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1.
Epigenetics Chromatin ; 15(1): 1, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34983624

RESUMO

The cause of nuclear shape abnormalities which are often seen in pre-neoplastic and malignant tissues is not clear. In this study we report that deformation of the nucleus can be induced by TGFß1 stimulation in several cell lines including Huh7. In our results, the upregulated histone H3.3 expression downstream of SMAD signaling contributed to TGFß1-induced nuclear deformation, a process of which requires incorporation of the nuclear envelope (NE) proteins lamin B1 and SUN1. During this process, the NE constitutively ruptured and reformed. Contrast to lamin B1 which was relatively stationary around the nucleus, the upregulated lamin A was highly mobile, clustering at the nuclear periphery and reintegrating into the nucleoplasm. The chromatin regions that lost NE coverage formed a supra-nucleosomal structure characterized by elevated histone H3K27me3 and histone H1, the formation of which depended on the presence of lamin A. These results provide evidence that shape of the nucleus can be modulated through TGFß1-induced compositional changes in the chromatin and nuclear lamina.


Assuntos
Histonas , Membrana Nuclear , Linhagem Celular Tumoral , Núcleo Celular , Humanos , Lâmina Nuclear , Isoformas de Proteínas
2.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34874453

RESUMO

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose , Fatores de Transcrição/metabolismo
3.
Biochim Biophys Acta Mol Cell Res ; 1869(1): 119161, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34655689

RESUMO

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Nuclear/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Animais , Células Cultivadas , Embrião de Galinha , Proteínas Intrinsicamente Desordenadas/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membrana Nuclear/ultraestrutura , Proteoma/genética , Proteoma/metabolismo , Peixe-Zebra
4.
Biochim Biophys Acta Biomembr ; 1864(1): 183780, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34547252

RESUMO

The general segregation of a molecular class, lipids, from the pathways of cellular communication, via endo-membranes, has resulted in the over-simplification and misconceptions in deciphering cell signalling mechanisms. Mechanisms in signal transduction and protein activation require targeting of proteins to membranous compartments with a specific localised morphology and dynamics that are dependent on their lipid composition. Many posttranslational events define cellular behaviours and without the active role of membranous compartments these events lead to various dysregulations of the signalling pathways. We summarise the key findings, using tools such as the rapalogue dimerisation, in the structural roles and signalling of the inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum.


Assuntos
Comunicação Celular/genética , Lipídeos de Membrana/genética , Fosfatidilinositóis/genética , Transdução de Sinais/genética , Membrana Nuclear/genética
5.
Nat Commun ; 12(1): 7216, 2021 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-34903738

RESUMO

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.


Assuntos
Neoplasias da Mama/patologia , Heterocromatina/metabolismo , Mecanotransdução Celular/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Citoesqueleto/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Adesões Focais/genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Metiltransferases/metabolismo , Camundongos , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Microambiente Tumoral
6.
Biophys J ; 120(21): 4698-4709, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34624272

RESUMO

Nuclear morphology is an important indicator of cell function. It is regulated by a variety of factors such as the osmotic pressure difference between the nucleoplasm and cytoplasm, cytoskeletal forces, elasticity of the nuclear envelope and chromosomes. Nucleus shape and size are typically quantified using multiple geometrical quantities that are not necessarily independent of one another. This interdependence makes it difficult to decipher the implications of changes in nuclear morphology. We resolved this by analyzing nucleus shapes of populations for multiple cell lines using a mechanics-based model. We deduced two independent nondimensional parameters, namely, flatness index and isometric scale factor. We show that nuclei in a cell population have similar flatness but variable scale factor. Furthermore, nuclei of different cell lines segregate according to flatness. Cellular perturbations using biochemical and biomechanical techniques suggest that the flatness index correlates with actin tension and the scale factor anticorrelates with elastic modulus of nuclear envelope. We argue that nuclear morphology measures such as volume, projected area, height etc., are subsumed by flatness and scale factor, which can unambiguously characterize nuclear morphology.


Assuntos
Núcleo Celular , Citoesqueleto , Actinas , Forma do Núcleo Celular , Citoplasma , Membrana Nuclear
7.
Elife ; 102021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34605406

RESUMO

Non-centrosomal microtubule-organizing centers (MTOCs) are pivotal for the function of multiple cell types, but the processes initiating their formation are unknown. Here, we find that the transcription factor myogenin is required in murine myoblasts for the localization of MTOC proteins to the nuclear envelope. Moreover, myogenin is sufficient in fibroblasts for nuclear envelope MTOC (NE-MTOC) formation and centrosome attenuation. Bioinformatics combined with loss- and gain-of-function experiments identified induction of AKAP6 expression as one central mechanism for myogenin-mediated NE-MTOC formation. Promoter studies indicate that myogenin preferentially induces the transcription of muscle- and NE-MTOC-specific isoforms of Akap6 and Syne1, which encodes nesprin-1α, the NE-MTOC anchor protein in muscle cells. Overexpression of AKAP6ß and nesprin-1α was sufficient to recruit endogenous MTOC proteins to the nuclear envelope of myoblasts in the absence of myogenin. Taken together, our results illuminate how mammals transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify AKAP6 as a novel NE-MTOC component in muscle cells.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Centro Organizador dos Microtúbulos/fisiologia , Células Musculares/metabolismo , Miogenina/metabolismo , Células 3T3 , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Células Musculares/citologia , Membrana Nuclear
8.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638534

RESUMO

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.


Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Laminopatias/patologia , Membrana Nuclear/metabolismo , Células 3T3 , Animais , Linhagem Celular , Fibroblastos/metabolismo , Imunofluorescência , Proteínas de Filamentos Intermediários/genética , Lamina Tipo A/genética , Lamina Tipo B/genética , Laminopatias/genética , Camundongos , Microscopia Confocal
9.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638576

RESUMO

Nuclear envelope (NE) and endoplasmic reticulum (ER) collaborate to control a multitude of nuclear and cytoplasmic actions. In this context, the transmembrane protein TMEM147 localizes to both NE and ER, and through direct and indirect interactions regulates processes as varied as production and transport of multipass membrane proteins, neuronal signaling, nuclear-shape, lamina and chromatin dynamics and cholesterol synthesis. Aiming to delineate the emerging multifunctionality of TMEM147 more comprehensively, we set as objectives, first, to assess potentially more fundamental effects of TMEM147 on the ER and, second, to identify significantly TMEM147-associated cell-wide protein networks and pathways. Quantifying curved and flat ER markers RTN4 and CLIMP63/CKAP4, respectively, we found that TMEM147 silencing causes area and intensity increases for both RTN4 and CLIMP63, and the ER in general, with a profound shift toward flat areas, concurrent with reduction in DNA condensation. Protein network and pathway analyses based on comprehensive compilation of TMEM147 interactors, targets and co-factors then served to manifest novel and established roles for TMEM147. Thus, algorithmically simplified significant pathways reflect TMEM147 function in ribosome binding, oxidoreductase activity, G protein-coupled receptor activity and transmembrane transport, while analysis of protein factors and networks identifies hub proteins and corresponding pathways as potential targets of TMEM147 action and of future functional studies.


Assuntos
Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Retículo Endoplasmático/ultraestrutura , Inativação Gênica , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas Nogo/metabolismo , Mapas de Interação de Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais
11.
Cells ; 10(9)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34571990

RESUMO

The endoplasmic reticulum (ER) is an organelle that is responsible for many essential subcellular processes. Interconnected narrow tubules at the periphery and thicker sheet-like regions in the perinuclear region are linked to the nuclear envelope. It is becoming apparent that the complex morphology and dynamics of the ER are linked to its function. Mutations in the proteins involved in regulating ER structure and movement are implicated in many diseases including neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis (ALS). The ER is also hijacked by pathogens to promote their replication. Bacteria such as Legionella pneumophila and Chlamydia trachomatis, as well as the Zika virus, bind to ER morphology and dynamics-regulating proteins to exploit the functions of the ER to their advantage. This review covers our understanding of ER morphology, including the functional subdomains and membrane contact sites that the organelle forms. We also focus on ER dynamics and the current efforts to quantify ER motion and discuss the diseases related to ER morphology and dynamics.


Assuntos
Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Retículo Endoplasmático/fisiologia , Animais , Citoesqueleto , Humanos , Lipídeos/biossíntese , Proteínas de Membrana/metabolismo , Microtúbulos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Membrana Nuclear/metabolismo , Relação Estrutura-Atividade
12.
Cells ; 10(9)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34572142

RESUMO

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.


Assuntos
Poro Nuclear/metabolismo , Pressão Osmótica/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina , Citoplasma/metabolismo , Humanos , Membrana Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Agregados Proteicos/fisiologia , Proteínas/metabolismo , Estresse Fisiológico/fisiologia
13.
Elife ; 102021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34586062

RESUMO

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.


Assuntos
Meiose , Proteínas de Membrana/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , /metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Stem Cell Res ; 56: 102539, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34560421

RESUMO

LAP1 is an inner nuclear membrane protein encoded by TOR1AIP1. A homozygous c.961C > T loss of function mutation in TOR1AIP1 that affects both isoforms of LAP1 was recently described. This mutation leads to the development of a severe multisystemic nuclear envelopathy syndrome. Here we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines derived from skin fibroblasts of two patients carrying the homozygous c.961C > T mutation. These novel lines can be used as a powerful tool to investigate the molecular mechanism by which LAP1 deficiency leads to the development of this severe hereditary disorder.


Assuntos
Células-Tronco Pluripotentes Induzidas , Códon sem Sentido/genética , Humanos , Mutação/genética , Membrana Nuclear , Isoformas de Proteínas/genética
15.
Dev Cell ; 56(18): 2562-2578.e3, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34407429

RESUMO

The cell nucleus is surrounded by a double membrane. The lipid packing and viscosity of membranes is critical for their function and is tightly controlled by lipid saturation. Circuits regulating the lipid saturation of the outer nuclear membrane (ONM) and contiguous endoplasmic reticulum (ER) are known. However, how lipid saturation is controlled in the inner nuclear membrane (INM) has remained enigmatic. Using INM biosensors and targeted genetic manipulations, we show that increased lipid unsaturation causes a reprogramming of lipid storage metabolism across the nuclear envelope (NE). Cells induce lipid droplet (LD) formation specifically from the distant ONM/ER, whereas LD formation at the INM is suppressed. In doing so, unsaturated fatty acids are shifted away from the INM. We identify the transcription circuits that topologically reprogram LD synthesis and identify seipin and phosphatidic acid as critical effectors. Our study suggests a detoxification mechanism protecting the INM from excess lipid unsaturation.


Assuntos
Gorduras Insaturadas/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Ácidos Fosfatídicos/metabolismo , Leveduras
16.
Curr Opin Cell Biol ; 72: 137-145, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34461580

RESUMO

During cell growth and motility in crowded tissues or interstitial spaces, cells must integrate multiple physical and biochemical environmental inputs. After a number of recent studies, the view of the nucleus as a passive object that cells have to drag along has become obsolete, placing the nucleus as a central player in sensing some of these inputs. In the present review, we will focus on changes in nuclear shape caused by external and internal forces. Depending on their magnitude, nuclear deformations can generate signaling events that modulate cell behavior and fate, or be a source of perturbations or even damage, having detrimental effects on cellular functions. On very large deformations, nuclear envelope rupture events become frequent, leading to uncontrolled nucleocytoplasmic mixing and DNA damage. We will also discuss the consequences of repeated compromised nuclear integrity, which can trigger DNA surveillance mechanisms, with critical consequences to cell fate and tissue homeostasis.


Assuntos
Núcleo Celular , Membrana Nuclear , Dano ao DNA , Transdução de Sinais
17.
PLoS Pathog ; 17(8): e1009679, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424922

RESUMO

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.


Assuntos
Herpes Simples/patologia , Herpesvirus Humano 2/fisiologia , Membrana Nuclear/patologia , Proteínas Virais/metabolismo , Liberação de Vírus , Animais , Capsídeo/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Células HeLa , Herpes Simples/metabolismo , Herpes Simples/virologia , Humanos , Membrana Nuclear/metabolismo , Membrana Nuclear/virologia , Fosforilação , Células Vero , Proteínas Virais/genética , Montagem de Vírus
18.
J Vis Exp ; (173)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34309603

RESUMO

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.


Assuntos
Núcleo Celular , Poro Nuclear , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Digitonina/metabolismo , Células HeLa , Humanos , Camundongos , Neurônios , Membrana Nuclear , Poro Nuclear/metabolismo
19.
Elife ; 102021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34286694

RESUMO

Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic (M) exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during M-exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon M-entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7's interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.


Assuntos
Proteína Quinase CDC2/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membrana Nuclear/metabolismo , Proteína Quinase CDC2/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos , Proteínas de Membrana , Microtúbulos/metabolismo , Mitose , Proteínas Nucleares , Telófase
20.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206382

RESUMO

Emerin is the inner nuclear membrane protein involved in maintaining the mechanical integrity of the nuclear membrane. Mutations in EMD encoding emerin cause Emery-Dreifuss muscular dystrophy (EDMD). Evidence is accumulating that emerin regulation of specific gene expression is associated with this disease, but the exact function of emerin has not been fully elucidated. Here, we show that emerin downregulates Signal transducer and activators of transcription 3 (STAT3) signaling, activated exclusively by Janus kinase (JAK). Deletion mutation experiments show that the lamin-binding domain of emerin is essential for the inhibition of STAT3 signaling. Emerin interacts directly and co-localizes with STAT3 in the nuclear membrane. Emerin knockdown induces STAT3 target genes Bcl2 and Survivin to increase cell survival signals and suppress hydrogen peroxide-induced cell death in HeLa cells. Specifically, downregulation of BAF or lamin A/C increases STAT3 signaling, suggesting that correct-localized emerin, by assembling with BAF and lamin A/C, acts as an intrinsic inhibitor against STAT3 signaling. In C2C12 cells, emerin knockdown induces STAT3 target gene, Pax7, and activated abnormal myoblast proliferation associated with muscle wasting in skeletal muscle homeostasis. Our results indicate that emerin downregulates STAT3 signaling by inducing retention of STAT3 and delaying STAT3 signaling in the nuclear membrane. This mechanism provides clues to the etiology of emerin-related muscular dystrophy and may be a new therapeutic target for treatment.


Assuntos
Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células A549 , Núcleo Celular/metabolismo , Sobrevivência Celular , Regulação da Expressão Gênica , Humanos , Janus Quinases/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/etiologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Mutação , Ligação Proteica , Fator de Transcrição STAT3/genética , Fatores de Transcrição/metabolismo
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