NTF2 (rAAV2-NTF2). Control rats were treated with rAAV2 only. Rat retinal capillary endothelial cells (RRCECs) were infected with rAAV2-NTF2, or with a vector expressing siRNA targeted against NTF2, to assess the effects of overexpression and inhibition of NTF2 on vascular endothelial growth factor (VEGF) expression (mRNA and protein). There was a strong trend for patients with DR to have lower blood NTF2 levels compared to those who did not have DR (0.10+/-0.01 versus 0.20+/-0.08, p=0.079). There was significantly less retinal blood vessel leakage in diabetic rats infected with rAAV2-NTF2 compared to controls (16.5+/-2.9 versus 24.7+/-7.3, p=0.039). These rats exhibited normal retinal vasculature and blood-retinal barrier function. VEGF expression was inhibited by NTF2 overexpression and stimulated by NTF2 inhibition, (protein [0.41+/-0.05 versus 0.23+/-0.06] and mRNA [0.37+/-0.04 versus 0.23+/-0.06] p<0.01 for all). These finding suggest that NTF2 is a potential mediator of retinal vasculature integrity. NTF2 may act by altering VEGF expression, thereby influencing the development of DR in patients with diabetes mellitus.
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