Organisation and role of actin microfilaments during cellular growth and morphogenesis in the moss Physcomitrella patens

ABSTRACT The network of actin cytoskeleton is composed of actin filaments (F-actin) that are made by polymerisation of actin monomers and actin binding proteins. It is required for growth and morphogenesis of eukaryotic cells. The labelling of F-actin with constitutively expressed GFP-Talin (Kost et al., 1998) reveals the organisation of cellular actin networks in plants. Due to the lack of information on actin cytoskeleton through gametophytic development of the model moss plant Physcornitrella patens, stable transgenic lines overexpressing GFP-Talin were generated to detect F-actin structures. It is shown that the 35S promoter driven expression is not suitable for F-actin labelling in all cells. When it is replaced by the inducible heat-shock promoter Gmhsp17.3 from soybean, one hour mild heat stress at 37°C followed by recovery at 25°C is enough to induce efficient and transient labelling in all tissues without altering cellular morphology. The optimal observations of F-actin structures at different stages of moss development can be done between 12-18 hours after the induction. By using confocal microscopy, we demonstrate that stellated actin arrays were densely accumulated at the growing tip in regenerating protoplasts, apical protonemal cells and rhizoids and connected with a fine dispersed F-actin mesh. Following three-dimensional growth, the cortical star-like structures are widespread in the meristematic cells of developing bud and young gametophores. On the contrary, undulating networks of actin cables are found at the final stage of cell differentiation. During redifferentiation of mature leaf cells into protonemal filaments the rather stagnant web of actin cables is replaced by diffuse actin meshwork. In eukaryotes, nucleation of the actin monomers prior to their polymerization is driven by the seven-subunit ARP2/3 complex and formins. We cloned the gene encoding the ARP3 subunit of P. patens and generated arp3 mutants of the moss through gene disruption. The knockout of ARP3 affects the elongation of chloronemal cells and blocks further differentiation of caulonemal cells and rhizoids, and the gametophores are slightly stunted compared to wild-type. The arp mutants were created in the heat-shock inducible GFP-Talin strains allowing us to visualise a disorganised actin network and a lack of star-like actin cytoskeleton arrays. We conclude that ARP2/3 dependent nucleation of actin filaments is critical for the growth of filamentous cells, which in turn influences moss colonization. In complementation assays, the overexpression of Physcomitrella and Arab idopsis ARP3 genes in the moss arp3 mutant results in full recovery of wild type phenotype. In contrast the ARP3 subunit of fission yeast is not able to complement the moss arp3 mutant of moss indicating that regulation of the ARP2/3 dependent actin nucleation diverged in different kingdoms. RESUME Le réseau d'actine est composé de filaments de F-actine et d'un ensemble de protéines s'y attachant (Actin binding proteins). Le réseau d'actine est nécessaire à la croissance et à la morphogenèse de toutes les cellules eucaryotes. Chez les plantes, le marquage ainsi que l'étude de l'organisation du réseau d'actine ont été réalisés en utilisant une fusion GFP-Talin (Kost et al., 1998) exprimée sous le control d'un promoteur constitutif. Afin d'étudier les structures F-actine dans les cellules de Physcomitrella Patens et pour combler le manque d'information sur le développement des gamétophores, des lignées transgéniques stables surexprimant GFP-Talin ont été crées. Nous avons démontré que l'utilisation du promoteur 35S est inadéquate pour le marquage complet et homogène des filaments d'actine dans toutes les cellules de P. patens. Par contre, l'utilisation du promoteur inductible Gmhsp17.3 nous a permis de réaliser un marquage transitoire et général dans tous les tissus de la mousse. Une heure de choc thermique à 37°C suivis d'un temps de récupération de 12-18h à 25°C sont les conditions optimales (sans dommages cellulaires) pour l'observation des structures F-actine à différentes étapes de développement de la mousse. En utilisant la microscopie confocale, nous avons observé l'existence de structures F-actine accumulées en forme d'étoiles. Ces structures, qui sont liées au réseau de microfilaments d'actine, ont été observées dans les protoplastes en régénération, les cellules des protonema apicales ainsi que dans les rhizoïdes. En suivant la croissance tridimensionnelle, ces structures en étoiles ont été observées dans les cellules meristématiques des bourgeons et des jeunes gamétophores. Par contre, dans les cellules différentiées ces structures laissent place à des réseaux de câbles épais. Nous avons également remarqué que durant la redifferentiation des cellules foliaires le réseau de câbles de F-actine est remplacé par un réseau de F-actine diffus. Dans les cellules eucaryotes, la nucléation des filaments d'actirie précédant leur polymérisation est contrôlé par sept sous unités du complexe ARP2/3 et par des formines. Nous avons isolé le gène codant pour la sous unité ARP3 de P. patens et nous avons crée des mutants arp3 par intégration ciblée (Knockout). L'élongation des cellules chloronema est clairement affectée dans les mutants arp3. La différentiation des caulonemata et des rhizoïdes est bloquée et les gametophores sont légèrement plus courts comparé au type sauvage. A fin d'étudier l'organisation des filaments d'actines dans les mutants arp3, nous avons aussi réalisé un arp3-knockout dans la lignée Hsp-GFP-Talin. La nouvelle lignée générée nous a permis de visualiser une désorganisation du réseau d'actine et une absence complète de structures de F-actine accumulée en forme d'étoiles. Les résultats obtenus nous amènent à conclure que la nucléation (ARP2/3 dépendante) des filaments d'actine est indispensable à la croissance des cellules filamenteuses. Par conséquent, les filaments d'actine semblent avoir un rôle dans la colonisation des milieux par les mousses. Nous avons également procédé à des essais de complémentation du mutant arp3. La surexpression des gènes ARP3 de Physcomitrella et d'Arabidopsis dans les cellules du mutant arp3 rétabli complètement le phénotype WT. Par contre, le gène ARP3 des levures n'est pas suffisant pour complémenter la même mutation dans les cellules de mousses. Ce résultat démontre que les mécanismes de régulation de la nucléation des filaments d'actine (ARP2/3 dépendante) sont différents entre les différents groupes d'eucaryotes.

Valoración quirúrgica del 5º radio

Este trabajo hace un repaso de la biomecánica y patomecánica del pie, además de describir la etiologíay la clínica del quintus varus y del 5º dedo en martillo. El autor describe un test valorativo pre- y post-quirúrgico para el 5º radio, así como las técnicasquirúrgicas de 5º radio habituales.

Soporte funcional del primer radio: revisión de varios casos clínicos

Los autores presentan una clasificación más actual de las patologías del pie, basándose en tres aspectos: morfológico, funcional y degenerativo. Esta nueva clasificación entendemos que reemplazaría a las que se han venido utilizando hasta el momento las cuales a menudo no son útiles para los podólogos. Asimismo en este artículo se describe el proceso de aplicación del soporte funcional del primer radio, las características de los materiales utilizados en su confección y varios casos clínicos, de diferente etiología a los cuales se le ha aplicado este tipo de soporte.

Deformación en flexión plantar del primer radio

La posición del primer radio tanto desde el plano frontal como desde el plano sagital, es de vital importancia para la consecución de una correcta y adecuada marcha, así como para mantener la estructura anatómica del pie durante la bipedestación estática. La deformidad en flexión plantar del primer radio modifica estos parámetros creando mecanismos compensatorios que pueden afectar a otras unidades funcionales articulares, tanto del mediopié como del retropié, lo que puede dar lugar a un patrón de metatarsalgia de difícil valoración al tener que contemplar y analizar varios diagnósticos y sus respectivos patrones mecánicos: el equino de columna lateral, el retropié varo compensado y el primer radio plantarflexionado flexible. Se presenta un caso clínico para su análisis conjunto.

Molecular crime and cellular punishment: active detoxification of misfolded and aggregated proteins in the cell by the chaperone and protease networks.

Labile or mutation-sensitised proteins may spontaneously convert into aggregation-prone conformations that may be toxic and infectious. This hazardous behavior, which can be described as a form of "molecular criminality", can be actively counteracted in the cell by a network of molecular chaperone and proteases. Similar to law enforcement agents, molecular chaperones and proteases can specifically identify, apprehend, unfold and thus neutralize "criminal" protein conformers, allowing them to subsequently refold into harmless functional proteins. Irreversibly damaged polypeptides that have lost the ability to natively refold are preferentially degraded by highly controlled ATP-consuming proteases. Damaged proteins that escape proteasomal degradation can also be "incarcerated" into dense amyloids, "evicted" from the cell, or internally "exiled" to the lysosome to be hydrolysed and recycled. Thus, remarkable parallels exist between molecular and human forms of criminality, as well as in the cellular and social responses to various forms of crime. Yet, differences also exist: whereas programmed death is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom chosen by lawless societies. Significantly, there is no cellular equivalent for the role of familial care and of education in general, which is so crucial to the proper shaping of functional persons in the society. Unlike in the cell, humanism introduces a bias against radical solutions such as capital punishment, favouring crime prevention, reeducation and social reinsertion of criminals.

Targeting Cancer Stem-like Cells as an Approach to Defeating Cellular Heterogeneity in Ewing Sarcoma.

Plasticity in cancer stem-like cells (CSC) may provide a key basis for cancer heterogeneity and therapeutic response. In this study, we assessed the effect of combining a drug that abrogates CSC properties with standard-of-care therapy in a Ewing sarcoma family tumor (ESFT). Emergence of CSC in this setting has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. In the present work, primary ESFT from four patients containing CD133(+) CSC subpopulations ranging from 3% to 17% of total tumor cells were subjected to treatment with enoxacin, doxorubicin, or both drugs. Primary ESFT CSC and bulk tumor cells displayed divergent responses to standard-of-care chemotherapy and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin, which enhances miRNA maturation by stimulating TARBP2 function, induced apoptosis but only in ESFT spheres. In combination, the two drugs markedly depleted CSCs and strongly reduced primary ESFTs in xenograft assays. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low-toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. Conclusions In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.

Flow cytometry in the analysis of cellular populations

Flow cytometry has become a valuable tool in cell biology. By analyzing large number of cells individually using light-scatter and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate cells and to distinguish among different cell stages and structures using multiple staining. In addition to high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of researchers in this technique. This chapter gives an overview of the principles of flow cytometry and examples of the application ofthe technique.

Cellular source and mechanisms of high transcriptome complexity in the Mammalian testis.

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexity in major mammalian organs have been scarce. Here, using extensive RNA-seq data, we show that transcription of the genome is substantially more widespread in the testis than in other organs across representative mammals. Furthermore, we reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding and long noncoding RNA genes but also poorly conserves intergenic sequences, suggesting that it may not be of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data suggest that this prevalent transcription, which most likely promoted the birth of new genes during evolution, is facilitated by an overall permissive chromatin in these germ cells that results from extensive chromatin remodeling.

Síndrome del dolor regional complejo tipo I en las fracturas del extremo distal del radio

Introducción: Las fracturas distales de radio (FDR) presentan diferentes resultadosfuncionales siendo desfavorables entre el 24% y el 31% de los casos. Una de lascomplicaciones más frecuentes es el síndrome del dolor regional complejo tipo I(SDRC-I), cuyo origen es desconocido. Sólo el 20-30% de los pacientes recuperarán sugrado de funcionalidad anterior.Objetivos: Estimar la prevalencia de SDRC-I tras una FDR de acuerdo a cuatro tipos detratamiento.Métodos: estudio de una serie de casos retrospectivos. La población de referencia hansido los 391 trabajadores afiliados a una Mutua en Cataluña y diagnosticados deFDR tras una lesión por accidente laboral durante lo años 2008-2010.Los casos han sido trabajadores accidentados que tras una FDR desarrollaron el SDRCI.Como variable dependiente se ha utilizado el diagnóstico de SDRC-I; como variablesindependientes el tipo de tratamiento, la edad, el sexo, la ocupación y el mecanismo dela lesión.Resultados: el 19,7% de la muestra ha desarrollado SDRC-I, siendo esta prevalenciasuperior en los pacientes intervenidos mediante la técnica de tracción bipolar o conagujas de Kirschner; 29,17% y 30,56% respectivamente, que para aquellos en los quese ha realizado un tratamiento conservador o una intervención quirúrgica con placa en Ty tornillos; 15,92% y 15,79%.Conclusiones El tratamiento conservador y la técnica quirúrgica de fijación con placaen T y tornillos han dado una menor prevalencia de SDRC-I.Los mecanismos de lesión asociados a una mayor fuerza presentan fracturas másinestables y requieren un tratamiento quirúrgico. Los tratamientos conservadores sonlos más usados en edades mayores relacionados con fracturas estables.A nivel clínico se extrae una recomendación profiláctica con Vitamina C para evitar laaparición de SDRC-I sobre los pacientes de edad comprendida entre los 36 y los 55años, con mecanismo lesional de caída o caída desde altura, intervenidos con agujas de Kirschner o tracción bipolar.