The nuclear matrix: a critical appraisal.

It is becoming increasingly clear that the cell nucleus is a highly structurized organelle. Because of its tight compartmentalization, it is generally believed that a framework must exist, responsible for maintaining such a spatial organization. Over the last twenty years many investigations have been devoted to identifying the nuclear framework. Structures isolated by different techniques have been obtained in vitro and are variously referred to as nuclear matrix, nucleoskeleton or nuclear scaffold. Many different functions, such as DNA replication and repair, mRNA transcription, processing and transport have been described to occur in close association with these structures. However, there is still much debate as to whether or not any of these preparations corresponds to a nuclear framework that exists in vivo. In this article we summarize the most commonly-used methods for obtaining preparations of nuclear frameworks and we also stress the possible artifacts that can be created in vitro during the isolation procedures. Emphasis is placed also on the protein composition of the frameworks as well as on some possible signalling functions that have been recently described to occur in tight association with the nuclear matrix.

Extracellular matrix and platelet function in patients with musculocontractural Ehlers-Danlos syndrome caused by mutations in the CHST14 gene.

We report on a consanguineous, Afghani family with two sisters affected with characteristic facial features, multiple contractures, progressive joint and skin laxity, hemorrhagic diathesis following minor trauma and multisystem fragility-related manifestations suggestive of a diagnosis of musculocontractural Ehlers-Danlos syndrome (EDS). This novel form of connective tissue disorder was recently reported in patients of Japanese, Turkish, and Indian descent who were formerly classified as having EDS type VIB and has now been recognized to be a part of spectrum including patients previously classified as having adducted thumb-clubfoot syndrome. We identified a previously unreported mutation in the CHST14 gene, which codes for the enzyme dermatan 4-O-sulfotransferase. We discuss the prenatal presentation, detailed clinical manifestations, and neurological findings in two sisters with this newly described musculocontractural EDS-CHST14 type. We demonstrate that fibroblasts from one of our patients produce more chondroitin sulfate than normal and show lower than normal deposition of collagens I and II and fibrillin 1-containing microfibrills. These findings suggest that the imbalance in the glycosaminoglycan content in developing tissues might interfere with normal deposition of other extracellular matrix components and ultimately contribute to the development of the phenotype observed in these patients. Furthermore, we ruled out the contribution of intrinsic platelet factors to the bleeding diathesis observed in some affected individuals. © 2012 Wiley Periodicals, Inc.

Early functional differentiation in the chick embryonic disc: interaction between mechanical activity and extracellular matrix

The mechanical behaviour of ectodermal cells in the area opaca and the supracellular organization of fibronectin in the adjacent extracellular matrix were studied in whole chick blastoderms developing in vitro. The pattern of spontaneous mechanical activity and its modification by immunoglobulins against fibronectin were determined using a real-time image-analysis system. The pattern of fibronectin was studied using immunocytochemical techniques. It was found that the ectodermal cells in the area opaca actively develop a radially oriented contraction, which leads to a distension of the area pellucida from which the embryo develops. Abnormally increased tension resulted in perturbations of gastrulation and neurulation. An optimized mechanical equilibrium within the blastoderm seems to be necessary for normal development. Anti-fibronectin antibodies applied to the basal side of the blastoderm led rapidly and reversibly to an increase of tension in the contracted cells. This observation indicates that modifications of the extracellular matrix can be transmitted to cytoskeletal elements within adjacent cells. The extracellular matrix of the area opaca contains fibronectin arranged in radially oriented fibrils. This orientation corresponds to the direction of migration of the mesodermal cells. Interestingly, the radial pattern of fibronectin is found in the regions where the ectodermal cells are contracted and develop radially oriented forces. This observation suggests that the supracellular assembly of the extracellular materials could be influenced by the mechanical activity of adjacent cells. Possible modulations of the supracellular organization of extracellular matrix by other factors, e.g. diffusible metabolites, is also discussed. The presence of characteristically organized extracellular matrix components, of spatially differentiated cell activities and of reciprocal interactions between them makes the young chick blastoderm an excellent system for physiological studies of the coordinated cellular activities that lead to changes in form, complexity and function.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion.

Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA-) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.

Multivariate QST-FST comparisons: a neutrality test for the evolution of the g matrix in structured populations.

Neutrality tests in quantitative genetics provide a statistical framework for the detection of selection on polygenic traits in wild populations. However, the existing method based on comparisons of divergence at neutral markers and quantitative traits (Q(st)-F(st)) suffers from several limitations that hinder a clear interpretation of the results with typical empirical designs. In this article, we propose a multivariate extension of this neutrality test based on empirical estimates of the among-populations (D) and within-populations (G) covariance matrices by MANOVA. A simple pattern is expected under neutrality: D = 2F(st)/(1 - F(st))G, so that neutrality implies both proportionality of the two matrices and a specific value of the proportionality coefficient. This pattern is tested using Flury's framework for matrix comparison [common principal-component (CPC) analysis], a well-known tool in G matrix evolution studies. We show the importance of using a Bartlett adjustment of the test for the small sample sizes typically found in empirical studies. We propose a dual test: (i) that the proportionality coefficient is not different from its neutral expectation [2F(st)/(1 - F(st))] and (ii) that the MANOVA estimates of mean square matrices between and among populations are proportional. These two tests combined provide a more stringent test for neutrality than the classic Q(st)-F(st) comparison and avoid several statistical problems. Extensive simulations of realistic empirical designs suggest that these tests correctly detect the expected pattern under neutrality and have enough power to efficiently detect mild to strong selection (homogeneous, heterogeneous, or mixed) when it is occurring on a set of traits. This method also provides a rigorous and quantitative framework for disentangling the effects of different selection regimes and of drift on the evolution of the G matrix. We discuss practical requirements for the proper application of our test in empirical studies and potential extensions.

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells.

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1 h at 37 degrees C to determine whether or not such an incubation might result in the redistribution of nuclear polypeptides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH4)2SO4 as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37 degrees C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37 degrees C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.

Adherence of Abiotrophia defectiva and Granulicatella species to fibronectin: is there a link with endovascular infections?

During a 6-year period, we isolated three Abiotrophia defectiva, six Granulicatella adiacens and two G. 'para-adiacens' strains from clinical specimens. All A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas the Granulicatella spp. strains were isolated from immunosuppressed patients with primary bacteremia. As the capacity of bacteria to adhere to the host extracellular matrix (ECM) has been implicated in the pathogenesis of endovascular infection, we investigated the ability of A. defectiva and Granulicatella spp. isolates to bind different ECM components immobilized in microtiter plates. Adherence tests showed a strong attachment of A. defectiva strains to fibronectin, whereas Granulicatella spp. strains were not adherent. The poor adherence of Granulicatella spp. strains to the ECM could be correlated with a lower propensity to induce endocarditis.

Junctional adhesion molecule-2 (JAM-2) promotes lymphocyte transendothelial migration.

The molecular mechanisms underlying lymphocyte extravasation remain poorly characterized. We have recently identified junctional adhesion molecule-2 (JAM-2), and have shown that antibodies to JAM-2 stain high endothelial venules (HEVs) within lymph nodes and Peyer patches of adult mice. Here we show that mouse lymphocytes migrate in greater numbers across monolayers of endothelioma cells transfected with JAM-2. The significance of these findings to an understanding of both normal and pathologic lymphocyte extravasation prompted us to clone the human homologue of JAM-2. We herein demonstrate that an anti-JAM-2 antibody, or a soluble JAM-2 molecule, blocks the transmigration of primary human peripheral blood leukocytes across human umbilical vein endothelial cells expressing endogenous JAM-2. Furthermore, we show that JAM-2 is expressed on HEVs in human tonsil and on a subset of human leukocytes, suggesting that JAM-2 plays a central role in the regulation of transendothelial migration.

Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines.

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

Molecular mechanisms and effects of a rasgap-derived peptide on metastatic progression

Cancer is the second leading cause of mortality worldwide. Cancer progression leads to metastasis formation, which accounts for more than ninety percent of cancer-related death. Metastases are more difficult to be surgically removed because of their invasive behavior and shape. In addition, during their transformation journey, they become more and more resistant to anticancer drugs. Significant improvements have been achieved in therapy against cancer in recent years but targeting the metastatic cascade remains the Achilles heel of the cure against cancer. A First step in the metastatic process is the escape of cancer cells from the primary tumor site. This involves an increase in cell motility and the concomitant ability to clear a path through the extracellular matrix. From a therapeutic point of view, inhibition of cell migration is a logical approach to develop anti-metastatic drugs. Our lab previously developed a cell permeable peptide derived from a caspase-3-generaied fragment of the RasGAP protein called TAT-RasGAP317-326. This peptide efficiently and specifically sensitizes cancer cells to chemotherapy- and radiotherapy-induced ceil death, which allows decreasing the anticancer drug doses and eventually their associated side- effects. In the present study we discovered that TAT-RasGAP317.326 also increases cell adhesion which was associated with inhibition of cell migration and invasion into the extracellular matrix. The ability of TAT-RasGAP317.326 to increase ceil adhesion involves the dramatic depolymerization of actin cytoskekton together with redistribution of focal adhesions. We found that the inhibitory effects on migration were mediated by a RhoGAP tumor and metastasis suppressor cailed DLC1 (Deleted in Liver Cancer 1). Moreover. DEC 1 was found to be a direct RasGAP-interacting protein and this interaction requires the RasGAP tryptophan 317 residue, the very first RasGAP residue of TAT-RasGAP317.326. We then evaluated the roie of RasGAP fragments in the in vivo metastatic cascade. We found that breast cancer cells overexpressing the parental RasGAP fragment, to which the TAT-RasGAP317.326 peptide belongs, have a markedly decreased ability to form lung metastases. Unfortunately, we were not able to recapitulate these an ti-metastatic effects when TAT-RasGAP317.326 was injected. However, we later understood that this was due to the fact that TAT-RasGAP317.326 was not properly delivered to the primary tumors. Further work, aimed at better understanding of how TAT-RasGAP317.326 functions, revealed that the ten amino acid TAT-RasGAP317.326 peptide could, be narrowed down to a three amino acid TAT-RasGAP317.329 peptide while keeping its sensitizer activity. In parallel, investigations on the RasGAP-DLCl binding indicated that the arginine linger of the DLC1 GAP domain is required for this interaction, which suggests that TAT-RasGAP317.326 modulates the GAP activity of DLC1. Additional work should be performed to fully elucidate its mechanism of action and render TAT-RasGAP317.326 usable as a tool to fight cancer on two fronts, by improving chemotherapy and preventing metastatic progression. - Le cancer est la deuxième cause de mortalité dans le monde. La formation de métastases est la dernière étape de la progression cancéreuse et représente plus du nonante pour cent des morts induites par le cancer. De par leur morphologie et comportement invasifs, ii est difficile d'avoir recours à la chirurgie pour exciser des métastases. De plus, les cellules cancéreuses en progression deviennent souvent de plus en plus résistantes aux drogues anticancéreuses. Ces dernières années, des avancements significatifs ont contribué à l'amélioration de la lutte contre le cancer. Néanmoins, pouvoir cibler spécifiquement la cascade métastatique demeure cependant le talon d'Achille des thérapies anticancéreuses. Une première étape dans ie processus métastatique est l'évasion des cellules cancéreuses du site de la tumeur primaire. Ceci requiert une augmentation de la motiliié cellulaire couplée à la capacité de se frayer un chemin au sein de la matrice extracelluiaire. D'un point de vue thérapeutique, inhiber la migration cellulaire est une approche attrayante. Notre laboratoire a développé un peptide, nommé TAT-RasGAP317.326 dérivé d'un fragment qui est lui-même le résultat du clivage de la protéine RasGAP par la caspase-3. Ce peptide est capable de pénétrer les cellules cancéreuses et de les sensibiliser spécifiquement à la mort induite par la radiothérapie et la chimiothérapie. La finalité des effets de ce peptide est de pouvoir diminuer les doses des traitements anti-cancéreux et donc des effets secondaires qu'ils engendrent. Dans cette étude, nous avons découvert que TAT-RasGAP317.326 augmente l'adhésion des cellules et inhibe la migration cellulaire ainsi que l'invasion des cellules à travers une matrice extracellulaire. La capacité de TAT-RasGAP317.326 à induire l'adhésion repose sur ia dépolymérisation du cytosquelette d'actine associée à une redistribution des points d'ancrage cellulaire. Nous avons découvert que l'inhibition de ia migration par TAT-RasGAP317.326 nécessitait la présence d'un suppresseur de tumeur et de métastases appelé DLC1 (Deleted in Liver Cancer l), qui par ailleurs s'avère aussi être une protéine RhoGAP. De plus, nous avons aussi trouvé que DLC1 était un partenaire d'interaction de RasGAP et que cette interaction s'effectuait via l'acide aminé tryptophane 317 de RasGAP. qui s'avère être le premier acide aminé du peptide TAT-RasGAP317.326. Nous avons ensuite évalué le rôle joué par certains fragments de RasGAP dans le processus de métastatisation. Dans ce contexte, des cellules de cancer du sein qui sur-expriment un fragment de RasGAP contenant la séquence TAT-RasGAP317.326 ont vu leur potentiel métastatique diminuer drastiquerment. Malheureusement, aucun effet anti-métastatique n'a été obtenu après injection de TAT-RasGAP317.326 dans les souris. Cependant, nous avons réalisé rétrospectivement que TAT-RasGAP317.326 n'était pas correctement délivré à la tumeur primaire, ce qui nous empêche de tirer des conclusions sur le rôle anti-métastatique de ce peptide. La suite de cette étude visant à mieux comprendre comment TAT-RasGAP317.326 agit, a mené à la découverte que les dix acides aminés de TAT-RasGAP317.326 pouvaient être réduits à trois acides aminés, TAT-RasGAP317.329, tout en gardant l'effet sensibilisateur à la chimiothérapie. En visant à élucider le mode d'interaction entre RasGAP et DLC1, nous avons découvert qu'un acide aminé nécessaire à l'activité GAP de DLC1 était requis pour lier RasGAP, ce qui laisse présager que TAT-RasGAp317.32c, module i'activité GAP de DLC1. Des travaux supplémentaires doivent encore être effectués pour complètement élucider les mécanismes d'action de TAT-RasGAP317.326 et afin de pouvoir l'utiliser comme un outil pour combattre le cancer sur deux fronts, en améliorant les chimiothérapies et en inhibant la formation de métastases.

Urokinase receptor (uPAR, CD87) is a platelet receptor important for kinetics and TNF-induced endothelial adhesion in mice.

BACKGROUND: Urokinase plasminogen activator receptor (uPAR, CD87) is a widely distributed 55-kD, glycoprotein I-anchored surface receptor. On binding of its ligand uPA, it is known to increase leukocyte adhesion and traffic. Using genetically deficient mice, we explored the role of uPAR in platelet kinetics and TNF-induced platelet consumption. METHODS AND RESULTS: Anti-uPAR antibody stained platelets from normal (+/+) but not from uPAR-/- mice, as seen by fluorescence-activated cell sorter analysis. 51Cr-labeled platelets from uPAR-/- donors survived longer than those from +/+ donors when injected into a +/+ recipient. Intratracheal TNF injection induced thrombocytopenia and a platelet pulmonary localization, pronounced in +/+ but absent in uPAR-/- mice. Aprotinin, a plasmin inhibitor, decreased TNF-induced thrombocytopenia. TNF injection markedly reduced the survival and increased the pulmonary localization of 51Cr-labeled platelets from +/+ but not from uPAR-/- donors, indicating that it is the platelet uPAR that is critical for their response to TNF. As seen by electron microscopy, TNF injection increased the number of platelets and polymorphonuclear neutrophils (PMNs) in the alveolar capillaries of +/+ mice, whereas in uPAR-/- mice, platelet trapping was insignificant and PMN trapping was slightly reduced. Platelets within alveolar capillaries of TNF-injected mice were activated, as judged from their shape, and this was evident in +/+ but not in uPAR-/- mice. CONCLUSIONS: These results demonstrate for the first time the critical role of platelet uPAR for kinetics as well as for activation and endothelium adhesion associated with inflammation.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.

Effective empirical treatment is of paramount importance to improve the outcome of patients with Staphylococcus aureus bacteraemia. We aimed to evaluate a PCR-based rapid diagnosis of methicillin resistance (GeneXpert MRSA) after early detection of S. aureus bacteraemia using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients with a first episode of S. aureus bacteraemia identified using MALDI-TOF MS were randomized in a prospective interventional open study between October 2010 and August 2012. In the control group, antibiotic susceptibility testing was performed after MALDI-TOF MS identification on blood culture pellets. In the intervention group, a GeneXpert MRSA was performed after S. aureus identification. The primary outcome was the performance of GeneXpert MRSA directly on blood cultures. We then assessed the impact of early diagnosis of methicillin resistance on the empirical treatment. In all, 197 episodes of S. aureus bacteraemia were included in the study, of which 106 were included in the intervention group. Median time from MALDI-TOF MS identification to GeneXpert MRSA result was 97 min (range 25-250). Detection of methicillin resistance using GeneXpert MRSA had a sensitivity of 99% and a specificity of 100%. There was less unnecessary coverage of MRSA in the intervention group (17.1% versus 29.2%, p 0.09). GeneXpert MRSA was highly reliable in diagnosing methicillin resistance when performed directly on positive blood cultures. This could help to avoid unnecessary prescriptions of anti-MRSA agents and promote the introduction of earlier adequate coverage in unsuspected cases.

Unavailability of CD147 leads to selective erythrocyte trapping in the spleen.

Adhesive interactions with stromal cells and the extracellular matrix are essential for the differentiation and migration of hematopoietic progenitors. In the erythrocytic lineage, a number of adhesion molecules are expressed in the developing erythrocytes and are thought to play a role in the homing and maturation of erythrocytic progenitors. However, many of these molecules are lost during the final developmental stages leading to mature erythrocytes. One of the adhesion molecules that remains expressed in mature, circulating erythrocytes is CD147. This study shows that blockade of this molecule on the cell surface by treatment with F(ab')(2) fragments of anti-CD147 monoclonal antibody disrupts the circulation of erythrocytes, leading to their selective trapping in the spleen. Consequently, mice develop an anemia, and de novo, erythropoietin-mediated erythropoiesis in the spleen. In contrast, these changes were not seen in mice similarly treated with another antierythrocyte monoclonal antibody with a different specificity. These results suggest that the CD147 expressed on erythrocytes likely plays a critical role in the recirculation of mature erythrocytes from the spleen into the general circulation. (Blood. 2001;97:3984-3988)