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Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner.

Lowe AR, Tang JH, Yassif J, Graf M, Huang WY, Groves JT, Weis K, Liphardt JT - Elife (2015)

Bottom Line: A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153.Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired.RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153.

View Article: PubMed Central - PubMed

Affiliation: Institute for Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom.

ABSTRACT
Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.

No MeSH data available.


Related in: MedlinePlus

Distribution of count values.Histogram of the count values for four conditions (wild type or Δ15370% cells with and without RanGTP) with a bin size of five molecules. Numbers of pores analyzed, means, and standard deviations are given.DOI:http://dx.doi.org/10.7554/eLife.04052.015
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fig4s1: Distribution of count values.Histogram of the count values for four conditions (wild type or Δ15370% cells with and without RanGTP) with a bin size of five molecules. Numbers of pores analyzed, means, and standard deviations are given.DOI:http://dx.doi.org/10.7554/eLife.04052.015

Mentions: Although dSTORM microscopy is able to localize populations of molecules and detect relative changes in their spatial arrangements, it does not allow absolute numbers of molecules to be estimated. We thus used a single-molecule photobleach step-counting assay (Leake et al., 2006) to estimate the numbers of impβ molecules displaced by nuclear RanGTP at the NPC. Digitonin-permeabilized nuclei from wild type and Δ15370% cells were incubated with impβ-mCherry with or without RanGTP and then fixed, yielding nuclear pores that can be imaged at the basal surface of the nucleus as bright spots (Figure 4A). Under the appropriate imaging conditions, discrete single-molecule photobleaching steps can be resolved in the fluorescence bleaching traces of impβ-mCherry at the pores (Figure 4B). The photobleaching fluorescence step-size, x, for a specific NPC, can be calculated by taking the first peak of the power spectrum of the pairwise difference distribution of the bleaching trace (Figure 4C). From the fluorescence step-size and initial intensity, ΔI, of the pore, the relative amount of impβ molecules at a single NPC can be measured. Because of potential systematic errors in determining absolute numbers of impβ molecules with this technique (e.g., homo-FRET, incomplete mCherry maturation), relative analysis of impβ levels was performed by defining the impβ signal of the wild type–RanGTP condition as 100% (Figure 4D). Wild type NPCs in the absence of RanGTP contained the greatest number of impβ molecules (70 bleach steps counted), whereas RanGTP caused a 27% decrease in impβ levels (Figure 4A, Figure 4—figure supplement 1, Table 1). In Δ15370% nuclei, we found a 34% drop in the amount of impβ per pore without RanGTP and a 33% drop with RanGTP. These results suggest that most of the impβ molecules that are displaced from the NPC by nuclear RanGTP are those that are bound to Nup153.10.7554/eLife.04052.014Figure 4.Photobleach step-counting of impβ at the NPC.


Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner.

Lowe AR, Tang JH, Yassif J, Graf M, Huang WY, Groves JT, Weis K, Liphardt JT - Elife (2015)

Distribution of count values.Histogram of the count values for four conditions (wild type or Δ15370% cells with and without RanGTP) with a bin size of five molecules. Numbers of pores analyzed, means, and standard deviations are given.DOI:http://dx.doi.org/10.7554/eLife.04052.015
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4375889&req=5

fig4s1: Distribution of count values.Histogram of the count values for four conditions (wild type or Δ15370% cells with and without RanGTP) with a bin size of five molecules. Numbers of pores analyzed, means, and standard deviations are given.DOI:http://dx.doi.org/10.7554/eLife.04052.015
Mentions: Although dSTORM microscopy is able to localize populations of molecules and detect relative changes in their spatial arrangements, it does not allow absolute numbers of molecules to be estimated. We thus used a single-molecule photobleach step-counting assay (Leake et al., 2006) to estimate the numbers of impβ molecules displaced by nuclear RanGTP at the NPC. Digitonin-permeabilized nuclei from wild type and Δ15370% cells were incubated with impβ-mCherry with or without RanGTP and then fixed, yielding nuclear pores that can be imaged at the basal surface of the nucleus as bright spots (Figure 4A). Under the appropriate imaging conditions, discrete single-molecule photobleaching steps can be resolved in the fluorescence bleaching traces of impβ-mCherry at the pores (Figure 4B). The photobleaching fluorescence step-size, x, for a specific NPC, can be calculated by taking the first peak of the power spectrum of the pairwise difference distribution of the bleaching trace (Figure 4C). From the fluorescence step-size and initial intensity, ΔI, of the pore, the relative amount of impβ molecules at a single NPC can be measured. Because of potential systematic errors in determining absolute numbers of impβ molecules with this technique (e.g., homo-FRET, incomplete mCherry maturation), relative analysis of impβ levels was performed by defining the impβ signal of the wild type–RanGTP condition as 100% (Figure 4D). Wild type NPCs in the absence of RanGTP contained the greatest number of impβ molecules (70 bleach steps counted), whereas RanGTP caused a 27% decrease in impβ levels (Figure 4A, Figure 4—figure supplement 1, Table 1). In Δ15370% nuclei, we found a 34% drop in the amount of impβ per pore without RanGTP and a 33% drop with RanGTP. These results suggest that most of the impβ molecules that are displaced from the NPC by nuclear RanGTP are those that are bound to Nup153.10.7554/eLife.04052.014Figure 4.Photobleach step-counting of impβ at the NPC.

Bottom Line: A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153.Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired.RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153.

View Article: PubMed Central - PubMed

Affiliation: Institute for Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom.

ABSTRACT
Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.

No MeSH data available.


Related in: MedlinePlus