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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

Localization precision.(A) Calibration of EMCCD camera for photon conversion factor. (B) Histogram of calculated localization precisions from dSTORM image data. The median value of localization precision (positional error) is 12 nm.DOI:http://dx.doi.org/10.7554/eLife.04052.008
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fig2s1: Localization precision.(A) Calibration of EMCCD camera for photon conversion factor. (B) Histogram of calculated localization precisions from dSTORM image data. The median value of localization precision (positional error) is 12 nm.DOI:http://dx.doi.org/10.7554/eLife.04052.008

Mentions: Having detected two kinetically distinct impβ pools within the pore, we sought to characterize their spatial distribution and identify the nucleoporins they were binding. We were especially interested in the RanGTP-sensitive impβ pool, since RanGTP drives active transport and RanGTP-induced alterations of pore organization could therefore be relevant to active transport. We directly imaged and localized individual Cy5 or Alexa647 dye-labeled impβ molecules within the pore using dSTORM super-resolution localization microscopy (Heilemann et al., 2008; van de Linde et al., 2011) (Figure 2A–D; mean spatial precision, σx,y of 12 nm, Figure 2—figure supplement 1). By directly labeling impβ with a fluorescent reporter, we removed additional localization uncertainty error (commonly referred to as linkage error) associated with the antibody labeling methods normally used for super-resolution or electron microscopy.10.7554/eLife.04052.007Figure 2.Super-resolution imaging of Alexa647-labeled impβ in digitonin-permeabilized HeLa cells.


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)

Localization precision.(A) Calibration of EMCCD camera for photon conversion factor. (B) Histogram of calculated localization precisions from dSTORM image data. The median value of localization precision (positional error) is 12 nm.DOI:http://dx.doi.org/10.7554/eLife.04052.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4375889&req=5

fig2s1: Localization precision.(A) Calibration of EMCCD camera for photon conversion factor. (B) Histogram of calculated localization precisions from dSTORM image data. The median value of localization precision (positional error) is 12 nm.DOI:http://dx.doi.org/10.7554/eLife.04052.008
Mentions: Having detected two kinetically distinct impβ pools within the pore, we sought to characterize their spatial distribution and identify the nucleoporins they were binding. We were especially interested in the RanGTP-sensitive impβ pool, since RanGTP drives active transport and RanGTP-induced alterations of pore organization could therefore be relevant to active transport. We directly imaged and localized individual Cy5 or Alexa647 dye-labeled impβ molecules within the pore using dSTORM super-resolution localization microscopy (Heilemann et al., 2008; van de Linde et al., 2011) (Figure 2A–D; mean spatial precision, σx,y of 12 nm, Figure 2—figure supplement 1). By directly labeling impβ with a fluorescent reporter, we removed additional localization uncertainty error (commonly referred to as linkage error) associated with the antibody labeling methods normally used for super-resolution or electron microscopy.10.7554/eLife.04052.007Figure 2.Super-resolution imaging of Alexa647-labeled impβ in digitonin-permeabilized HeLa cells.

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