scholarly journals An APEX2 proximity ligation method for mapping interactions with the nuclear lamina

2020 ◽  
Vol 220 (1) ◽  
Author(s):  
Joseph R. Tran ◽  
Danielle I. Paulson ◽  
James J. Moresco ◽  
Stephen A. Adam ◽  
John R. Yates ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Membrane ◽  
Regulatory Protein ◽  
Nuclear Lamina ◽  
Inner Nuclear Membrane ◽  
Proximity Ligation ◽  
Lamin B1 ◽  
Ligation Method ◽  
Rna And Dna

The nuclear lamina (NL) is a meshwork found beneath the inner nuclear membrane. The study of the NL is hindered by the insolubility of the meshwork and has driven the development of proximity ligation methods to identify the NL-associated/proximal proteins, RNA, and DNA. To simplify and improve temporal labeling, we fused APEX2 to the NL protein lamin-B1 to map proteins, RNA, and DNA. The identified NL-interacting/proximal RNAs show a long 3′ UTR bias, a finding consistent with an observed bias toward longer 3′ UTRs in genes deregulated in lamin-null cells. A C-rich motif was identified in these 3′ UTR. Our APEX2-based proteomics identifies a C-rich motif binding regulatory protein that exhibits altered localization in lamin-null cells. Finally, we use APEX2 to map lamina-associated domains (LADs) during the cell cycle and uncover short, H3K27me3-rich variable LADs. Thus, the APEX2-based tools presented here permit identification of proteomes, transcriptomes, and genome elements associated with or proximal to the NL.

scholarly journals Characterization of A 54-kD protein of the inner nuclear membrane: evidence for cell cycle-dependent interaction with the nuclear lamina.

1991 ◽  
Vol 114 (3) ◽  
pp. 389-400 ◽  
Author(s):  
S M Bailer ◽  
H M Eppenberger ◽  
G Griffiths ◽  
E A Nigg
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Cultured Cells ◽  
Nuclear Lamina ◽  
Biochemical Properties ◽  
Inner Nuclear Membrane ◽  
Cycle Dependent ◽  
Mitotic Phosphorylation

Using a mAb (R-7), we have characterized a 54-kD protein of the chicken nuclear envelope. Based on its biochemical properties and subnuclear distribution p54 is likely to be an integral membrane component specific to the inner nuclear membrane. Fractionation experiments indicate that p54 interacts, directly or indirectly, with the nuclear lamina, and analysis of p54 in cultured cells suggests that this interaction is controlled by cell cycle-dependent posttranslational modification, most likely phosphorylation. Modification of p54 results in a slightly reduced electrophoretic mobility, and it converts the protein from a detergent-resistant to a detergent-extractable form. Detergent solubilization of p54 can be induced in vivo by treating isolated nuclei or nuclear envelopes with highly purified cdc2 kinase, one of the most prominent kinases active in mitotic cells. These results suggest that mitotic phosphorylation of p54 might contribute to control nuclear envelope dynamics during mitosis in vivo.

scholarly journals The versatility of Ascorbate Peroxidase-aided mapping uncovers insights of the nuclear lamina interactions and function

2020 ◽  
Author(s):  
Joseph R. Tran ◽  
Danielle I. Paulson ◽  
James J. Moresco ◽  
Stephen A. Adam ◽  
John R. Yates ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Nuclear Lamina ◽  
Sequence Motif ◽  
Proximity Ligation ◽  
Lamin B1 ◽  
Histone Lysine ◽  
And Function ◽  
Systematic Identification ◽  
Rna And Dna

AbstractThe nuclear lamina (NL) is a proteinaceous network found beneath the inner nuclear membrane. The NL is linked to a number of dynamic cellular activities including chromatin organization, transcription and RNA/protein trafficking through nuclear pores. Our understanding of the NL has been hindered in part by the general insolubility and low extractability of proteins from this region. This has spurred the development of proximity ligation methods that label proteins and/or DNA near the NL for systematic identification (Bar et al., 2018; Chen et al., 2018b; Guelen et al., 2008; Roux et al., 2012). To simplify labeling and improve temporal resolution, we fused APEX2 (Hung et al., 2014; Lam et al., 2015) to the nuclear lamina protein lamin-B1 to map proteins, RNA and DNA associated with the NL. We show that APEX2 labeling of the NL is robust and requires as little as 20 seconds. In addition to identifying the NL proteome, this method revealed NL-proximal RNA species that were largely spliced. These NL-proximal RNAs show a bias toward long 3’ UTRs, suggesting an RNA-regulatory role of the NL. This is further supported by the finding of a bias toward longer 3’ UTRs in genes deregulated in lamin-null cells. Interestingly, these RNAs share a sequence motif in their 3’ UTRs. Finally, we demonstrate that the APEX2 method can reliably map lamina-associated domains (LADs) at different stages of the cell cycle, revealing a variability of short LADs regions enriched for histone lysine 27 trimethylation (H3K27me3). Thus the APEX2 method report here is a useful addition to the molecular toolbox for the study of the NL and permits the identification of proteome, transcriptome, and genome elements associated with this nuclear substructure.

scholarly journals The nuclear lamina is a hub for the nuclear function of Jacob

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Sebastian Samer ◽  
Rajeev Raman ◽  
Gregor Laube ◽  
Michael R. Kreutz ◽  
Anna Karpova
Keyword(s):  
Nuclear Membrane ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Lamina ◽  
Proximity Ligation Assay ◽  
Immunogold Electron Microscopy ◽  
Inner Nuclear Membrane ◽  
Proximity Ligation ◽  
Export Signal

AbstractJacob is a synapto-nuclear messenger protein that couples NMDAR activity to CREB-dependent gene expression. In this study, we investigated the nuclear distribution of Jacob and report a prominent targeting to the nuclear envelope that requires NMDAR activity and nuclear import. Immunogold electron microscopy and proximity ligation assay combined with STED imaging revealed preferential association of Jacob with the inner nuclear membrane where it directly binds to LaminB1, an intermediate filament and core component of the inner nuclear membrane (INM). The association with the INM is transient; it involves a functional nuclear export signal in Jacob and a canonical CRM1-RanGTP-dependent export mechanism that defines the residing time of the protein at the INM. Taken together, the data suggest a stepwise redistribution of Jacob within the nucleus following nuclear import and prior to nuclear export.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

2020 ◽  
Vol 477 (14) ◽  
pp. 2715-2720
Author(s):  
Susana Castro-Obregón
Keyword(s):  
Cellular Senescence ◽  
Nuclear Membrane ◽  
Chromatin Structure ◽  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
Chimeric Protein ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

scholarly journals Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

2021 ◽  
Author(s):  
Laura A Murray-Nerger ◽  
Joshua L Justice ◽  
Pranav Rekapalli ◽  
Josiah E Hutton ◽  
Ileana M Cristea
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Posttranslational Modifications ◽  
Cell Cycle Progression ◽  
Nuclear Periphery ◽  
Virus Production ◽  
Nuclear Lamina ◽  
Regulatory Function ◽  
Herpesvirus Type ◽  
Lamin B1

Abstract The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.

scholarly journals Internuclear exchange of an inner nuclear membrane protein (p55) in heterokaryons: in vivo evidence for the interaction of p55 with the nuclear lamina.

1990 ◽  
Vol 111 (6) ◽  
pp. 2225-2234 ◽  
Author(s):  
L Powell ◽  
B Burke
Keyword(s):  
Nuclear Membrane ◽  
Lateral Diffusion ◽  
Nuclear Lamina ◽  
Membrane System ◽  
Mouse Cell ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Inner Nuclear Membrane ◽  
Pore Complexes

The movement between nuclei of an integral protein of the inner nuclear membrane has been studied in rat/mouse and rat/hamster heterokaryons. This protein, p55, was found to equilibrate between nuclei over a period of approximately 6 h in the absence of new protein synthesis. When rat/mouse heterokaryons were constructed using an undifferentiated murine embryonal carcinoma (P19), which lacks lamins A and C, no accumulation of p55 in the mouse cell nucleus was observed. However, P19 nuclei could be rendered competent to accumulate p55 by transfecting the parent cells with human lamin A before cell fusion, supporting the notion that p55 may interact with the nuclear lamina. Since p55 does not appear to be able to dissociate from the nuclear membrane, it is concluded that this exchange between nuclei does not occur in the aqueous phase and instead is probably membrane mediated. It is proposed that this protein may be free to move between the inner and outer nuclear membranes via the continuities at the nuclear pore complexes and that transfer between nuclei occurs via lateral diffusion through the peripheral ER, which appears to form a single continuous membrane system in these heterokaryons. One implication of these observations is that accumulation of at least some integral proteins in the inner nuclear membrane may be mediated by interactions with other nuclear components and may not require a single defined targeting sequence.

scholarly journals Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.

1988 ◽  
Vol 107 (6) ◽  
pp. 2029-2036 ◽  
Author(s):  
A Senior ◽  
L Gerace
Keyword(s):  
Membrane Proteins ◽  
Nuclear Membrane ◽  
Immunofluorescence Microscopy ◽  
Nuclear Lamina ◽  
Integral Membrane Proteins ◽  
Inner Nuclear Membrane ◽  
Attachment Sites ◽  
Nonionic Detergent ◽  
Pore Complexes ◽  
Integral Proteins

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.

Nesprin isoforms: are they inside or outside the nucleus?

2010 ◽  
Vol 38 (1) ◽  
pp. 278-280 ◽  
Author(s):  
Glenn E. Morris ◽  
K. Natalie Randles
Keyword(s):  
Membrane Proteins ◽  
Experimental Evidence ◽  
Nuclear Membrane ◽  
Nuclear Import ◽  
Nuclear Lamina ◽  
Cellular Organization ◽  
Inner Nuclear Membrane

The giant isoforms of nesprins 1 and 2 are emerging as important players in cellular organization, particularly in the positioning of nuclei, and possibly other organelles, within the cytoplasm. The experimental evidence suggests that nesprins also occur at the inner nuclear membrane, where they interact with the nuclear lamina. In this paper, we consider whether this is consistent with current ideas about nesprin anchorage and about mechanisms for nuclear import of membrane proteins.

scholarly journals Visualization of the Nuclear Lamina in Mouse Anterior Pituitary Cells and Immunocytochemical Detection of Lamin A/C by Quick-freeze Freeze-substitution Electron Microscopy

2005 ◽  
Vol 53 (4) ◽  
pp. 497-507 ◽  
Author(s):  
Takao Senda ◽  
Akiko Iizuka-Kogo ◽  
Atsushi Shimomura
Keyword(s):  
Electron Microscopy ◽  
Nuclear Membrane ◽  
Anterior Pituitary ◽  
Freeze Substitution ◽  
Nuclear Lamina ◽  
Immunogold Electron Microscopy ◽  
Lamin A ◽  
Pituitary Cells ◽  
Inner Nuclear Membrane ◽  
Anterior Pituitary Cells

We examined the nuclear lamina in the quickly frozen anterior pituitary cells by electron microscopic techniques combined with freeze substitution, deep etching, and immunocytochemistry and compared it with that in the chemically fixed cells. By quick-freeze freeze-substitution electron microscopy, an electron-lucent layer, as thick as 20 nm, was revealed just inside the inner nuclear membrane, whereas in the conventionally glutaraldehyde-fixed cells the layer was not seen. By quick-freeze deep-etch electron microscopy, we could not distinguish definitively the layer corresponding to the nuclear lamina in either fresh unfixed or glutaraldehyde-fixed cells. Immunofluorescence microscopy showed that lamin A/C in the nucleus was detected in the acetone-fixed cells and briefly in paraformaldehyde-fixed cells but not in the cells with prolonged paraformaldehyde fixation. Nuclear localization of lamin A/C was revealed by immunogold electron microscopy also in the quickly frozen and freeze-substituted cells, but not in the paraformaldehyde-fixed cells. Lamin A/C was localized mainly in the peripheral nucleoplasm within 60 nm from the inner nuclear membrane, which corresponded to the nuclear lamina. These results suggest that the nuclear lamina can be preserved both ultrastructurally and immunocytochemically by quick-freezing fixation, rather than by conventional chemical fixation.

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