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Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

Krull S, Dörries J, Boysen B, Reidenbach S, Magnius L, Norder H, Thyberg J, Cordes VC - EMBO J. (2010)

Bottom Line: HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain.RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin.These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.

ABSTRACT
Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

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RNAi-mediated Tpr knockdown in HeLa cells. (A) Confocal IFM of Tpr at day 4 after transfection with different Tpr siRNAs (Ib3, IV2, IV4) or target-less control siRNAs. Only traces of Tpr staining are seen in most cells after Tpr RNAi; bright nuclear rim staining shown as reference is visible only in cells that remained untransfected. For occasionally observed dots of residual Tpr staining at otherwise largely Tpr-deficient NEs, see Supplementary Figure S6. Bar: 20 μm. (B1) SDS–PAGE and Coomassie staining of serial dilutions of whole-protein extracts from non-transfected cells (Ctrl 2), and cells treated with Tpr siRNAs (Ib3, III4, III5, IV2, IV4) or transfection reagent alone (Ctrl 1), showing that the bulk of cellular proteins remains unaffected by siRNA treatment. (B2) Immunoblotting of identical loadings as in panel B1. Incubations with anti-Tpr and anti-Nup98 (asterisks: unrelated cross-reactions) were on different halves of the same membrane. Efficient Tpr knockdown was achieved with all Tpr siRNAs without eliciting distinct effects on other NPC proteins, including Nup93, Nup107, Nup133, and gp210 (not shown). Cells transfected with III4 and III5, however, were later found to not allow for a normal PV-infection process, so that these siRNAs were not used further. Of the infection-compatible siRNAs, Ib3 and IV4 were used for all subsequent PV-infection experiments in parallel. (C) TEM of non-transfected cells, and of cell populations at day 4 after treatment with transfection reagent alone, and transfection with Tpr siRNAs Ib3, or non-target control siRNAs (Ctrl 3). NPCs juxtaposed to heterochromatic or nucleolar material (arrows) in control cells remained characterized by HEZs but mostly lacked such exclusion zones after Tpr RNAi. Bars: 200 nm; same magnification for all the images.
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f5: RNAi-mediated Tpr knockdown in HeLa cells. (A) Confocal IFM of Tpr at day 4 after transfection with different Tpr siRNAs (Ib3, IV2, IV4) or target-less control siRNAs. Only traces of Tpr staining are seen in most cells after Tpr RNAi; bright nuclear rim staining shown as reference is visible only in cells that remained untransfected. For occasionally observed dots of residual Tpr staining at otherwise largely Tpr-deficient NEs, see Supplementary Figure S6. Bar: 20 μm. (B1) SDS–PAGE and Coomassie staining of serial dilutions of whole-protein extracts from non-transfected cells (Ctrl 2), and cells treated with Tpr siRNAs (Ib3, III4, III5, IV2, IV4) or transfection reagent alone (Ctrl 1), showing that the bulk of cellular proteins remains unaffected by siRNA treatment. (B2) Immunoblotting of identical loadings as in panel B1. Incubations with anti-Tpr and anti-Nup98 (asterisks: unrelated cross-reactions) were on different halves of the same membrane. Efficient Tpr knockdown was achieved with all Tpr siRNAs without eliciting distinct effects on other NPC proteins, including Nup93, Nup107, Nup133, and gp210 (not shown). Cells transfected with III4 and III5, however, were later found to not allow for a normal PV-infection process, so that these siRNAs were not used further. Of the infection-compatible siRNAs, Ib3 and IV4 were used for all subsequent PV-infection experiments in parallel. (C) TEM of non-transfected cells, and of cell populations at day 4 after treatment with transfection reagent alone, and transfection with Tpr siRNAs Ib3, or non-target control siRNAs (Ctrl 3). NPCs juxtaposed to heterochromatic or nucleolar material (arrows) in control cells remained characterized by HEZs but mostly lacked such exclusion zones after Tpr RNAi. Bars: 200 nm; same magnification for all the images.

Mentions: Furthermore, it had been shown that activation of the RNAi machinery does not necessarily trigger antiviral responses, and siRNA-transfected cells can remain susceptible for subsequent PV infection (Gitlin et al, 2002). Nonetheless, to avoid innate cellular immune responses that could impair PV infection (e.g., Ida-Hosonuma et al, 2005), we first screened different Tpr siRNAs for high knockdown efficiencies without off-target effects unrelated to Tpr deficiency (data not shown). Several pre-selected siRNAs, complementary to non-overlapping ORF segments of the Tpr mRNA, caused a clear knockdown of Tpr protein levels at day 4 after transfection (Figure 5A). The mean intensities of residual Tpr staining in the transfected cells could be as low as 4–6% as determined by IFM, whereas residual Tpr levels in immunoblots of total cell extracts commonly ranged between 10% and less than 20% (Figure 5B). TEM of such cell populations allowed sporadic detection of perpendicularly sectioned NPCs that were juxtaposed to heterochromatic or nucleolar material but now mostly lacked distinct exclusion zones. This indicated that Tpr might at least play a role in the establishment of the few HEZs seen in non-infected HeLa cells. By contrast, even though correspondingly positioned NPCs were similarly sparse in simultaneous controls, they were still characterized by distinct HEZs (Figure 5C; see also Supplementary Text to Figure 5, and Supplementary Figure S6).


Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

Krull S, Dörries J, Boysen B, Reidenbach S, Magnius L, Norder H, Thyberg J, Cordes VC - EMBO J. (2010)

RNAi-mediated Tpr knockdown in HeLa cells. (A) Confocal IFM of Tpr at day 4 after transfection with different Tpr siRNAs (Ib3, IV2, IV4) or target-less control siRNAs. Only traces of Tpr staining are seen in most cells after Tpr RNAi; bright nuclear rim staining shown as reference is visible only in cells that remained untransfected. For occasionally observed dots of residual Tpr staining at otherwise largely Tpr-deficient NEs, see Supplementary Figure S6. Bar: 20 μm. (B1) SDS–PAGE and Coomassie staining of serial dilutions of whole-protein extracts from non-transfected cells (Ctrl 2), and cells treated with Tpr siRNAs (Ib3, III4, III5, IV2, IV4) or transfection reagent alone (Ctrl 1), showing that the bulk of cellular proteins remains unaffected by siRNA treatment. (B2) Immunoblotting of identical loadings as in panel B1. Incubations with anti-Tpr and anti-Nup98 (asterisks: unrelated cross-reactions) were on different halves of the same membrane. Efficient Tpr knockdown was achieved with all Tpr siRNAs without eliciting distinct effects on other NPC proteins, including Nup93, Nup107, Nup133, and gp210 (not shown). Cells transfected with III4 and III5, however, were later found to not allow for a normal PV-infection process, so that these siRNAs were not used further. Of the infection-compatible siRNAs, Ib3 and IV4 were used for all subsequent PV-infection experiments in parallel. (C) TEM of non-transfected cells, and of cell populations at day 4 after treatment with transfection reagent alone, and transfection with Tpr siRNAs Ib3, or non-target control siRNAs (Ctrl 3). NPCs juxtaposed to heterochromatic or nucleolar material (arrows) in control cells remained characterized by HEZs but mostly lacked such exclusion zones after Tpr RNAi. Bars: 200 nm; same magnification for all the images.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: RNAi-mediated Tpr knockdown in HeLa cells. (A) Confocal IFM of Tpr at day 4 after transfection with different Tpr siRNAs (Ib3, IV2, IV4) or target-less control siRNAs. Only traces of Tpr staining are seen in most cells after Tpr RNAi; bright nuclear rim staining shown as reference is visible only in cells that remained untransfected. For occasionally observed dots of residual Tpr staining at otherwise largely Tpr-deficient NEs, see Supplementary Figure S6. Bar: 20 μm. (B1) SDS–PAGE and Coomassie staining of serial dilutions of whole-protein extracts from non-transfected cells (Ctrl 2), and cells treated with Tpr siRNAs (Ib3, III4, III5, IV2, IV4) or transfection reagent alone (Ctrl 1), showing that the bulk of cellular proteins remains unaffected by siRNA treatment. (B2) Immunoblotting of identical loadings as in panel B1. Incubations with anti-Tpr and anti-Nup98 (asterisks: unrelated cross-reactions) were on different halves of the same membrane. Efficient Tpr knockdown was achieved with all Tpr siRNAs without eliciting distinct effects on other NPC proteins, including Nup93, Nup107, Nup133, and gp210 (not shown). Cells transfected with III4 and III5, however, were later found to not allow for a normal PV-infection process, so that these siRNAs were not used further. Of the infection-compatible siRNAs, Ib3 and IV4 were used for all subsequent PV-infection experiments in parallel. (C) TEM of non-transfected cells, and of cell populations at day 4 after treatment with transfection reagent alone, and transfection with Tpr siRNAs Ib3, or non-target control siRNAs (Ctrl 3). NPCs juxtaposed to heterochromatic or nucleolar material (arrows) in control cells remained characterized by HEZs but mostly lacked such exclusion zones after Tpr RNAi. Bars: 200 nm; same magnification for all the images.
Mentions: Furthermore, it had been shown that activation of the RNAi machinery does not necessarily trigger antiviral responses, and siRNA-transfected cells can remain susceptible for subsequent PV infection (Gitlin et al, 2002). Nonetheless, to avoid innate cellular immune responses that could impair PV infection (e.g., Ida-Hosonuma et al, 2005), we first screened different Tpr siRNAs for high knockdown efficiencies without off-target effects unrelated to Tpr deficiency (data not shown). Several pre-selected siRNAs, complementary to non-overlapping ORF segments of the Tpr mRNA, caused a clear knockdown of Tpr protein levels at day 4 after transfection (Figure 5A). The mean intensities of residual Tpr staining in the transfected cells could be as low as 4–6% as determined by IFM, whereas residual Tpr levels in immunoblots of total cell extracts commonly ranged between 10% and less than 20% (Figure 5B). TEM of such cell populations allowed sporadic detection of perpendicularly sectioned NPCs that were juxtaposed to heterochromatic or nucleolar material but now mostly lacked distinct exclusion zones. This indicated that Tpr might at least play a role in the establishment of the few HEZs seen in non-infected HeLa cells. By contrast, even though correspondingly positioned NPCs were similarly sparse in simultaneous controls, they were still characterized by distinct HEZs (Figure 5C; see also Supplementary Text to Figure 5, and Supplementary Figure S6).

Bottom Line: HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain.RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin.These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.

ABSTRACT
Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

Show MeSH
Related in: MedlinePlus