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1.
Exp Dermatol ; 32(5): 620-631, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36695185

RESUMEN

Skin ageing is an intricate physiological process affected by intrinsic and extrinsic factors. There is a demand to understand how the skin changes with age and photoexposure in individuals with Fitzpatrick skin types I-III due to accelerated photoageing and the risk of cutaneous malignancies. To assess the structural impact of intrinsic and extrinsic ageing, we analysed 14 skin parameters from the photoprotected buttock and photoexposed dorsal forearm of young and ageing females with Fitzpatrick skin types II-III (n = 20) using histomorphic techniques. Whilst the minimum viable epidermis (Emin ) remained constant (Q > 0.05), the maximum viable epidermis (Emax ) was decreased by both age and photoexposure (Q ≤ 0.05), which suggests that differences in epidermal thickness are attributed to changes in the dermal-epidermal junction (DEJ). Changes in Emax were not affected by epidermal cell proliferation. For the first time, we investigated the basal keratinocyte morphology with age and photoexposure. Basal keratinocytes had an increased cell size, cellular height and a more columnar phenotype in photoexposed sites of young and ageing individuals (Q ≤ 0.05), however no significant differences were observed with age. Some of the most striking changes were observed in the DEJ, and a decrease in the interdigitation index was observed with both age and photoexposure (Q ≤ 0.001), accompanied by a decreased height of rête ridges and dermal papilla. Interestingly, young photoexposed skin was comparable to ageing skin across many parameters, and we hypothesise that this is due to accelerated photoageing. This study highlights the importance of skin care education and photoprotection from an early age.


Asunto(s)
Envejecimiento de la Piel , Enfermedades de la Piel , Femenino , Humanos , Piel/patología , Epidermis/fisiología , Enfermedades de la Piel/patología
2.
J Anat ; 234(4): 438-455, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30740672

RESUMEN

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.


Asunto(s)
Piel , Ingeniería de Tejidos/métodos , Membrana Basal/citología , Membrana Basal/ultraestructura , Diferenciación Celular , Células Cultivadas , Dermis/citología , Dermis/ultraestructura , Epidermis/ultraestructura , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro/métodos , Queratinocitos/metabolismo , Microscopía Electrónica , Piel/anatomía & histología , Piel/ultraestructura
3.
Semin Cell Dev Biol ; 29: 169-79, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24374011

RESUMEN

LINC (Linker of Nucleoskeleton and Cytoskeleton) complex is an evolutionary conserved structure that spans the entire nuclear envelope (NE), and integrates the nuclear interior with the cytoskeleton, in order to support a diverse array of fundamental biological processes. Key components of the LINC complex are the nesprins (Nuclear Envelope SPectrin Repeat proteINS) that were initially described as large integral NE proteins. However, nesprin genes are complex and generate many variants, which occupy various sub-cellular compartments suggesting additional functions. Hence, the potential involvement of nesprins in disease has expanded immensely on what we already know. That is, nesprins are implicated in diseases such as cancer, myopathies, arthrogryposis, neurological disorders and hearing loss. Here we review nesprins by providing an in depth account of their structure, molecular interactions and cellular functions with relevance to their potential roles in disease. Specifically, we speculate about possible pathomechanisms underlying nesprin-associated diseases.


Asunto(s)
Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Envejecimiento Prematuro/genética , Animales , Proteínas del Citoesqueleto , Citoesqueleto , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinesinas/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedades Musculoesqueléticas/genética , Neoplasias/genética , Enfermedades Neurodegenerativas/genética , Membrana Nuclear/patología , Proteínas Nucleares/metabolismo
4.
Cells ; 13(11)2024 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-38891038

RESUMEN

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.


Asunto(s)
Invasividad Neoplásica , Proteína Disulfuro Isomerasas , Humanos , Línea Celular Tumoral , Proteína Disulfuro Isomerasas/metabolismo , Femenino , Regulación hacia Abajo/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Membrana Nuclear/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Péptidos y Proteínas de Señalización Intracelular
5.
Eur J Immunol ; 42(8): 2132-41, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22736282

RESUMEN

Cytotoxic T lymphocytes (CTLs) kill tumorigenic and virally infected cells by targeted secretion of lytic granule contents. The precise point at which secretion occurs is directed by the centrosome docking at the immunological synapse (IS). The centrosome is highly dynamic in CTLs, lagging behind the nucleus in the uropod of migrating CTLs, but translocating across the entire length of the cell to dock at the IS when a target cell is recognized. While in most cell types, the centrosome is always closely associated with the nuclear membrane, in CTLs, it often appears to be dissociated from the nucleus, both in migrating cells and when forming an IS. We asked whether this dissociation is required for CTL killing, by expressing GFP-BICD2-NT-nesprin-3, which tethers the centrosome to the nucleus irreversibly. Immunofluorescence microscopy revealed that the centrosome polarized successfully to the central supramolecular activation complex (cSMAC) of the synapse in GFP-BICD2-NT-nesprin-3-expressing CTLs, with the centrosome and nucleus migrating together to the IS. CTLs in which the centrosome was "glued" to the nucleus were able to dock and release granules at the IS as effectively as mock-treated cells. These data demonstrate that CTL cytotoxicity is independent of centrosomal dissociation from the nuclear envelope.


Asunto(s)
Núcleo Celular/metabolismo , Centrosoma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Animales , Polaridad Celular , Células Cultivadas , Técnicas de Transferencia de Gen , Sinapsis Inmunológicas , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Membrana Nuclear , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , Linfocitos T Citotóxicos/ultraestructura
6.
Cell Mol Life Sci ; 69(20): 3493-509, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22653047

RESUMEN

Nesprins-1/-2/-3/-4 are nuclear envelope proteins, which connect nuclei to the cytoskeleton. The largest nesprin-1/-2 isoforms (termed giant) tether F-actin through their N-terminal actin binding domain (ABD). Nesprin-3, however, lacks an ABD and associates instead to plectin, which binds intermediate filaments. Nesprins are integrated into the outer nuclear membrane via their C-terminal KASH-domain. Here, we show that nesprin-1/-2 ABDs physically and functionally interact with nesprin-3. Thus, both ends of nesprin-1/-2 giant are integrated at the nuclear surface: via the C-terminal KASH-domain and the N-terminal ABD-nesprin-3 association. Interestingly, nesprin-2 ABD or KASH-domain overexpression leads to increased nuclear areas. Conversely, nesprin-2 mini (contains the ABD and KASH-domain but lacks the massive nesprin-2 giant rod segment) expression yields smaller nuclei. Nuclear shrinkage is further enhanced upon nesprin-3 co-expression or microfilament depolymerization. Our findings suggest that multivariate intermolecular nesprin interactions with the cytoskeleton form a lattice-like filamentous network covering the outer nuclear membrane, which determines nuclear size.


Asunto(s)
Actinas/metabolismo , Núcleo Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Western Blotting , Núcleo Celular/ultraestructura , Células Cultivadas , Proteínas del Citoesqueleto , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Genes Dominantes , Humanos , Inmunoprecipitación , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Plásmidos , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética
7.
Cell Mol Life Sci ; 68(9): 1593-610, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-20922455

RESUMEN

Cell polarization is a fundamental process underpinning organismal development, and tissue homeostasis, which requires an orchestrated interplay of nuclear, cytoskeletal, and centrosomal structures. The underlying molecular mechanisms, however, still remain elusive. Here we report that kinesin-1/nesprin-2/SUN-domain macromolecular assemblies, spanning the entire nuclear envelope (NE), function in cell polarization by anchoring cytoskeletal structures to the nuclear lamina. Nesprin-2 forms complexes with the kinesin-1 motor protein apparatus by associating with and recruiting kinesin light chain 1 (KLC1) to the outer nuclear membrane. Similar to nesprin-2, KLC1 requires lamin A/C for proper NE localization. The depletion of nesprin-2 or KLC1, or the uncoupling of nesprin-2/SUN-domain protein associations impairs cell polarization during wounding and dislodges the centrosome from the NE. In addition nesprin-2 loss has profound effects on KLC1 levels, the cytoskeleton, and Golgi apparatus organization. Collectively these data show that NE-associated proteins are pivotal determinants of cell architecture and polarization.


Asunto(s)
Centrosoma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Membrana Nuclear/metabolismo , Animales , Línea Celular , Polaridad Celular , Chlorocebus aethiops/metabolismo , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Regiones de Fijación a la Matriz , Ratones , Proteínas del Tejido Nervioso/metabolismo
8.
J Biol Chem ; 285(45): 34932-8, 2010 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-20801886

RESUMEN

Nesprins and emerin are structural nuclear envelope proteins that tether nuclei to the cytoskeleton. In this work, we identified the cytoskeleton-associated α-N/E-catenins as novel nesprin-2-binding partners. The association involves the C termini of nesprin-2 giant and α-N/E-catenins. α-E/T/N-catenins are known primarily for their roles in cadherin-mediated cell-cell adhesion. Here, we show that, in addition, α-catenin forms complexes with nesprin-2 that include ß-catenin and emerin. We demonstrate that the depletion of nesprin-2 reduces both the amount of active ß-catenin inside the nucleus and T-cell factor/lymphoid-enhancing factor-dependent transcription. Taken together, these findings suggest novel nesprin-2 functions in cellular signaling. Moreover, we propose that, in contrast to emerin, nesprin-2 is a positive regulator of the Wnt signaling pathway.


Asunto(s)
Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , alfa Catenina/metabolismo , Animales , Células COS , Adhesión Celular/fisiología , Chlorocebus aethiops , Humanos , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas Wnt/genética , alfa Catenina/genética , beta Catenina/genética , beta Catenina/metabolismo
9.
Cells ; 10(5)2021 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-34066027

RESUMEN

Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.


Asunto(s)
Biomimética , Epidermis/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Fenómenos Biomecánicos , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Núcleo Celular , Proliferación Celular , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Hidrogeles/química , Técnicas In Vitro , Mecanotransducción Celular , Lámina Nuclear/metabolismo , Ósmosis , Presión Osmótica , Presión , Piel/patología , Estrés Mecánico
10.
Genes (Basel) ; 12(9)2021 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34573277

RESUMEN

Autism spectrum disorder (ASD) is a group of neurological and developmental disabilities characterised by clinical and genetic heterogeneity. The current study aimed to expand ASD genotyping by investigating potential associations with SYNE2 mutations. Specifically, the disease-causing variants of SYNE2 in 410 trios manifesting neurodevelopmental disorders using whole-exome sequencing were explored. The consequences of the identified variants were studied at the transcript level using quantitative polymerase chain reaction (qPCR). For validation, immunofluorescence and immunoblotting were performed to analyse mutational effects at the protein level. The compound heterozygous variants of SYNE2 (NM_182914.3:c.2483T>G; p.(Val828Gly) and NM_182914.3:c.2362G>A; p.(Glu788Lys)) were identified in a 4.5-year-old male, clinically diagnosed with autism spectrum disorder, developmental delay and intellectual disability. Both variants reside within the nesprin-2 giant spectrin repeat (SR5) domain and are predicted to be highly damaging using in silico tools. Specifically, a significant reduction of nesprin-2 giant protein levels is revealed in patient cells. SYNE2 transcription and the nuclear envelope localisation of the mutant proteins was however unaffected as compared to parental control cells. Collectively, these data provide novel insights into the cardinal role of the nesprin-2 giant in neurodevelopment and suggest that the biallelic hypomorphic SYNE2 mutations may be a new cause of intellectual disability and ASD.


Asunto(s)
Trastorno del Espectro Autista/genética , Discapacidad Intelectual/genética , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Células Cultivadas , Niño , Heterocigoto , Humanos , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Mutación Missense , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Dominios Proteicos , Transporte de Proteínas
11.
Biochim Biophys Acta ; 1783(12): 2415-26, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18845190

RESUMEN

SUN-domain proteins form a novel and conserved family of inner nuclear membrane (INM) proteins, which establish physical connections between the nucleoplasm and the cytoskeleton. In the current study, we provide evidence that within the nuclear envelope (NE) Sun1 proteins form highly immobile oligomeric complexes in interphase cells. By performing inverse fluorescence recovery after photobleaching analysis, we demonstrate in vivo that both perinuclear and nucleoplasmic Sun1 segments are essential for maintenance of Sun1 immobility at the NE. Our data in particular underline the self-association properties of the C-terminal coiled-coil Sun1 segment, the ability of which to form dimers and tetramers is demonstrated. Furthermore, the Sun1 tertiary structure involves interchain disulfide bonds that might contribute to higher homo-oligomer formation, although the overall dynamics of the Sun1 C-terminus remains unaffected when the cysteins involved are mutated. While a major Sun1 pool colocalizes with nuclear pore complex proteins, a large fraction of the Sun1 protein assemblies colocalize with immunoreactive foci of Sun2, another SUN-domain paralogue at the NE. We demonstrate that the Sun1 coiled-coil domain permits these heterophilic associations with Sun2. Sun1 therefore provides a non-dynamic platform for the formation of different macromolecular assemblies at the INM. Our data support a model in which SUN-protein-containing multi-variate complexes may provide versatile outer nuclear membrane attachment sites for cytoskeletal filaments.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/fisiología , Secuencia de Aminoácidos , Western Blotting , Núcleo Celular/ultraestructura , Reactivos de Enlaces Cruzados , Disulfuros/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Membrana Nuclear/ultraestructura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos
12.
Biochem Soc Trans ; 36(Pt 6): 1368-72, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19021557

RESUMEN

Over the last few years, several novel proteins have been identified that facilitate the physical integration of the nucleus with the cytoplasmic compartment. The majority belong to the evolutionarily conserved KASH [klarsicht/ANC-1 (anchorage 1)/SYNE (synaptic nuclear envelope protein) homology]-domain family, which function primarily as exclusive outer nuclear membrane scaffolds that associate with the cytoskeleton, the centrosome and the motor protein apparatus. In the present paper, we propose a novel model, which may explain why these proteins also determine nuclear architecture. Moreover, we discuss further nuclear membrane-tethering devices, which indicate collectively the presence of specific molecular mechanisms that organize the cytoplasmic-nuclear membrane interface in mammalian cells.


Asunto(s)
Citoesqueleto/metabolismo , Membrana Nuclear/metabolismo , Proteínas/química , Proteínas/metabolismo , Animales , Transporte Biológico , Humanos , Estructura Terciaria de Proteína
13.
Mol Biol Cell ; 16(7): 3411-24, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15843432

RESUMEN

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 alpha-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.


Asunto(s)
Lamina Tipo A/biosíntesis , Proteínas de Microfilamentos/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Membrana Nuclear/metabolismo , Proteínas Nucleares/biosíntesis , Animales , Western Blotting , Células COS , Caenorhabditis elegans , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Drosophila melanogaster , Genes Dominantes , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Técnicas In Vitro , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Modelos Genéticos , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Timopoyetinas/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos
14.
Methods Mol Biol ; 1840: 295-306, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30141052

RESUMEN

The genome in eukaryotic cells is encased by two intricate and interconnected concentric membranes, which together with the underlying nuclear lamina form the nuclear envelope (NE). Two fundamental macromolecular structures are embedded within the nuclear envelope: the nuclear pore (NPC) and the LINC complex. The former perforates the nucleus controlling biomolecule trafficking between the nucleoplasm and the cytoplasm, while the latter integrates the nucleus via the cytoskeleton to the extracellular matrix. LINC complex structural and functional integrity is of utmost importance for various fundamental cellular functions. Mechanical forces are relayed into the nuclear interior via the LINC complex, which controls lamina organization, chromosome dynamics, and genome organization and stability. Thus, LINC constituents play pivotal roles in cellular architecture including organelle positioning, cell movement, tissue assembly, organ homeostasis, and organismal aging. The LINC complex oligomeric core contains several multi-isomeric, multifunctional, and often tissue-specific proteins. Therefore, for a proper functional analysis, genetic mouse models are an invaluable resource. Herein, we focus on the LINC complex roles in the skin and describe methods that enable the successful isolation of primary embryonic fibroblast and newborn skin cells, which can be then investigated functionally in vitro.


Asunto(s)
Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Piel/metabolismo , Animales , Línea Celular , Movimiento Celular , Células Cultivadas , Fibroblastos/metabolismo , Queratinocitos/metabolismo , Ratones
15.
J Invest Dermatol ; 137(10): 2157-2167, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28595999

RESUMEN

The maintenance of a proper nuclear architecture and three-dimensional organization of the genes, enhancer elements, and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by a marked decrease in expression of several nuclear envelope-associated components (Lamin B1, Lamin A/C, Sun1, Nesprin-3, Plectin) compared with controls. Furthermore, chromatin immunoprecipitation-quantitative PCR assay showed enrichment of p63 on Sun1, Syne3, and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks trimethylation on lysine 27 of histone H3, trimethylation on lysine 9 of histone H3, and heterochromatin protein 1-alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription toward the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture, and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression program in the epidermis.


Asunto(s)
Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Queratinocitos/metabolismo , Fosfoproteínas/genética , Transactivadores/genética , Animales , Diferenciación Celular , Núcleo Celular/metabolismo , Epidermis/patología , Humanos , Queratinocitos/patología , Ratones , Modelos Animales , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Fosfoproteínas/biosíntesis , ARN/genética , Transactivadores/biosíntesis , Factores de Transcripción/genética , Transcripción Genética
16.
Methods Mol Biol ; 1411: 221-32, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27147045

RESUMEN

Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.


Asunto(s)
Western Blotting , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Animales , Western Blotting/métodos , Línea Celular , Proteínas del Citoesqueleto , Humanos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Peso Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Isoformas de Proteínas
17.
Int J Cell Biol ; 2012: 736524, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22518138

RESUMEN

Nesprin-1 is a giant tail-anchored nuclear envelope protein composed of an N-terminal F-actin binding domain, a long linker region formed by multiple spectrin repeats and a C-terminal transmembrane domain. Based on this structure, it connects the nucleus to the actin cytoskeleton. Earlier reports had shown that Nesprin-1 binds to nuclear envelope proteins emerin and lamin through C-terminal spectrin repeats. These repeats can also self-associate. We focus on the N-terminal Nesprin-1 sequences and show that they interact with Nesprin-3, a further member of the Nesprin family, which connects the nucleus to the intermediate filament network. We show that upon ectopic expression of Nesprin-3 in COS7 cells, which are nearly devoid of Nesprin-3 in vitro, vimentin filaments are recruited to the nucleus and provide evidence for an F-actin interaction of Nesprin-3 in vitro. We propose that Nesprins through interactions amongst themselves and amongst the various Nesprins form a network around the nucleus and connect the nucleus to several cytoskeletal networks of the cell.

18.
Eur J Cell Biol ; 91(8): 614-28, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22555292

RESUMEN

Emery-Dreifuss muscular dystrophy (EDMD) is a late onset-disease characterized by skeletal muscle wasting and heart defects with associated risk of sudden death. The autosomal dominant form of the disease is caused by mutations in the LMNA gene encoding LaminA and C, the X-linked form results from mutations in the gene encoding the inner nuclear membrane protein Emerin (STA). Both Emerin and LaminA/C interact with the nuclear envelope proteins Nesprin-1 and -2 and mutations in genes encoding C-terminal isoforms of Nesprin-1 and -2 have also been implicated in EDMD. Here we analyse primary fibroblasts from patients affected by either Duchenne muscular dystrophy (DMD) or Emery-Dreifuss muscular dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) that in addition to the disease causing mutations harbour mutations in the Nesprin-1 gene and in the SUN1 and SUN2 gene, respectively. SUN proteins together with the Nesprins form the core of the LINC complex which connects the nucleus with the cytoskeleton. The mutations are accompanied by changes in cell adhesion, cell migration, senescence, and stress response, as well as in nuclear shape and nuclear envelope composition which are changes characteristic for laminopathies. Our results point to a potential influence of mutations in components of the LINC complex on the clinical outcome and the molecular pathology in the patients.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Fibroblastos/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Emery-Dreifuss/genética , Adhesión Celular , Movimiento Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patología , Forma del Núcleo Celular , Senescencia Celular , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Proteínas del Citoesqueleto , Femenino , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patología , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Estrés Fisiológico , Transfección , Cicatrización de Heridas
19.
Nucleus ; 3(2): 172-86, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22198684

RESUMEN

Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.


Asunto(s)
Diferenciación Celular , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Cicatrización de Heridas , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Adhesiones Focales/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Mutación , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de Heridas/efectos de los fármacos
20.
Mol Biol Cell ; 21(2): 354-68, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-19940018

RESUMEN

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.


Asunto(s)
Núcleo Celular/enzimología , Retículo Endoplásmico/enzimología , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Actinas/metabolismo , Aminoácidos/metabolismo , Apoptosis , Autofagia , Línea Celular , Núcleo Celular/ultraestructura , Supervivencia Celular , Colesterol/metabolismo , Retículo Endoplásmico/ultraestructura , Humanos , Laminas/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas de Complejo Poro Nuclear/metabolismo , Fenotipo , Proteínas Recombinantes de Fusión/metabolismo , Estrés Fisiológico , Transfección , Vacuolas/metabolismo , Vacuolas/ultraestructura , Receptor de Lamina B
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