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Probing the nucleoporin FG repeat network defines structural and functional features of the nuclear pore complex.

Stelter P, Kunze R, Fischer J, Hurt E - J. Cell Biol. (2011)

Bottom Line: Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport.In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport.Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemie-Zentrum der Universität Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport. In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport. Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

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Recruitment of Dyn2 to eDID-modified FG repeat Nups can cause inhibition of nuclear import and export processes. (A) Analysis of nuclear mRNA export after 2-h galactose induction of Dyn2 expression in the indicated eDID-FG repeat NUP strains harboring an empty GAL plasmid (left) or pGAL-DYN2 (right). Poly(A)+ RNA was analyzed by in situ hybridization with a Cy3-labeled oligo d(T) probe, and DNA was stained with DAPI. (B) Time course of nuclear poly(A)+ accumulation in logarithmically growing NSP1-eDID dyn2Δ cells expressing Dyn2 from integrated GAL::DYN2. Cells were grown in YPR to an OD of ∼0.5 before Dyn2 expression was induced by adding 2% galactose. After the indicated time points, samples were taken and fixed by adding 4% formaldehyde to the culture medium. Samples were further processed for in situ poly(A)+ hybridization (see Materials and methods). (C) Analysis of nuclear import of Pho4-GFP in the dyn2Δ and NSP1-eDID dyn2Δ strains lacking or harboring the chromosomal integrated GAL::DYN2. Cells were grown in high phosphate medium followed by 2-h galactose induction of the GAL::DYN2 and switched to low phosphate medium. Pho4-GFP import was analyzed after 1 h in the fluorescence microscope. Bars, 5 µm.
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fig2: Recruitment of Dyn2 to eDID-modified FG repeat Nups can cause inhibition of nuclear import and export processes. (A) Analysis of nuclear mRNA export after 2-h galactose induction of Dyn2 expression in the indicated eDID-FG repeat NUP strains harboring an empty GAL plasmid (left) or pGAL-DYN2 (right). Poly(A)+ RNA was analyzed by in situ hybridization with a Cy3-labeled oligo d(T) probe, and DNA was stained with DAPI. (B) Time course of nuclear poly(A)+ accumulation in logarithmically growing NSP1-eDID dyn2Δ cells expressing Dyn2 from integrated GAL::DYN2. Cells were grown in YPR to an OD of ∼0.5 before Dyn2 expression was induced by adding 2% galactose. After the indicated time points, samples were taken and fixed by adding 4% formaldehyde to the culture medium. Samples were further processed for in situ poly(A)+ hybridization (see Materials and methods). (C) Analysis of nuclear import of Pho4-GFP in the dyn2Δ and NSP1-eDID dyn2Δ strains lacking or harboring the chromosomal integrated GAL::DYN2. Cells were grown in high phosphate medium followed by 2-h galactose induction of the GAL::DYN2 and switched to low phosphate medium. Pho4-GFP import was analyzed after 1 h in the fluorescence microscope. Bars, 5 µm.

Mentions: Strikingly, induction of GAL::DYN2 expression induced a lethal or extremely slow growth phenotype in several of the strains that harbored such an eDID-modified FG Nup. Specifically, Dyn2 expression was lethal to cells expressing Nsp1-eDID, Nup49-eDID, Nup57-eDID, Nup2-eDID, and Nup1-eDID but was not toxic in cells expressing Nup116-eDID or Nup159ΔDID-eDID (Fig. 1 B, galactose). The growth inhibition (and also the mRNA transport defects; Fig. 2 and Fig. S3 B) was not always fully complemented by coexpression of the respective wild-type Nup (Fig. S3). These data suggest some dominant effect of these Nup-eDID constructs with respect to growth and/or nucleocytoplasmic transport.


Probing the nucleoporin FG repeat network defines structural and functional features of the nuclear pore complex.

Stelter P, Kunze R, Fischer J, Hurt E - J. Cell Biol. (2011)

Recruitment of Dyn2 to eDID-modified FG repeat Nups can cause inhibition of nuclear import and export processes. (A) Analysis of nuclear mRNA export after 2-h galactose induction of Dyn2 expression in the indicated eDID-FG repeat NUP strains harboring an empty GAL plasmid (left) or pGAL-DYN2 (right). Poly(A)+ RNA was analyzed by in situ hybridization with a Cy3-labeled oligo d(T) probe, and DNA was stained with DAPI. (B) Time course of nuclear poly(A)+ accumulation in logarithmically growing NSP1-eDID dyn2Δ cells expressing Dyn2 from integrated GAL::DYN2. Cells were grown in YPR to an OD of ∼0.5 before Dyn2 expression was induced by adding 2% galactose. After the indicated time points, samples were taken and fixed by adding 4% formaldehyde to the culture medium. Samples were further processed for in situ poly(A)+ hybridization (see Materials and methods). (C) Analysis of nuclear import of Pho4-GFP in the dyn2Δ and NSP1-eDID dyn2Δ strains lacking or harboring the chromosomal integrated GAL::DYN2. Cells were grown in high phosphate medium followed by 2-h galactose induction of the GAL::DYN2 and switched to low phosphate medium. Pho4-GFP import was analyzed after 1 h in the fluorescence microscope. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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fig2: Recruitment of Dyn2 to eDID-modified FG repeat Nups can cause inhibition of nuclear import and export processes. (A) Analysis of nuclear mRNA export after 2-h galactose induction of Dyn2 expression in the indicated eDID-FG repeat NUP strains harboring an empty GAL plasmid (left) or pGAL-DYN2 (right). Poly(A)+ RNA was analyzed by in situ hybridization with a Cy3-labeled oligo d(T) probe, and DNA was stained with DAPI. (B) Time course of nuclear poly(A)+ accumulation in logarithmically growing NSP1-eDID dyn2Δ cells expressing Dyn2 from integrated GAL::DYN2. Cells were grown in YPR to an OD of ∼0.5 before Dyn2 expression was induced by adding 2% galactose. After the indicated time points, samples were taken and fixed by adding 4% formaldehyde to the culture medium. Samples were further processed for in situ poly(A)+ hybridization (see Materials and methods). (C) Analysis of nuclear import of Pho4-GFP in the dyn2Δ and NSP1-eDID dyn2Δ strains lacking or harboring the chromosomal integrated GAL::DYN2. Cells were grown in high phosphate medium followed by 2-h galactose induction of the GAL::DYN2 and switched to low phosphate medium. Pho4-GFP import was analyzed after 1 h in the fluorescence microscope. Bars, 5 µm.
Mentions: Strikingly, induction of GAL::DYN2 expression induced a lethal or extremely slow growth phenotype in several of the strains that harbored such an eDID-modified FG Nup. Specifically, Dyn2 expression was lethal to cells expressing Nsp1-eDID, Nup49-eDID, Nup57-eDID, Nup2-eDID, and Nup1-eDID but was not toxic in cells expressing Nup116-eDID or Nup159ΔDID-eDID (Fig. 1 B, galactose). The growth inhibition (and also the mRNA transport defects; Fig. 2 and Fig. S3 B) was not always fully complemented by coexpression of the respective wild-type Nup (Fig. S3). These data suggest some dominant effect of these Nup-eDID constructs with respect to growth and/or nucleocytoplasmic transport.

Bottom Line: Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport.In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport.Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemie-Zentrum der Universität Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport. In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain-interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport. Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

Show MeSH