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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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Nup153 and Nup98 appear largely degraded upon PV infection whereas the coiled-coil rod domain of protein Tpr remains intact. (A) SDS–PAGE and Coomassie staining of whole-protein extracts from HeLa cells before and at 2–12 h post-infection, showing the bulk of cellular proteins unaffected by PV-induced proteolysis. The extracts were from the same HeLa cell cultures analysed by TEM in Figure 1. (B) Immunoblotting of extracts used in panel A, showing that lamin A and lamin C, like lamin B (not shown), remain unaffected. This indicates that rearrangements within the nuclear lamina or its NE attachments, but not lamin proteolysis, are required for the NE upfolding observed. (C) Epitope sites of Nup153, Nup98, and Tpr antibodies indicated by arrowheads. Different antibodies targeting the same protein are numbered as in panels D and E. (D, E) Immunoblotting of Nup153, Nup98, and Tpr, using cell extracts shown in panel A; (see also Supplementary Figure S3). Target regions are given in parentheses; Δ indicates mAbs for which actual epitopes within defined protein segments are unknown. (D) At 8–12 h post-infection, when HEZs are visible at almost all NPCs, Nup153 (full-length proteins marked by arrows) appears largely degraded, except for a small segment (double-asterisk) comprising at least part of the Tpr-binding region. Additionally, only minor amounts of a 120-kDa degradation product (asterisk) and some unspecific cross-reactions (u) are seen with some Nup153 antibodies. Nup98 is degraded more rapidly, with only its C-terminal domain (asterisk) resisting proteolysis slightly longer. (E) Whereas Tpr's C-terminal domain is being degraded around 8 h post-infection, its entire rod domain (double-asterisk) withstands proteolysis. The membrane marked 1622–1640 was first incubated with rb-anti-Tpr-4 (2063–2084), then stripped and re-incubated with gp-anti-Tpr-3 (1622–1640).
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f3: Nup153 and Nup98 appear largely degraded upon PV infection whereas the coiled-coil rod domain of protein Tpr remains intact. (A) SDS–PAGE and Coomassie staining of whole-protein extracts from HeLa cells before and at 2–12 h post-infection, showing the bulk of cellular proteins unaffected by PV-induced proteolysis. The extracts were from the same HeLa cell cultures analysed by TEM in Figure 1. (B) Immunoblotting of extracts used in panel A, showing that lamin A and lamin C, like lamin B (not shown), remain unaffected. This indicates that rearrangements within the nuclear lamina or its NE attachments, but not lamin proteolysis, are required for the NE upfolding observed. (C) Epitope sites of Nup153, Nup98, and Tpr antibodies indicated by arrowheads. Different antibodies targeting the same protein are numbered as in panels D and E. (D, E) Immunoblotting of Nup153, Nup98, and Tpr, using cell extracts shown in panel A; (see also Supplementary Figure S3). Target regions are given in parentheses; Δ indicates mAbs for which actual epitopes within defined protein segments are unknown. (D) At 8–12 h post-infection, when HEZs are visible at almost all NPCs, Nup153 (full-length proteins marked by arrows) appears largely degraded, except for a small segment (double-asterisk) comprising at least part of the Tpr-binding region. Additionally, only minor amounts of a 120-kDa degradation product (asterisk) and some unspecific cross-reactions (u) are seen with some Nup153 antibodies. Nup98 is degraded more rapidly, with only its C-terminal domain (asterisk) resisting proteolysis slightly longer. (E) Whereas Tpr's C-terminal domain is being degraded around 8 h post-infection, its entire rod domain (double-asterisk) withstands proteolysis. The membrane marked 1622–1640 was first incubated with rb-anti-Tpr-4 (2063–2084), then stripped and re-incubated with gp-anti-Tpr-3 (1622–1640).

Mentions: We hence studied these and other proteins by immunoblotting of total HeLa proteins, collected at different time points after PV infection. SDS–PAGE showed that the bulk of cellular proteins remained unaffected by PV-induced proteolysis (Figure 3A). Furthermore, immunoblotting showed that lamins (Figure 3B) and most NPC-core proteins were left unharmed (Supplementary Figure S3). This included those of the Nup160 subcomplex, which represent the fundament for direct or indirect NPC anchorage of other NPC-associated proteins such as Nup98, Nup153, and Tpr (e.g., Vasu et al, 2001). By contrast, most FG-repeat nucleoporins (Figure 3C and D, and Supplementary Figure S3) had their FG-repeat domains removed. Tpr was also partially degraded (Figure 3C and E).


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)

Nup153 and Nup98 appear largely degraded upon PV infection whereas the coiled-coil rod domain of protein Tpr remains intact. (A) SDS–PAGE and Coomassie staining of whole-protein extracts from HeLa cells before and at 2–12 h post-infection, showing the bulk of cellular proteins unaffected by PV-induced proteolysis. The extracts were from the same HeLa cell cultures analysed by TEM in Figure 1. (B) Immunoblotting of extracts used in panel A, showing that lamin A and lamin C, like lamin B (not shown), remain unaffected. This indicates that rearrangements within the nuclear lamina or its NE attachments, but not lamin proteolysis, are required for the NE upfolding observed. (C) Epitope sites of Nup153, Nup98, and Tpr antibodies indicated by arrowheads. Different antibodies targeting the same protein are numbered as in panels D and E. (D, E) Immunoblotting of Nup153, Nup98, and Tpr, using cell extracts shown in panel A; (see also Supplementary Figure S3). Target regions are given in parentheses; Δ indicates mAbs for which actual epitopes within defined protein segments are unknown. (D) At 8–12 h post-infection, when HEZs are visible at almost all NPCs, Nup153 (full-length proteins marked by arrows) appears largely degraded, except for a small segment (double-asterisk) comprising at least part of the Tpr-binding region. Additionally, only minor amounts of a 120-kDa degradation product (asterisk) and some unspecific cross-reactions (u) are seen with some Nup153 antibodies. Nup98 is degraded more rapidly, with only its C-terminal domain (asterisk) resisting proteolysis slightly longer. (E) Whereas Tpr's C-terminal domain is being degraded around 8 h post-infection, its entire rod domain (double-asterisk) withstands proteolysis. The membrane marked 1622–1640 was first incubated with rb-anti-Tpr-4 (2063–2084), then stripped and re-incubated with gp-anti-Tpr-3 (1622–1640).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2876962&req=5

f3: Nup153 and Nup98 appear largely degraded upon PV infection whereas the coiled-coil rod domain of protein Tpr remains intact. (A) SDS–PAGE and Coomassie staining of whole-protein extracts from HeLa cells before and at 2–12 h post-infection, showing the bulk of cellular proteins unaffected by PV-induced proteolysis. The extracts were from the same HeLa cell cultures analysed by TEM in Figure 1. (B) Immunoblotting of extracts used in panel A, showing that lamin A and lamin C, like lamin B (not shown), remain unaffected. This indicates that rearrangements within the nuclear lamina or its NE attachments, but not lamin proteolysis, are required for the NE upfolding observed. (C) Epitope sites of Nup153, Nup98, and Tpr antibodies indicated by arrowheads. Different antibodies targeting the same protein are numbered as in panels D and E. (D, E) Immunoblotting of Nup153, Nup98, and Tpr, using cell extracts shown in panel A; (see also Supplementary Figure S3). Target regions are given in parentheses; Δ indicates mAbs for which actual epitopes within defined protein segments are unknown. (D) At 8–12 h post-infection, when HEZs are visible at almost all NPCs, Nup153 (full-length proteins marked by arrows) appears largely degraded, except for a small segment (double-asterisk) comprising at least part of the Tpr-binding region. Additionally, only minor amounts of a 120-kDa degradation product (asterisk) and some unspecific cross-reactions (u) are seen with some Nup153 antibodies. Nup98 is degraded more rapidly, with only its C-terminal domain (asterisk) resisting proteolysis slightly longer. (E) Whereas Tpr's C-terminal domain is being degraded around 8 h post-infection, its entire rod domain (double-asterisk) withstands proteolysis. The membrane marked 1622–1640 was first incubated with rb-anti-Tpr-4 (2063–2084), then stripped and re-incubated with gp-anti-Tpr-3 (1622–1640).
Mentions: We hence studied these and other proteins by immunoblotting of total HeLa proteins, collected at different time points after PV infection. SDS–PAGE showed that the bulk of cellular proteins remained unaffected by PV-induced proteolysis (Figure 3A). Furthermore, immunoblotting showed that lamins (Figure 3B) and most NPC-core proteins were left unharmed (Supplementary Figure S3). This included those of the Nup160 subcomplex, which represent the fundament for direct or indirect NPC anchorage of other NPC-associated proteins such as Nup98, Nup153, and Tpr (e.g., Vasu et al, 2001). By contrast, most FG-repeat nucleoporins (Figure 3C and D, and Supplementary Figure S3) had their FG-repeat domains removed. Tpr was also partially degraded (Figure 3C and E).

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