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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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Perinuclear accumulation of material in cells expressing Nsp1-eDID with bound Dyn2. (A) Perinuclear accumulation of poly(A)+ RNA in the NSP1-eDID cells harboring the chromosomal integrated GAL::DYN2 after shift from raffinose to galactose medium that was induced (2 h in galactose) and uninduced (0 h in galactose). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. (B) Analysis of nuclear export of specific mRNAs encoding actin and SSA1 proteins in the NSP1-eDID dyn2Δ strain harboring the integrated GAL::DYN2 after shift from raffinose to galactose medium. 0 h in galactose (uninduced); 2 h in galactose (induced). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. For the detection of SSA1 mRNA, cells were shifted to 37°C for 30 min before cells were fixed and further processed. (C) Transmission EM of thin-sectioned yeast cells expressing the eDID-modified FG repeat domain of Nsp1 with bound Dyn2. (top) Overview micrographs of representative cells are shown derived from strains dyn2Δ + GAL::DYN2 and NSP1-eDID dyn2Δ + GAL::DYN2 in an uninduced and Dyn2-induced condition. (bottom) Higher magnification EM micrographs showing strain NSP1-eDID dyn2Δ + GAL::DYN2 in the Dyn2-induced condition. NPC, nuclear pore complex (filled arrowhead); NE, nuclear envelope; N, nucleus; C, cytoplasm. White arrowheads indicate electron-dense particles accumulating in the perinuclear region with some particles close to an NPC. Bars: (A and B) 5 µm; (C) 250 nm.
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fig3: Perinuclear accumulation of material in cells expressing Nsp1-eDID with bound Dyn2. (A) Perinuclear accumulation of poly(A)+ RNA in the NSP1-eDID cells harboring the chromosomal integrated GAL::DYN2 after shift from raffinose to galactose medium that was induced (2 h in galactose) and uninduced (0 h in galactose). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. (B) Analysis of nuclear export of specific mRNAs encoding actin and SSA1 proteins in the NSP1-eDID dyn2Δ strain harboring the integrated GAL::DYN2 after shift from raffinose to galactose medium. 0 h in galactose (uninduced); 2 h in galactose (induced). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. For the detection of SSA1 mRNA, cells were shifted to 37°C for 30 min before cells were fixed and further processed. (C) Transmission EM of thin-sectioned yeast cells expressing the eDID-modified FG repeat domain of Nsp1 with bound Dyn2. (top) Overview micrographs of representative cells are shown derived from strains dyn2Δ + GAL::DYN2 and NSP1-eDID dyn2Δ + GAL::DYN2 in an uninduced and Dyn2-induced condition. (bottom) Higher magnification EM micrographs showing strain NSP1-eDID dyn2Δ + GAL::DYN2 in the Dyn2-induced condition. NPC, nuclear pore complex (filled arrowhead); NE, nuclear envelope; N, nucleus; C, cytoplasm. White arrowheads indicate electron-dense particles accumulating in the perinuclear region with some particles close to an NPC. Bars: (A and B) 5 µm; (C) 250 nm.

Mentions: Next, we wanted to find out why cell growth stops when Dyn2 is recruited to a distinct group of eDID-modified FG Nups. We hypothesized that this could be a result of a blockage of nucleocytoplasmic transport caused by corrupting the FG repeat network in the active transport channel with Dyn2 molecules. Strikingly, nuclear mRNA export was massively inhibited after an induction of pGAL-DYN2 expression in the nonviable NSP1-eDID, NSP1-eDID(2), NUP49-eDID, NUP57-eDID, NUP1-eDID, and NUP2-eDID cells but not in the viable NUP116-eDID, NUP159ΔDID-eDID, and NUP159ΔDID-eDID(2) cells (Fig. 2 A). The onset of this defect is very fast and could already be well observed after a 45-min shift to galactose medium (note that it requires ∼20–30 min to initiate GAL promoter-induced protein expression) and, hence, is indicative of a direct transport inhibition rather than an NPC assembly defect (Fig. 2 B). Both, poly(A)+ RNA as well as specific mRNAs (e.g., actin and SSA1 heat shock mRNAs) that accumulate in the nucleus tend to cluster in foci that are close to the nuclear envelope (Fig. 2, A and B; and Fig. 3).


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)

Perinuclear accumulation of material in cells expressing Nsp1-eDID with bound Dyn2. (A) Perinuclear accumulation of poly(A)+ RNA in the NSP1-eDID cells harboring the chromosomal integrated GAL::DYN2 after shift from raffinose to galactose medium that was induced (2 h in galactose) and uninduced (0 h in galactose). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. (B) Analysis of nuclear export of specific mRNAs encoding actin and SSA1 proteins in the NSP1-eDID dyn2Δ strain harboring the integrated GAL::DYN2 after shift from raffinose to galactose medium. 0 h in galactose (uninduced); 2 h in galactose (induced). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. For the detection of SSA1 mRNA, cells were shifted to 37°C for 30 min before cells were fixed and further processed. (C) Transmission EM of thin-sectioned yeast cells expressing the eDID-modified FG repeat domain of Nsp1 with bound Dyn2. (top) Overview micrographs of representative cells are shown derived from strains dyn2Δ + GAL::DYN2 and NSP1-eDID dyn2Δ + GAL::DYN2 in an uninduced and Dyn2-induced condition. (bottom) Higher magnification EM micrographs showing strain NSP1-eDID dyn2Δ + GAL::DYN2 in the Dyn2-induced condition. NPC, nuclear pore complex (filled arrowhead); NE, nuclear envelope; N, nucleus; C, cytoplasm. White arrowheads indicate electron-dense particles accumulating in the perinuclear region with some particles close to an NPC. Bars: (A and B) 5 µm; (C) 250 nm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Perinuclear accumulation of material in cells expressing Nsp1-eDID with bound Dyn2. (A) Perinuclear accumulation of poly(A)+ RNA in the NSP1-eDID cells harboring the chromosomal integrated GAL::DYN2 after shift from raffinose to galactose medium that was induced (2 h in galactose) and uninduced (0 h in galactose). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. (B) Analysis of nuclear export of specific mRNAs encoding actin and SSA1 proteins in the NSP1-eDID dyn2Δ strain harboring the integrated GAL::DYN2 after shift from raffinose to galactose medium. 0 h in galactose (uninduced); 2 h in galactose (induced). The indicated RNA was detected by in situ hybridization using appropriate Cy3-labeled RNA probes. DNA was stained with DAPI. For the detection of SSA1 mRNA, cells were shifted to 37°C for 30 min before cells were fixed and further processed. (C) Transmission EM of thin-sectioned yeast cells expressing the eDID-modified FG repeat domain of Nsp1 with bound Dyn2. (top) Overview micrographs of representative cells are shown derived from strains dyn2Δ + GAL::DYN2 and NSP1-eDID dyn2Δ + GAL::DYN2 in an uninduced and Dyn2-induced condition. (bottom) Higher magnification EM micrographs showing strain NSP1-eDID dyn2Δ + GAL::DYN2 in the Dyn2-induced condition. NPC, nuclear pore complex (filled arrowhead); NE, nuclear envelope; N, nucleus; C, cytoplasm. White arrowheads indicate electron-dense particles accumulating in the perinuclear region with some particles close to an NPC. Bars: (A and B) 5 µm; (C) 250 nm.
Mentions: Next, we wanted to find out why cell growth stops when Dyn2 is recruited to a distinct group of eDID-modified FG Nups. We hypothesized that this could be a result of a blockage of nucleocytoplasmic transport caused by corrupting the FG repeat network in the active transport channel with Dyn2 molecules. Strikingly, nuclear mRNA export was massively inhibited after an induction of pGAL-DYN2 expression in the nonviable NSP1-eDID, NSP1-eDID(2), NUP49-eDID, NUP57-eDID, NUP1-eDID, and NUP2-eDID cells but not in the viable NUP116-eDID, NUP159ΔDID-eDID, and NUP159ΔDID-eDID(2) cells (Fig. 2 A). The onset of this defect is very fast and could already be well observed after a 45-min shift to galactose medium (note that it requires ∼20–30 min to initiate GAL promoter-induced protein expression) and, hence, is indicative of a direct transport inhibition rather than an NPC assembly defect (Fig. 2 B). Both, poly(A)+ RNA as well as specific mRNAs (e.g., actin and SSA1 heat shock mRNAs) that accumulate in the nucleus tend to cluster in foci that are close to the nuclear envelope (Fig. 2, A and B; and Fig. 3).

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
Related in: MedlinePlus