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The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.

Korfali N, Wilkie GS, Swanson SK, Srsen V, Batrakou DG, Fairley EA, Malik P, Zuleger N, Goncharevich A, de Las Heras J, Kelly DA, Kerr AR, Florens L, Schirmer EC - Mol. Cell Proteomics (2010)

Bottom Line: Several known proteins identified in both data sets have functions in chromatin organization and gene regulation.To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects.The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom.

ABSTRACT
A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

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mRFP fusions confirm NE targeting for several novel PBMC NETs. A, HT1080 cells expressing NETs fused to mRFP were directly fixed (left) or extracted with Triton X-100 prior to fixation (right). The NE is marked by lamin A in green so that yellow indicates co-localization of the NET at the NE. The directly fixed emerin image has part of an untransfected cell, confirming that none of the NET staining at the nuclear rim is due to bleed-through from the lamin A channel. Note that prefixation extraction affects morphology and sometimes leaves aggregated protein in the cytoplasm. The emerin control and new NET C20orf3 are retained after extraction, whereas the ER protein calreticulin is not. Scale bar, 10 μm. B, other NET-mRFP fusions were similarly pre-extracted and retained at the NE. Tmem41A is shown in COS-7 cells because it was not expressed in HT1080 cells, and Tmem126A is shown in Jurkat cells because it failed to be expressed in either HT1080 or COS-7 cells. Scale bar, 10 μm. C, inner versus outer nuclear membrane targeting. If a NET (red) is in the INM it should appear in the same plane as the nuclear basket protein Nup153 (green, left) and internal to the cytoplasmic filament protein Nup358 (green, right) using structured illumination microscopy. Characterized NET LAP2β and most new NETs tested appeared in the INM. One INM NET, METTL7A, did not resist Triton pre-extraction. Scale bars, 5 μm.
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Figure 4: mRFP fusions confirm NE targeting for several novel PBMC NETs. A, HT1080 cells expressing NETs fused to mRFP were directly fixed (left) or extracted with Triton X-100 prior to fixation (right). The NE is marked by lamin A in green so that yellow indicates co-localization of the NET at the NE. The directly fixed emerin image has part of an untransfected cell, confirming that none of the NET staining at the nuclear rim is due to bleed-through from the lamin A channel. Note that prefixation extraction affects morphology and sometimes leaves aggregated protein in the cytoplasm. The emerin control and new NET C20orf3 are retained after extraction, whereas the ER protein calreticulin is not. Scale bar, 10 μm. B, other NET-mRFP fusions were similarly pre-extracted and retained at the NE. Tmem41A is shown in COS-7 cells because it was not expressed in HT1080 cells, and Tmem126A is shown in Jurkat cells because it failed to be expressed in either HT1080 or COS-7 cells. Scale bar, 10 μm. C, inner versus outer nuclear membrane targeting. If a NET (red) is in the INM it should appear in the same plane as the nuclear basket protein Nup153 (green, left) and internal to the cytoplasmic filament protein Nup358 (green, right) using structured illumination microscopy. Characterized NET LAP2β and most new NETs tested appeared in the INM. One INM NET, METTL7A, did not resist Triton pre-extraction. Scale bars, 5 μm.

Mentions: After 30 h, cells were either directly fixed for 7 min in 3.7% formaldehyde or washed with PBS; then extracted for 1 min with 1% Triton X-100, 25 mm Tris, pH 8.0, 150 mm KOAc, 15 mm NaCl, 5 mm MgCl2; washed again with PBS; and then fixed with formaldehyde. For antibody staining, cells that were not pre-extracted were permeabilized for 6 min in 0.2% Triton X-100 after fixation. Cells were then blocked with 10% FBS, 200 mm glycine in PBS and incubated for 40 min at RT with relevant antibodies. DNA was visualized with Hoechst 33342 or 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and coverslips mounted in Fluoromount-G (EM Sciences). For structured illumination (OMX) microscopy, Alexa Fluor secondary antibodies (Molecular Probes) were used. Most images were obtained using a Nikon TE-2000 microscope equipped with a 1.45 numerical aperture 100× objective, Sedat quad filter set, and CoolSnapHQ High Speed Monochrome charge-coupled device camera (Photometrics). Structured illumination images (Fig. 4C) were taken on the OMX system at the University of Dundee microscopy facility (details described at http://microscopy.lifesci.dundee.ac.uk/omx/).


The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.

Korfali N, Wilkie GS, Swanson SK, Srsen V, Batrakou DG, Fairley EA, Malik P, Zuleger N, Goncharevich A, de Las Heras J, Kelly DA, Kerr AR, Florens L, Schirmer EC - Mol. Cell Proteomics (2010)

mRFP fusions confirm NE targeting for several novel PBMC NETs. A, HT1080 cells expressing NETs fused to mRFP were directly fixed (left) or extracted with Triton X-100 prior to fixation (right). The NE is marked by lamin A in green so that yellow indicates co-localization of the NET at the NE. The directly fixed emerin image has part of an untransfected cell, confirming that none of the NET staining at the nuclear rim is due to bleed-through from the lamin A channel. Note that prefixation extraction affects morphology and sometimes leaves aggregated protein in the cytoplasm. The emerin control and new NET C20orf3 are retained after extraction, whereas the ER protein calreticulin is not. Scale bar, 10 μm. B, other NET-mRFP fusions were similarly pre-extracted and retained at the NE. Tmem41A is shown in COS-7 cells because it was not expressed in HT1080 cells, and Tmem126A is shown in Jurkat cells because it failed to be expressed in either HT1080 or COS-7 cells. Scale bar, 10 μm. C, inner versus outer nuclear membrane targeting. If a NET (red) is in the INM it should appear in the same plane as the nuclear basket protein Nup153 (green, left) and internal to the cytoplasmic filament protein Nup358 (green, right) using structured illumination microscopy. Characterized NET LAP2β and most new NETs tested appeared in the INM. One INM NET, METTL7A, did not resist Triton pre-extraction. Scale bars, 5 μm.
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Related In: Results  -  Collection

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Figure 4: mRFP fusions confirm NE targeting for several novel PBMC NETs. A, HT1080 cells expressing NETs fused to mRFP were directly fixed (left) or extracted with Triton X-100 prior to fixation (right). The NE is marked by lamin A in green so that yellow indicates co-localization of the NET at the NE. The directly fixed emerin image has part of an untransfected cell, confirming that none of the NET staining at the nuclear rim is due to bleed-through from the lamin A channel. Note that prefixation extraction affects morphology and sometimes leaves aggregated protein in the cytoplasm. The emerin control and new NET C20orf3 are retained after extraction, whereas the ER protein calreticulin is not. Scale bar, 10 μm. B, other NET-mRFP fusions were similarly pre-extracted and retained at the NE. Tmem41A is shown in COS-7 cells because it was not expressed in HT1080 cells, and Tmem126A is shown in Jurkat cells because it failed to be expressed in either HT1080 or COS-7 cells. Scale bar, 10 μm. C, inner versus outer nuclear membrane targeting. If a NET (red) is in the INM it should appear in the same plane as the nuclear basket protein Nup153 (green, left) and internal to the cytoplasmic filament protein Nup358 (green, right) using structured illumination microscopy. Characterized NET LAP2β and most new NETs tested appeared in the INM. One INM NET, METTL7A, did not resist Triton pre-extraction. Scale bars, 5 μm.
Mentions: After 30 h, cells were either directly fixed for 7 min in 3.7% formaldehyde or washed with PBS; then extracted for 1 min with 1% Triton X-100, 25 mm Tris, pH 8.0, 150 mm KOAc, 15 mm NaCl, 5 mm MgCl2; washed again with PBS; and then fixed with formaldehyde. For antibody staining, cells that were not pre-extracted were permeabilized for 6 min in 0.2% Triton X-100 after fixation. Cells were then blocked with 10% FBS, 200 mm glycine in PBS and incubated for 40 min at RT with relevant antibodies. DNA was visualized with Hoechst 33342 or 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and coverslips mounted in Fluoromount-G (EM Sciences). For structured illumination (OMX) microscopy, Alexa Fluor secondary antibodies (Molecular Probes) were used. Most images were obtained using a Nikon TE-2000 microscope equipped with a 1.45 numerical aperture 100× objective, Sedat quad filter set, and CoolSnapHQ High Speed Monochrome charge-coupled device camera (Photometrics). Structured illumination images (Fig. 4C) were taken on the OMX system at the University of Dundee microscopy facility (details described at http://microscopy.lifesci.dundee.ac.uk/omx/).

Bottom Line: Several known proteins identified in both data sets have functions in chromatin organization and gene regulation.To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects.The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom.

ABSTRACT
A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

Show MeSH
Related in: MedlinePlus