Limits...
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.

Show MeSH

Related in: MedlinePlus

NPC-associated HEZs are no longer maintained after in vivo depletion of Tpr, resulting in NPC coating by heterochromatin. (A) After mock transfection (Ctrl 1) or treatment with transfection reagent alone (Ctrl 2), HeLa controls were PV-infected, harvested 9.5–10 h post-infection, and then analysed by TEM, in parallel to the specimens shown in panel B. The hyper-condensed chromatin masses contoured but did not trespass the borderlines of the NPC-associated HEZs (white arrows) that persisted in these cells. (B) Four days after transfection with Tpr siRNAs, HeLa cells were infected with PV and harvested 9.5–10 h later. Amassments of condensed chromatin were found across the entrance of most NPCs (black arrows). Beside HEZ loss in the Tpr-knockdown cells, the staining of the hyper-condensed chromatin often appeared slightly lighter than in parallel controls. Whether such decreased affinity for heavy metal stains correlates with altered chromatin condensation, and how this might be caused by Tpr deficiency, remains unknown. Bar: 500 nm; same magnification for all the images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2876962&req=5

f7: NPC-associated HEZs are no longer maintained after in vivo depletion of Tpr, resulting in NPC coating by heterochromatin. (A) After mock transfection (Ctrl 1) or treatment with transfection reagent alone (Ctrl 2), HeLa controls were PV-infected, harvested 9.5–10 h post-infection, and then analysed by TEM, in parallel to the specimens shown in panel B. The hyper-condensed chromatin masses contoured but did not trespass the borderlines of the NPC-associated HEZs (white arrows) that persisted in these cells. (B) Four days after transfection with Tpr siRNAs, HeLa cells were infected with PV and harvested 9.5–10 h later. Amassments of condensed chromatin were found across the entrance of most NPCs (black arrows). Beside HEZ loss in the Tpr-knockdown cells, the staining of the hyper-condensed chromatin often appeared slightly lighter than in parallel controls. Whether such decreased affinity for heavy metal stains correlates with altered chromatin condensation, and how this might be caused by Tpr deficiency, remains unknown. Bar: 500 nm; same magnification for all the images.

Mentions: At the cytological level, progression of chromatin condensation and its final expansion throughout the nucleus appeared similar too. However, whereas NPC-associated HEZs were omnipresent in the infected control cells, the majority of cells in the Tpr siRNA-treated populations lacked HEZs (Figure 7 and Supplementary Figure S7). In fact, in nuclei in which nuclear-peripheral chromatin had started to condense, such material was already found distributed laterally along the NE's inner face, concealing the NPCs' nuclear entrances. Also at time points when the condensed chromatin had filled larger areas of the nucleus, the NPCs of Tpr-deficient cells remained devoid of HEZs.


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)

NPC-associated HEZs are no longer maintained after in vivo depletion of Tpr, resulting in NPC coating by heterochromatin. (A) After mock transfection (Ctrl 1) or treatment with transfection reagent alone (Ctrl 2), HeLa controls were PV-infected, harvested 9.5–10 h post-infection, and then analysed by TEM, in parallel to the specimens shown in panel B. The hyper-condensed chromatin masses contoured but did not trespass the borderlines of the NPC-associated HEZs (white arrows) that persisted in these cells. (B) Four days after transfection with Tpr siRNAs, HeLa cells were infected with PV and harvested 9.5–10 h later. Amassments of condensed chromatin were found across the entrance of most NPCs (black arrows). Beside HEZ loss in the Tpr-knockdown cells, the staining of the hyper-condensed chromatin often appeared slightly lighter than in parallel controls. Whether such decreased affinity for heavy metal stains correlates with altered chromatin condensation, and how this might be caused by Tpr deficiency, remains unknown. Bar: 500 nm; same magnification for all the images.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: NPC-associated HEZs are no longer maintained after in vivo depletion of Tpr, resulting in NPC coating by heterochromatin. (A) After mock transfection (Ctrl 1) or treatment with transfection reagent alone (Ctrl 2), HeLa controls were PV-infected, harvested 9.5–10 h post-infection, and then analysed by TEM, in parallel to the specimens shown in panel B. The hyper-condensed chromatin masses contoured but did not trespass the borderlines of the NPC-associated HEZs (white arrows) that persisted in these cells. (B) Four days after transfection with Tpr siRNAs, HeLa cells were infected with PV and harvested 9.5–10 h later. Amassments of condensed chromatin were found across the entrance of most NPCs (black arrows). Beside HEZ loss in the Tpr-knockdown cells, the staining of the hyper-condensed chromatin often appeared slightly lighter than in parallel controls. Whether such decreased affinity for heavy metal stains correlates with altered chromatin condensation, and how this might be caused by Tpr deficiency, remains unknown. Bar: 500 nm; same magnification for all the images.
Mentions: At the cytological level, progression of chromatin condensation and its final expansion throughout the nucleus appeared similar too. However, whereas NPC-associated HEZs were omnipresent in the infected control cells, the majority of cells in the Tpr siRNA-treated populations lacked HEZs (Figure 7 and Supplementary Figure S7). In fact, in nuclei in which nuclear-peripheral chromatin had started to condense, such material was already found distributed laterally along the NE's inner face, concealing the NPCs' nuclear entrances. Also at time points when the condensed chromatin had filled larger areas of the nucleus, the NPCs of Tpr-deficient cells remained devoid of HEZs.

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