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1.
Cytometry A ; 89(6): 575-84, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27214589

RESUMO

Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting. © 2016 International Society for Advancement of Cytometry.


Assuntos
Hepatócitos/ultraestrutura , Células-Tronco Embrionárias Humanas/ultraestrutura , Gotículas Lipídicas/ultraestrutura , Análise de Célula Única/métodos , Biomarcadores/análise , Diferenciação Celular , Linhagem Celular , Espectroscopia Dielétrica/instrumentação , Espectroscopia Dielétrica/métodos , Eletroforese/instrumentação , Eletroforese/métodos , Hepatócitos/fisiologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Gotículas Lipídicas/fisiologia , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Análise de Célula Única/instrumentação , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos
2.
Curr Biol ; 34(1): 117-131.e5, 2024 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-38134935

RESUMO

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.


Assuntos
Proteínas de Ciclo Celular , Oócitos , Humanos , Feminino , Idoso , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Oócitos/metabolismo , Coesinas , Meiose , Centrômero/metabolismo , Cromátides/metabolismo , Segregação de Cromossomos
3.
Mol Cell Proteomics ; 10(1): M110.003129, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20876400

RESUMO

Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights.


Assuntos
Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animais , Diferenciação Celular , Fracionamento Celular , Linhagem Celular , Humanos , Espectrometria de Massas , Proteínas de Membrana/química , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/ultraestrutura , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/química , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes
4.
Commun Biol ; 6(1): 543, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-37202417

RESUMO

The role of the mechanical environment in defining tissue function, development and growth has been shown to be fundamental. Assessment of the changes in stiffness of tissue matrices at multiple scales has relied mostly on invasive and often specialist equipment such as AFM or mechanical testing devices poorly suited to the cell culture workflow.In this paper, we have developed a unbiased passive optical coherence elastography method, exploiting ambient vibrations in the sample that enables real-time noninvasive quantitative profiling of cells and tissues. We demonstrate a robust method that decouples optical scattering and mechanical properties by actively compensating for scattering associated noise bias and reducing variance. The efficiency for the method to retrieve ground truth is validated in silico and in vitro, and exemplified for key applications such as time course mechanical profiling of bone and cartilage spheroids, tissue engineering cancer models, tissue repair models and single cell. Our method is readily implementable with any commercial optical coherence tomography system without any hardware modifications, and thus offers a breakthrough in on-line tissue mechanical assessment of spatial mechanical properties for organoids, soft tissues and tissue engineering.


Assuntos
Técnicas de Imagem por Elasticidade , Vibração , Técnicas de Imagem por Elasticidade/métodos , Tomografia de Coerência Óptica/métodos , Cartilagem , Organoides
5.
Mol Cell Proteomics ; 9(12): 2571-85, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20693407

RESUMO

A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.


Assuntos
Genoma Humano , Leucócitos/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteoma , Animais , Western Blotting , Linhagem Celular , Humanos , Microscopia de Fluorescência , Ratos
6.
Biochem Soc Trans ; 39(6): 1742-6, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22103518

RESUMO

The nuclear envelope is a complex double membrane system that serves as a dynamic interface between the nuclear and cytoplasmic compartments. Among its many roles is to provide an anchor for gene regulatory proteins on its nucleoplasmic surface and for the cytoskeleton on its cytoplasmic surface. Both sets of anchors are proteins called NETs (nuclear envelope transmembrane proteins), embedded respectively in the inner or outer nuclear membranes. Several lines of evidence indicate that the nuclear envelope contributes to cell-cycle regulation. These contributions come from both inner and outer nuclear membrane NETs and appear to operate through several distinct mechanisms ranging from sequestration of gene-regulatory proteins to activating kinase cascades.


Assuntos
Ciclo Celular , Membrana Nuclear/metabolismo , Animais , Humanos , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Fatores de Transcrição/metabolismo
7.
J Cell Biol ; 174(5): 625-30, 2006 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-16943179

RESUMO

Previous evidence has indicated that an intact centrosome is essential for cell cycle progress and that elimination of the centrosome or depletion of individual centrosome proteins prevents the entry into S phase. To investigate the molecular mechanisms of centrosome-dependent cell cycle progress, we performed RNA silencing experiments of two centrosome-associated proteins, pericentriolar material 1 (PCM-1) and pericentrin, in primary human fibroblasts. We found that cells depleted of PCM-1 or pericentrin show lower levels of markers for S phase and cell proliferation, including cyclin A, Ki-67, proliferating cell nuclear antigen, minichromosome maintenance deficient 3, and phosphorylated retinoblastoma protein. Also, the percentage of cells undergoing DNA replication was reduced by >50%. At the same time, levels of p53 and p21 increased in these cells, and cells were predisposed to undergo senescence. Conversely, depletion of centrosome proteins in cells lacking p53 did not cause any cell cycle arrest. Inhibition of p38 mitogen-activated protein kinase rescued cell cycle activity after centrosome protein depletion, indicating that p53 is activated by the p38 stress pathway.


Assuntos
Antígenos/metabolismo , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Centrossomo/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Anticorpos , Antígenos/genética , Antígenos/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Ciclina A/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Replicação do DNA , Fibroblastos , Humanos , Antígeno Ki-67/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Proteína do Retinoblastoma/metabolismo , Fase S , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Cell Mol Life Sci ; 67(8): 1353-69, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20091084

RESUMO

Nuclear envelope complexity is expanding with respect to identification of protein components. Here we test the validity of proteomics results that identified 67 novel predicted nuclear envelope transmembrane proteins (NETs) from liver by directly comparing 30 as tagged fusions using targeting assays. This confirmed 21 as NETs, but 4 only targeted in certain cell types, underscoring the complexity of interactions that tether NETs to the nuclear envelope. Four NETs accumulated at the nuclear rim in normal fibroblasts but not in fibroblasts lacking lamin A, suggesting involvement of lamin A in tethering them in the nucleus. However, intriguingly, for the NETs tested alternative mechanisms for nuclear envelope retention could be found in Jurkat cells that normally lack lamin A. This study expands by a factor of three the number of liver NETs analyzed, bringing the total confirmed to 31, and shows that several have multiple mechanisms for nuclear envelope retention.


Assuntos
Lamina Tipo A/fisiologia , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animais , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Camundongos , Camundongos Knockout , Mioblastos/citologia , Mioblastos/metabolismo , Transporte Proteico , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
BMC Cell Biol ; 10: 28, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19383121

RESUMO

BACKGROUND: Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood. RESULTS: We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils. CONCLUSION: Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.


Assuntos
Diferenciação Celular , Centrossomo/química , Proteínas do Citoesqueleto/análise , Mioblastos/química , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mioblastos/citologia , Mioblastos/ultraestrutura
10.
Neuromuscul Disord ; 27(4): 338-351, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28214269

RESUMO

Reports of aberrant distribution for some nuclear envelope proteins in cells expressing a few Emery-Dreifuss muscular dystrophy mutations raised the possibility that such protein redistribution could underlie pathology and/or be diagnostic. However, this disorder is linked to 8 different genes encoding nuclear envelope proteins, raising the question of whether a particular protein is most relevant. Therefore, myoblast/fibroblast cultures from biopsy and tissue sections from a panel of nine Emery-Dreifuss muscular dystrophy patients (4 male, 5 female) including those carrying emerin and FHL1 (X-linked) and several lamin A (autosomal dominant) mutations were stained for the proteins linked to the disorder. As tissue-specific nuclear envelope proteins have been postulated to mediate the tissue-specific pathologies of different nuclear envelopathies, patient samples were also stained for several muscle-specific nuclear membrane proteins. Although linked proteins nesprin 1 and SUN2 and muscle-specific proteins NET5/Samp1 and Tmem214 yielded aberrant distributions in individual patient cells, none exhibited defects through the larger patient panel. Muscle-specific Tmem38A normally appeared in both the nuclear envelope and sarcoplasmic reticulum, but most patient samples exhibited a moderate redistribution favouring the sarcoplasmic reticulum. The absence of striking uniform defects in nuclear envelope protein distribution indicates that such staining will be unavailing for general diagnostics, though it remains possible that specific mutations exhibiting protein distribution defects might reflect a particular clinical variant. These findings further argue that multiple pathways can lead to the generally similar pathologies of this disorder while at the same time the different cellular phenotypes observed possibly may help explain the considerable clinical variation of EDMD.


Assuntos
Distrofia Muscular de Emery-Dreifuss/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Bancos de Tecidos , Adolescente , Adulto , Biomarcadores/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Músculo Esquelético , Distrofia Muscular de Emery-Dreifuss/patologia
11.
Sci Rep ; 7: 37541, 2017 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-28134251

RESUMO

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.


Assuntos
Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/metabolismo , Fígado/metabolismo , Junções Íntimas/patologia , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular , Hepatócitos/patologia , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Proteína da Zônula de Oclusão-1/metabolismo
12.
Neuromuscul Disord ; 25(2): 127-36, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25454731

RESUMO

Emery-Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterized by early contractures, slowly progressive muscular weakness and life-threatening cardiac arrhythmia that can develop into cardiomyopathy. In X-linked EDMD (EDMD1), female carriers are usually unaffected. Here we present a clinical description and in vitro characterization of a mildly affected EDMD1 female carrying the heterozygous EMD mutation c.174_175delTT; p.Y59* that yields loss of protein. Muscle tissue sections and cultured patient myoblasts exhibited a mixed population of emerin-positive and -negative cells; thus uneven X-inactivation was excluded as causative. Patient blood cells were predominantly emerin-positive, but considerable nuclear lobulation was observed in non-granulocyte cells - a novel phenotype in EDMD. Both emerin-positive and emerin-negative myoblasts exhibited spontaneous differentiation in tissue culture, though emerin-negative myoblasts were more proliferative than emerin-positive cells. The preferential proliferation of emerin-negative myoblasts together with the high rate of spontaneous differentiation in both populations suggests that loss of functional satellite cells might be one underlying mechanism for disease pathology. This could also account for the slowly developing muscle phenotype.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Distrofia Muscular de Emery-Dreifuss/patologia , Mioblastos/patologia , Adolescente , Adulto , Idade de Início , Antígenos CD , Autoantígenos/metabolismo , Cardiomiopatias/etiologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Criança , Saúde da Família , Feminino , Citometria de Fluxo , Humanos , Antígeno Ki-67/metabolismo , Lamina Tipo A/metabolismo , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/complicações , Distrofia Muscular de Emery-Dreifuss/genética , Mioblastos/metabolismo , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção , Adulto Jovem
13.
Nucleus ; 4(6): 460-77, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24213376

RESUMO

Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution.


Assuntos
Núcleo Celular/metabolismo , Membrana Nuclear/química , Especificidade de Órgãos/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Evolução Molecular , Humanos , Proteínas de Membrana/metabolismo , Modelos Animais , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Transdução de Sinais
14.
Biomicrofluidics ; 6(3): 34113, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23940503

RESUMO

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 µm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.

15.
Nucleus ; 3(6): 552-64, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22990521

RESUMO

One hypothesis to explain how mutations in the same nuclear envelope proteins yield pathologies focused in distinct tissues is that as yet unidentified tissue-specific partners mediate the disease pathologies. The nuclear envelope proteome was recently determined from leukocytes and muscle. Here the same methodology is applied to liver and a direct comparison of the liver, muscle and leukocyte data sets is presented. At least 74 novel transmembrane proteins identified in these studies have been directly confirmed at the nuclear envelope. Within this set, RT-PCR, western blot and staining of tissue cryosections confirms that the protein complement of the nuclear envelope is clearly distinct from one tissue to another. Bioinformatics reveals similar divergence between tissues across the larger data sets. For proteins acting in complexes according to interactome data, the whole complex often exhibited the same tissue-specificity. Other tissue-specific nuclear envelope proteins identified were known proteins with functions in signaling and gene regulation. The high tissue specificity in the nuclear envelope likely underlies the complex disease pathologies and argues that all organelle proteomes warrant re-examination in multiple tissues.


Assuntos
Membrana Nuclear/metabolismo , Proteoma/metabolismo , Animais , Western Blotting , Biologia Computacional , Humanos , Leucócitos/metabolismo , Leucócitos/patologia , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Especificidade de Órgãos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
PLoS One ; 6(4): e18762, 2011 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-21533191

RESUMO

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/-) cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Ciclo Celular , Linhagem Celular , Senescência Celular , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Interferência de RNA
17.
PLoS One ; 4(12): e8303, 2009 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-20011525

RESUMO

BACKGROUND: In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown. METHODOLOGY: In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of non-muscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis. CONCLUSIONS: Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bind centrosome proteins, and that this potential becomes activated during myoblast differentiation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Células Musculares/citologia , Células Musculares/metabolismo , Mioblastos/citologia , Animais , Fusão Celular , Linhagem Celular , Humanos , Camundongos , Microtúbulos/metabolismo , Modelos Biológicos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Membrana Nuclear/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Transporte Proteico
18.
Cell Div ; 1: 26, 2006 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-17109756

RESUMO

Centrosomes are frequently amplified in cancer cells. Increased numbers of centrosomes can give rise to multipolar spindles in mitosis, and thereby lead to the formation of aneuploid daughter cells. However, whether centrosome amplification is a cause or a consequence of cancer is unclear. In contrast, loss of a functional centrosome has been shown to lead to cell cycle arrest. In this review, the potential mechanisms underlying centrosome amplification and centrosome-dependent cell cycle regulation are discussed.

19.
Biol Reprod ; 67(6): 1726-33, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12444046

RESUMO

A series of experiments have been carried out to determine whether follicles secrete factors able to affect the growth and development of other, like-sized follicles. Late preantral mouse ovarian follicles were either cocultured or cultured in media conditioned by previously cultured follicles. In particular, the experiments examined whether follicles do secrete such factors, whether the level of FSH in the culture media can affect that process, and what the nature of such secretory factor(s) might be. First, pairs of follicles were cocultured across a polycarbonate membrane containing pores. This showed that communication between the follicles resulted in the stimulation of growth and that the stimulation was due, at least in part, to the production of secretory factor(s). In subsequent experiments, follicles were cultured in media that had been preconditioned by previously cultured follicles. The concentration of FSH in the cultures determined the effect of the conditioned media: conditioned media was stimulatory to follicle growth when levels of FSH remained high throughout the culture, but inhibitory when FSH levels were dropped midway through the cultures. Heat inactivation removed this inhibitory effect, showing that the factor was likely to be a protein; addition of follistatin to the conditioned media did not alter its effect, indicating that the factor was unlikely to be activin. We have shown through a series of culture experiments that mouse follicles secrete factor(s) that can affect the development of other like-sized follicles when cultured from the late preantral to Graafian stages. Furthermore, we have shown that the effect (or production) of such factors is dependent on the FSH environment of the follicles.


Assuntos
Substâncias de Crescimento/metabolismo , Substâncias de Crescimento/farmacologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Animais , Técnicas de Cocultura , Meios de Cultivo Condicionados , Técnicas de Cultura , Feminino , Hormônio Foliculoestimulante/análise , Folistatina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Folículo Ovariano/efeitos dos fármacos
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