RESUMEN
Many Lamin A-associated proteins (LAAP's) that are key constituents of the nuclear envelope (NE), assemble at the "core" domains of chromosomes during NE reformation and mitotic exit. However, the identity and function of the chromosomal core domains remain ill-defined. Here, we show that a distinct section of the core domain overlaps with the centromeres/kinetochores of chromosomes during mitotic telophase. The core domain can thus be demarcated into a kinetochore proximal core (KPC) on one side of the segregated chromosomes and the kinetochore distal core (KDC) on the opposite side, close to the central spindle. We next tested if centromere assembly is connected to NE re-formation. We find that centromere assembly is markedly perturbed after inhibiting the function of LMNA and the core-localized LAAPs, BANF1 and Emerin. We also find that the LAAPs exhibit multiple biochemical interactions with the centromere and inner kinetochore proteins. Consistent with this, normal mitotic progression and chromosome segregation was severely impeded after inhibiting LAAP function. Intriguingly, the inhibition of centromere function also interferes with the assembly of LAAP components at the core domain, suggesting a mutual dependence of LAAP and centromeres for their assembly at the core domains. Finally, we find that the localization of key proteins involved in the centromeric loading of CENP-A, including the Mis18 complex and HJURP were markedly affected in LAAP-inhibited cells. Our evidence points to a model where LAAP assembly at the core domain serves a key function in loading new copies of centromeric proteins during or immediately after mitotic exit.
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In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.
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Lamina Tipo A , Membrana Nuclear , Animales , Ratones , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismoRESUMEN
Vimentin is a Type III intermediate filament (VIF) cytoskeletal protein that regulates the mechanical and migratory behavior of cells. Its expression is considered to be a marker for the epithelial to mesenchymal transition (EMT) that takes place in tumor metastasis. However, the molecular mechanisms regulated by the expression of vimentin in the EMT remain largely unexplored. We created MCF7 epithelial cell lines expressing vimentin from a cumate-inducible promoter to address this question. When vimentin expression was induced in these cells, extensive cytoplasmic VIF networks were assembled accompanied by changes in the organization of the endogenous keratin intermediate filament networks and disruption of desmosomes. Significant reductions in intercellular forces by the cells expressing VIFs were measured by quantitative monolayer traction force and stress microscopy. In contrast, laser trapping micro-rheology revealed that the cytoplasm of MCF7 cells expressing VIFs was stiffer than the uninduced cells. Vimentin expression activated transcription of genes involved in pathways responsible for cell migration and locomotion. Importantly, the EMT related transcription factor TWIST1 was upregulated only in wild type vimentin expressing cells and not in cells expressing a mutant non-polymerized form of vimentin, which only formed unit length filaments (ULF). Taken together, our results suggest that vimentin expression induces a hybrid EMT correlated with the upregulation of genes involved in cell migration.
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The chromatin associated with the nuclear lamina (NL) is referred to as lamina-associated domains (LADs). Here, we present an adaptation of the tyramide-signal amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50 000 cells. The cTSA-seq mapped regions are composed of previously defined LADs and smaller chromatin regions that fall within the Hi-C defined B-compartment containing nuclear peripheral heterochromatin. We used cTSA-seq to map chromatin at or near the assembling NL in cultured cells progressing through early G1. cTSA-seq revealed that the distal ends of chromosomes are near or at the reassembling NL during early G1, a feature similar to those found in senescent cells. We expand the use of cTSA-seq to the mapping of chromatin at or near the NL from fixed-frozen mouse cerebellar tissue sections. This mapping reveals a general conservation of NL-associated chromatin and identifies global and local changes during cerebellar development. The cTSA-seq method reported here is useful for analyzing chromatin at or near the NL from small numbers of cells derived from both in vitro and in vivo sources.
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Cromatina , Mapeo Cromosómico , Lámina Nuclear , Análisis de Secuencia de ADN , Animales , Ratones , Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Heterocromatina/metabolismo , Lámina Nuclear/metabolismo , Análisis de Secuencia de ADN/métodos , Mapeo Cromosómico/métodosRESUMEN
Keratin intermediate filaments convey mechanical stability and protection against stress to epithelial cells. Keratins are essential for colon health, as seen in keratin 8 knockout (K8-/-) mice exhibiting a colitis phenotype. We hypothesized that keratins support the nuclear envelope and lamina in colonocytes. K8-/- colonocytes in vivo exhibit significantly decreased levels of lamins A/C, B1, and B2 in a colon-specific and cell-intrinsic manner. CRISPR/Cas9- or siRNA-mediated K8 knockdown in Caco-2 cells similarly decreased lamin levels, which recovered after reexpression of K8 following siRNA treatment. Nuclear area was not decreased, and roundness was only marginally increased in cells without K8. Down-regulation of K8 in adult K8flox/flox;Villin-CreERt2 mice following tamoxifen administration significantly decreased lamin levels at day 4 when K8 levels had reduced to 40%. K8 loss also led to reduced levels of plectin, LINC complex, and lamin-associated proteins. While keratins were not seen in the nucleoplasm without or with leptomycin B treatment, keratins were found intimately located at the nuclear envelope and complexed with SUN2 and lamin A. Furthermore, K8 loss in Caco-2 cells compromised nuclear membrane integrity basally and after shear stress. In conclusion, colonocyte K8 helps maintain nuclear envelope and lamina composition and contributes to nuclear integrity.
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Queratina-8 , Queratinas , Animales , Células CACO-2 , Colon/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Humanos , Queratina-8/genética , Queratinas/metabolismo , Lamina Tipo A/metabolismo , Ratones , Membrana Nuclear/metabolismo , Plectina/metabolismo , ARN Interferente Pequeño/metabolismo , TamoxifenoRESUMEN
Stereocilia are actin-based projections of hair cells that are arranged in a step like array, in rows of increasing height, and that constitute the mechanosensory organelle used for the senses of hearing and balance. In order to function properly, stereocilia must attain precise sizes in different hair cell types and must coordinately form distinct rows with varying lengths. Espins are actin-bundling proteins that have a well-characterized role in stereocilia formation; loss of function mutations in Espin result in shorter stereocilia and deafness in the jerker mouse. Here we describe the generation of an Espin overexpressing transgenic mouse line that results in longer first row stereocilia and discoordination of second-row stereocilia length. Furthermore, Espin overexpression results in the misregulation of other stereocilia factors including GNAI3, GPSM2, EPS8, WHRN, and MYO15A, revealing that GNAI3 and GPSM2 are dispensable for stereocilia overgrowth. Finally, using an in vitro actin polymerization assay we show that espin provides an anti-capping function that requires both the G-actin binding WH2 domain as well as either the C-terminal F-actin binding domain or the internal xAB actin-binding domain. Our results provide a novel function for Espins at the barbed ends of actin filaments distinct from its previous known function of actin bundling that may account for their effects on stereocilia growth.
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Actinas , Proteínas de Microfilamentos , Estereocilios , Animales , Ratones , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cilios/metabolismo , Polimerizacion , Estereocilios/patología , Proteínas de Microfilamentos/metabolismoRESUMEN
The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we conduct quantitative confocal imaging to show that the loss of A-type lamins tends to increase nuclear and cellular volume while the loss of B-type lamins behaves in the opposite manner. We use fluorescence recovery after photobleaching, atomic force microscopy, optical tweezer microrheology, and traction force microscopy to demonstrate that A-type lamins engage with both F-actin and vimentin intermediate filaments (VIFs) through the linker of nucleoskeleton and cytoskeleton (LINC) complexes to modulate cortical and cytoplasmic stiffness as well as cellular contractility in mouse embryonic fibroblasts (MEFs). In contrast, we show that B-type lamins predominantly interact with VIFs through LINC complexes to regulate cytoplasmic stiffness and contractility. We then propose a physical model mediated by the laminLINC complex that explains these distinct mechanical phenotypes (mechanophenotypes). To verify this model, we use dominant negative constructs and RNA interference to disrupt the LINC complexes that facilitate the interaction of the nucleus with the F-actin and VIF cytoskeletons and show that the loss of these elements results in mechanophenotypes like those observed in MEFs that lack A- or B-type lamin isoforms. Finally, we demonstrate that the loss of each lamin isoform softens the cell nucleus and enhances constricted cell migration but in turn increases migration-induced DNA damage. Together, our findings uncover distinctive roles for each of the four major lamin isoforms in maintaining nucleocytoskeletal interactions and cellular mechanics.
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Fibroblastos , Lámina Nuclear , Animales , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Ratones , Lámina Nuclear/metabolismo , Matriz Nuclear/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.
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Citoplasma/metabolismo , Filamentos Intermedios/metabolismo , Vimentina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Biopolímeros/metabolismo , Células Cultivadas , Tomografía con Microscopio Electrónico/métodos , Filamentos Intermedios/química , Ratones , Vimentina/químicaRESUMEN
Nuclear lamins are type V intermediate filament proteins that polymerize into complex filamentous meshworks at the nuclear periphery and in less structured forms throughout the nucleoplasm. Lamins interact with a wide range of nuclear proteins and are involved in numerous nuclear and cellular functions. Within the nucleus, they play roles in chromatin organization and gene regulation, nuclear shape, size, and mechanics, and the organization and anchorage of nuclear pore complexes. At the whole cell level, they are involved in the organization of the cytoskeleton, cell motility, and mechanotransduction. The expression of different lamin isoforms has been associated with developmental progression, differentiation, and tissue-specific functions. Mutations in lamins and their binding proteins result in over 15 distinct human diseases, referred to as laminopathies. The laminopathies include muscular (e.g., Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy), neurological (e.g., microcephaly), and metabolic (e.g., familial partial lipodystrophy) disorders as well as premature aging diseases (e.g., Hutchinson-Gilford Progeria and Werner syndromes). How lamins contribute to the etiology of laminopathies is still unknown. In this review article, we summarize major recent findings on the structure, organization, and multiple functions of lamins in nuclear and more global cellular processes.
RESUMEN
Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D-structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna-/- and Lmnb1-/- fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.
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Simulación por Computador , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Poro Nuclear/metabolismo , Animales , Línea Celular , Embrión de Mamíferos/ultraestructura , Fibroblastos/ultraestructura , Lamina Tipo A/genética , Lamina Tipo B/genética , Ratones , Ratones Noqueados , Poro Nuclear/genética , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
The nuclear lamina (NL) is a meshwork found beneath the inner nuclear membrane. The study of the NL is hindered by the insolubility of the meshwork and has driven the development of proximity ligation methods to identify the NL-associated/proximal proteins, RNA, and DNA. To simplify and improve temporal labeling, we fused APEX2 to the NL protein lamin-B1 to map proteins, RNA, and DNA. The identified NL-interacting/proximal RNAs show a long 3' UTR bias, a finding consistent with an observed bias toward longer 3' UTRs in genes deregulated in lamin-null cells. A C-rich motif was identified in these 3' UTR. Our APEX2-based proteomics identifies a C-rich motif binding regulatory protein that exhibits altered localization in lamin-null cells. Finally, we use APEX2 to map lamina-associated domains (LADs) during the cell cycle and uncover short, H3K27me3-rich variable LADs. Thus, the APEX2-based tools presented here permit identification of proteomes, transcriptomes, and genome elements associated with or proximal to the NL.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas/metabolismo , Enzimas Multifuncionales/metabolismo , Lámina Nuclear/metabolismo , Mapeo de Interacción de Proteínas , Regiones no Traducidas 3'/genética , Secuencia de Bases , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Endonucleasas/química , Células HCT116 , Células HEK293 , Humanos , Células K562 , Lamina Tipo B/metabolismo , Enzimas Multifuncionales/química , Dominios Proteicos , Proteoma/metabolismo , ARN/metabolismo , Empalme del ARN/genéticaRESUMEN
Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration.
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Núcleo Celular/metabolismo , Daño del ADN , Vimentina/metabolismo , Animales , Movimiento Celular , Colágeno/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/metabolismo , Ratones , Microscopía de Fuerza Atómica , Microscopía Confocal , Necrosis/metabolismoRESUMEN
The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels (MSCs), without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through MSCs and consequent changes to histone modification state and chromatin-based nuclear rigidity.
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Heterocromatina/metabolismo , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Animales , Fenómenos Biomecánicos/fisiología , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Forma de la Célula/fisiología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Heterocromatina/fisiología , Histonas/metabolismo , Humanos , Lamina Tipo A/metabolismo , Mecanorreceptores/metabolismoRESUMEN
The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels, without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through mechanosensitive ion channels and consequent changes to histone modification state and chromatin-based nuclear rigidity.
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Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of tissue injury associated with inflammatory bowel diseases (IBDs). The pathological effects of PMNs are largely attributed to the release of soluble mediators and reactive oxygen species (ROS). We identified what we believe is a new, ROS-independent mechanism whereby activated tissue-infiltrating PMNs release microparticles armed with proinflammatory microRNAs (miR-23a and miR-155). Using IBD clinical samples, and in vitro and in vivo injury models, we show that PMN-derived miR-23a and miR-155 promote accumulation of double-strand breaks (DSBs) by inducing lamin B1-dependent replication fork collapse and inhibition of homologous recombination (HR) by targeting HR-regulator RAD51. DSB accumulation in injured epithelium led to impaired colonic healing and genomic instability. Targeted inhibition of miR-23a and miR-155 in cultured intestinal epithelial cells and in acutely injured mucosa decreased the detrimental effects of PMNs and enhanced tissue healing responses, suggesting that this approach can be used in therapies aimed at resolution of inflammation, in wound healing, and potentially to prevent neoplasia.
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Colitis/metabolismo , Colon/lesiones , Inestabilidad Genómica , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cicatrización de Heridas , Animales , Colitis/patología , Colon/metabolismo , Colon/patología , Roturas del ADN de Doble Cadena , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , MicroARNs/metabolismo , Neutrófilos/patología , Recombinasa Rad51/metabolismoRESUMEN
Keratin intermediate filaments (IFs) are the major cytoskeletal component in epithelial cells. The dynamics of keratin IFs have been described to depend mostly on the actin cytoskeleton, but the rapid transport of fully polymerized keratin filaments has not been reported. In this work, we used a combination of photoconversion experiments and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 genome editing to study the role of microtubules and microtubule motors in keratin filament transport. We found that long keratin filaments, like other types of IFs, are transported along microtubules by kinesin-1. Our data revealed that keratin and vimentin are nonconventional kinesin-1 cargoes because their transport did not require kinesin light chains, which are a typical adapter for kinesin-dependent cargo transport. Furthermore, we found that the same domain of the kinesin heavy chain tail is involved in keratin and vimentin IF transport, strongly suggesting that multiple types of IFs move along microtubules using an identical mechanism.-Robert, A., Tian, P., Adam, S. A., Kittisopikul, M., Jaqaman, K., Goldman, R. D., Gelfand, V. I. Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.
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Filamentos Intermedios/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Cinesinas/fisiología , Microtúbulos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Vimentina/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Cinesinas/antagonistas & inhibidores , Ratones , Ratones Noqueados , Microscopía FluorescenteRESUMEN
While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a "cytological ruler" for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription "hot zones." Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression.
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Mapeo Cromosómico , Simulación por Computador , Regulación de la Expresión Génica/fisiología , Genoma Humano , Lámina Nuclear/metabolismo , Transcripción Genética/fisiología , Estudio de Asociación del Genoma Completo , Humanos , Células K562 , Lámina Nuclear/genéticaRESUMEN
Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed "blebs" are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson-Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity.
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Heterocromatina/metabolismo , Histonas/metabolismo , Laminas/metabolismo , Membrana Nuclear/ultraestructura , Animales , Células Cultivadas , Células HeLa , Heterocromatina/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Laminas/genética , Mecanotransducción Celular/efectos de los fármacos , Ratones , Membrana Nuclear/efectos de los fármacos , Progeria/genética , Procesamiento Proteico-PostraduccionalRESUMEN
SUMMARYThe nucleoskeleton is an important structural feature of the metazoan nucleus and is involved in the regulation of genome expression and maintenance. The nuclear lamins are intermediate filament proteins that form a peripheral nucleoskeleton in concert with other lamin-associated proteins. Several other proteins normally found in the cytoskeleton have also been identified in the nucleus, but, as will be discussed here, their roles in forming a nucleoskeleton have not been elucidated. Nevertheless, mutations in lamins and lamin-associated proteins cause a spectrum of diseases, making them interesting targets for future research.
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Matriz Nuclear/fisiología , Humanos , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
The cell nucleus must continually resist and respond to intercellular and intracellular mechanical forces to transduce mechanical signals and maintain proper genome organization and expression. Altered nuclear mechanics is associated with many human diseases, including heart disease, progeria, and cancer. Chromatin and nuclear envelope A-type lamin proteins are known to be key nuclear mechanical components perturbed in these diseases, but their distinct mechanical contributions are not known. Here we directly establish the separate roles of chromatin and lamin A/C and show that they determine two distinct mechanical regimes via micromanipulation of single isolated nuclei. Chromatin governs response to small extensions (<3 µm), and euchromatin/heterochromatin levels modulate the stiffness. In contrast, lamin A/C levels control nuclear strain stiffening at large extensions. These results can be understood through simulations of a polymeric shell and cross-linked polymer interior. Our results provide a framework for understanding the differential effects of chromatin and lamin A/C in cell nuclear mechanics and their alterations in disease.