scholarly journals Lamin A/C–dependent Localization of Nesprin-2, a Giant Scaffolder at the Nuclear Envelope

2005 ◽  
Vol 16 (7) ◽  
pp. 3411-3424 ◽  
Author(s):  
Thorsten Libotte ◽  
Hafida Zaim ◽  
Sabu Abraham ◽  
V. C. Padmakumar ◽  
Maria Schneider ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Normal Skin ◽  
Basal Layer ◽  
Dominant Negative ◽  
Actin Binding ◽  
Lamin A ◽  
Common Region ◽  
Lamin B

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of α-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

scholarly journals Mechanisms of allelic and clinical heterogeneity of lamin A/C phenotypes

2018 ◽  
Vol 50 (9) ◽  
pp. 694-704 ◽  
Author(s):  
Jelena Perovanovic ◽  
Eric P. Hoffman
Keyword(s):  
Muscular Dystrophy ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Structural Model ◽  
Terminal Differentiation ◽  
Dominant Negative ◽  
Single Amino Acid ◽  
Lamin A ◽  
Gain Of Function ◽  
Heterochromatin Formation

Mutations in the lamin A/C ( LMNA) gene cause a broad range of clinical syndromes that show tissue-restricted abnormalities of post mitotic tissues, such as muscle, nerve, heart, and adipose tissue. Mutations in other nuclear envelope proteins cause clinically overlapping disorders. The majority of mutations are dominant single amino acid changes (toxic protein produced by the single mutant gene), and patients are heterozygous with both normal and abnormal proteins. Experimental support has been provided for different models of cellular pathogenesis in nuclear envelope diseases, including changes in heterochromatin formation at the nuclear membrane (epigenomics), changes in the timing of steps during terminal differentiation of cells, and structural abnormalities of the nuclear membrane. These models are not mutually exclusive and may be important in different cells at different times of development. Recent experiments using fusion proteins of normal and mutant lamin A/C proteins fused to a bacterial adenine methyltransferase (DamID) provided compelling evidence of mutation-specific perturbation of epigenomic imprinting during terminal differentiation. These gain-of-function properties include lineage-specific ineffective genomic silencing during exit from the cell cycle (heterochromatinization), as well as promiscuous initiation of silencing at incorrect places in the genome. To date, these findings have been limited to a few muscular dystrophy and lipodystrophy LMNA mutations but seem shared with a distinct nuclear envelope disease, emerin-deficient muscular dystrophy. The dominant-negative structural model and gain-of-function epigenomic models for distinct LMNA mutations are not mutually exclusive, and it is likely that both models contribute to aspects of the many complex clinical phenotypes observed.

Alterations of nuclear envelope and chromatin organization in mandibuloacral dysplasia, a rare form of laminopathy

2005 ◽  
Vol 23 (2) ◽  
pp. 150-158 ◽  
Author(s):  
Ilaria Filesi ◽  
Francesca Gullotta ◽  
Giovanna Lattanzi ◽  
Maria Rosaria D'Apice ◽  
Cristina Capanni ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Protein A ◽  
Nuclear Architecture ◽  
Mendelian Inheritance ◽  
Premature Aging ◽  
Lamin A ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Epigenetic Events ◽  
Mandibuloacral Dysplasia

Autosomal recessive mandibuloacral dysplasia [mandibuloacral dysplasia type A (MADA); Online Mendelian Inheritance in Man (OMIM) no. 248370 ] is caused by a mutation in LMNA encoding lamin A/C. Here we show that this mutation causes accumulation of the lamin A precursor protein, a marked alteration of the nuclear architecture and, hence, chromatin disorganization. Heterochromatin domains are altered or completely lost in MADA nuclei, consistent with the finding that heterochromatin-associated protein HP1β and histone H3 methylated at lysine 9 and their nuclear envelope partner protein lamin B receptor (LBR) are delocalized and solubilized. Both accumulation of lamin A precursor and chromatin defects become more severe in older patients. These results strongly suggest that altered chromatin remodeling is a key event in the cascade of epigenetic events causing MADA and could be related to the premature-aging phenotype.

scholarly journals The Vaccinia-related Kinases Phosphorylate the N′ Terminus of BAF, Regulating Its Interaction with DNA and Its Retention in the Nucleus

2006 ◽  
Vol 17 (5) ◽  
pp. 2451-2464 ◽  
Author(s):  
R. Jeremy Nichols ◽  
Matthew S. Wiebe ◽  
Paula Traktman
Keyword(s):  
Drosophila Melanogaster ◽  
Cell Division ◽  
Nuclear Envelope ◽  
Temperature Sensitivity ◽  
Nuclear Membrane ◽  
Essential Role ◽  
Nuclear Architecture ◽  
Nuclear Chromatin ◽  
N Terminus ◽  
Protein Barrier

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N′ terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture.

scholarly journals Induction of a Massive Endoplasmic Reticulum and Perinuclear Space Expansion by Expression of Lamin B Receptor Mutants and the Related Sterol Reductases TM7SF2 and DHCR7

2010 ◽  
Vol 21 (2) ◽  
pp. 354-368 ◽  
Author(s):  
Monika Zwerger ◽  
Thorsten Kolb ◽  
Karsten Richter ◽  
Iakowos Karakesisoglou ◽  
Harald Herrmann
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Cell Lines ◽  
Nuclear Membrane ◽  
Cultured Cells ◽  
Membrane Separation ◽  
Reductase Activity ◽  
Nuclear Pore ◽  
Lamin B ◽  
Lamin B Receptor

Lamin B receptor (LBR) is an inner nuclear membrane protein involved in tethering the nuclear lamina and the underlying chromatin to the nuclear envelope. In addition, LBR exhibits sterol reductase activity. Mutations in the LBR gene cause two different human diseases: Pelger-Huët anomaly and Greenberg skeletal dysplasia, a severe chrondrodystrophy causing embryonic death. Our study aimed at investigating the effect of five LBR disease mutants on human cultured cells. Three of the tested LBR mutants caused a massive compaction of chromatin coincidental with the formation of a large nucleus-associated vacuole (NAV) in several human cultured cell lines. Live cell imaging and electron microscopy revealed that this structure was generated by the separation of the inner and outer nuclear membrane. During NAV formation, nuclear pore complexes and components of the linker of nucleoskeleton and cytoskeleton complex were lost in areas of membrane separation. Concomitantly, a large number of smaller vacuoles formed throughout the cytoplasm. Notably, forced expression of the two structurally related sterol reductases transmembrane 7 superfamily member 2 and 7-dehydrocholesterol reductase caused, even in their wild-type form, a comparable phenotype in susceptible cell lines. Hence, LBR mutant variants and sterol reductases can severely interfere with the regular organization of the nuclear envelope and the endoplasmic reticulum.

scholarly journals The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum.

1988 ◽  
Vol 107 (2) ◽  
pp. 397-406 ◽  
Author(s):  
R Stick ◽  
B Angres ◽  
C F Lehner ◽  
E A Nigg
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Membrane Vesicles ◽  
Soluble Form ◽  
Lamin A ◽  
Cell Fractionation ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Nuclear Lamin ◽  
Mitotic Cells

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.

scholarly journals SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

2014 ◽  
Vol 204 (7) ◽  
pp. 1099-1109 ◽  
Author(s):  
Yagmur Turgay ◽  
Lysie Champion ◽  
Csaba Balazs ◽  
Michael Held ◽  
Alberto Toso ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Dominant Negative ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Microtubule Depolymerization ◽  
Nuclear Envelope Breakdown ◽  
And Migration ◽  
Delayed Removal

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN–KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.

scholarly journals Lamin A, lamin B, and lamin B receptor analogues in yeast.

1989 ◽  
Vol 108 (6) ◽  
pp. 2069-2082 ◽  
Author(s):  
S D Georgatos ◽  
I Maroulakou ◽  
G Blobel
Keyword(s):  
Nuclear Envelope ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Structural Similarity ◽  
Integral Membrane Protein ◽  
Lamin A ◽  
Nuclear Fraction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Lamin B ◽  
Lamin B Receptor

Previous studies have shown that turkey erythrocyte lamin B is anchored to the nuclear envelope via a 58-kD integral membrane protein termed p58 or lamin B receptor (Worman H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). We now identify a p58 analogue in the yeast Saccharomyces cerevisiae. Turkey erythrocyte lamin B binds to yeast urea-extracted nuclear envelopes with high affinity, associating predominantly with a 58-kD polypeptide. This yeast polypeptide is recognized by polyclonal antibodies against turkey p58, partitions entirely with the nuclear fraction, remains membrane bound after urea extraction of the nuclear envelopes, and is structurally similar to turkey p58 by peptide mapping criteria. Using polyclonal antibodies against turkey erythrocyte lamins A and B, we also identify two yeast lamin forms. The yeast lamin B analogue has a molecular mass of 66 kD and is structurally related to erythrocyte lamin B. Moreover, the yeast lamin B analogue partitions exclusively with the nuclear envelope fraction, is quantitatively removed from the envelopes by urea extraction, and binds to turkey lamin A and vimentin. As many higher eukaryotic lamin B forms, the yeast analogue is chemically heterogeneous comprising two serologically related species with different charge characteristics. Antibodies against turkey lamin A detect a 74-kD yeast protein, slightly larger than the turkey lamin A. It is more abundant than the yeast lamin B analogue and partitions between a soluble cytoplasmic fraction and a nuclear envelope fraction. The yeast lamin A analogue can be extracted from the nuclear envelope by urea, shows structural similarity to turkey and rat lamin A, and binds to isolated turkey lamin B. These data indicate that analogues of typical nuclear lamina components (lamins A and B, as well as lamin B receptor) are present in yeast and behave as their vertebrate counterparts.

scholarly journals Human Adenovirus Type 5 Infection Leads to Nuclear Envelope Destabilization and Membrane Permeability Independently of Adenovirus Death Protein

2021 ◽  
Vol 22 (23) ◽  
pp. 13034
Author(s):  
Søren Pfitzner ◽  
Jens B. Bosse ◽  
Helga Hofmann-Sieber ◽  
Felix Flomm ◽  
Rudolph Reimer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Membrane Permeability ◽  
Nuclear Membrane ◽  
Membrane Damage ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Lamin A ◽  
Infected Cells ◽  
Adenovirus Type 5

The human adenovirus type 5 (HAdV5) infects epithelial cells of the upper and lower respiratory tract. The virus causes lysis of infected cells and thus enables spread of progeny virions to neighboring cells for the next round of infection. The mechanism of adenovirus virion egress across the nuclear barrier is not known. The human adenovirus death protein (ADP) facilitates the release of virions from infected cells and has been hypothesized to cause membrane damage. Here, we set out to answer whether ADP does indeed increase nuclear membrane damage. We analyzed the nuclear envelope morphology using a combination of fluorescence and state-of-the-art electron microscopy techniques, including serial block-face scanning electron microscopy and electron cryo-tomography of focused ion beam-milled cells. We report multiple destabilization phenotypes of the nuclear envelope in HAdV5 infection. These include reduction of lamin A/C at the nuclear envelope, large-scale membrane invaginations, alterations in double membrane separation distance and small-scale membrane protrusions. Additionally, we measured increased nuclear membrane permeability and detected nuclear envelope lesions under cryoconditions. Unexpectedly, and in contrast to previous hypotheses, ADP did not have an effect on lamin A/C reduction or nuclear permeability.

Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly

1999 ◽  
Vol 112 (6) ◽  
pp. 977-987 ◽  
Author(s):  
P. Collas
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Cdc2 Kinase ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Nuclear Envelope Breakdown ◽  
Simultaneous Inhibition ◽  
Integral Proteins

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis.

scholarly journals Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

2011 ◽  
Vol 22 (18) ◽  
pp. 3306-3317 ◽  
Author(s):  
Li-Chuan Tseng ◽  
Rey-Huei Chen
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Chromatin Binding ◽  
Temporal Control ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Nuclear Envelope Assembly ◽  
Nuclear Membrane Protein

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

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