RESUMEN
The adaptive in vivo mechanisms underlying the switch in Salmonella enterica lifestyles from the infectious form to a dormant form remain unknown. We employed Caenorhabditis elegans as a heterologous host to understand the temporal dynamics of Salmonella pathogenesis and to identify its lifestyle form in vivo. We discovered that Salmonella exists as sessile aggregates, or in vivo biofilms, in the persistently infected C. elegans gut. In the absence of in vivo biofilms, Salmonella killed the host more rapidly by actively inhibiting innate immune pathways. Regulatory cross-talk between two major Salmonella pathogenicity islands, SPI-1 and SPI-2, was responsible for biofilm-induced changes in host physiology during persistent infection. Thus, biofilm formation is a survival strategy in long-term infections, as prolonging host survival is beneficial for the parasitic lifestyle.
Asunto(s)
Biopelículas , Caenorhabditis elegans/microbiología , Inmunidad Innata/fisiología , Salmonella/fisiología , Animales , Biomarcadores/metabolismo , Caenorhabditis elegans/crecimiento & desarrollo , Intestinos/parasitología , Larva/microbiología , Salmonella/metabolismo , Salmonella/patogenicidad , VirulenciaRESUMEN
Syncytial architecture is an evolutionarily-conserved feature of the germline of many species and plays a crucial role in their fertility. However, the mechanism supporting syncytial organization is largely unknown. Here, we identify a corset-like actomyosin structure within the syncytial germline of Caenorhabditis elegans, surrounding the common rachis. Using laser microsurgery, we demonstrate that actomyosin contractility within this structure generates tension both in the plane of the rachis surface and perpendicular to it, opposing membrane tension. Genetic and pharmacological perturbations, as well as mathematical modeling, reveal a balance of forces within the gonad and show how changing the tension within the actomyosin corset impinges on syncytial germline structure, leading, in extreme cases, to sterility. Thus, our work highlights a unique tissue-level cytoskeletal structure, and explains the critical role of actomyosin contractility in the preservation of a functional germline.
Asunto(s)
Actomiosina/metabolismo , Células Germinativas/metabolismo , Células Gigantes/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Corriente Citoplasmática , Gónadas/metabolismo , Modelos Biológicos , Miosinas/metabolismoRESUMEN
Cell fate determination in the early mammalian embryo is regulated by multiple mechanisms. Recently, genes involved in vesicular trafficking have been shown to play an important role in cell fate choice, although the regulation of their expression remains poorly understood. Here we demonstrate for the first time that multiple endocytosis associated genes (EAGs) are repressed through a novel, dual mechanism in mouse embryonic stem cells (mESCs). This involves the action of the Polycomb Repressive Complex, PRC2, as well as post-transcriptional regulation by the ESC-specific cell cycle-regulating (ESCC) family of microRNAs. This repression is relieved upon differentiation. Forced expression of EAGs in mESCs results in a decrease in pluripotency, highlighting the importance of dual repression in cell fate regulation. We propose that endocytosis is critical for cell fate choice, and dual repression may function to tightly regulate levels of endocytic genes.
Asunto(s)
Endocitosis/genética , MicroARNs/genética , Células Madre Embrionarias de Ratones/citología , Proteínas del Grupo Polycomb/metabolismo , Animales , Ratones , Transcripción GenéticaRESUMEN
Classical cadherins are well known for their essential function in mediating cell-cell adhesion via their extra-cellular cadherin domains and intra-cellular connections to the actin cytoskeleton [1-3]. There is evidence, however, of adhesion-independent cadherin clusters existing outside of cell-cell junctions [4-6]. What function, if any, these clusters have is not known. HMR-1, the sole classical cadherin in Caenorhabditis elegans, plays essential roles during gastrulation, blastomere polarity establishment, and epidermal morphogenesis [7-11]. To elucidate the physiological roles of non-junctional cadherin, we analyzed HMR-1 in the C. elegans zygote, which is devoid of neighbors. We show that non-junctional clusters of HMR-1 form during the one-cell polarization stage and associate with F-actin at the cortex during episodes of cortical flow. Non-junctional HMR-1 clusters downregulate RHO-1 activity and inhibit accumulation of non-muscle myosin II (NMY-2) at the anterior cortex. We found that HMR-1 clusters impede cortical flows and play a role in preserving the integrity of the actomyosin cortex, preventing it from splitting in two. Importantly, we uncovered an inverse relationship between the amount of HMR-1 at the cell surface and the rate of cytokinesis. The effect of HMR-1 clusters on cytokinesis is independent of their effect on NMY-2 levels, and is also independent of their extra-cellular domains. Thus, in addition to their canonical role in inter-cellular adhesion, HMR-1 clusters regulate RHO-1 activity and NMY-2 level at the cell surface, reinforce the stability of the actomyosin cortex, and resist its movement to influence cell-shape dynamics.
Asunto(s)
Actomiosina/metabolismo , Cadherinas/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Cigoto/metabolismo , Actinas/metabolismo , Actomiosina/genética , Animales , Cadherinas/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Adhesión Celular , Citocinesis , Regulación de la Expresión Génica , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.
Asunto(s)
Cadherinas/metabolismo , Uniones Intercelulares , Biofisica , Línea Celular , Citoesqueleto/metabolismo , Humanos , Cinética , Membrana Dobles de Lípidos , Transducción de SeñalRESUMEN
Adherens junctions, broadly defined as attachment sites in which cadherin adhesion receptors connect the actin cytoskeletons of neighboring animal cells, are multi-tasking by nature. In addition to mediating cell-cell adhesion and providing the tissue with mechanical continuity and barrier function, they maintain polarity, are sites of mechanosensing and signaling, and they regulate actomyosin dynamics and can thus generate forces to drive morphogenesis. Here we propose that the key to performing such diverse tasks is the integration within the cadherin adhesome of functional modules that evolved independently to perform other duties within the cell, and we discuss three such functional modules: force transmission, actin dynamics regulation, and contractile force generation. We compare each module to a more ancient cellular structure with similar function, identify shared components, and speculate on how the module was integrated into the cadherin adhesome.
Asunto(s)
Uniones Adherentes/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular , Humanos , Transducción de SeñalRESUMEN
In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly.
Asunto(s)
Actinas/metabolismo , Actomiosina/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , CitocinesisRESUMEN
Correct positioning of the cell division machinery is crucial for genomic stability and cell fate determination. The fission yeast Schizosaccharomyces pombe, like animal cells, divides using an actomyosin ring and is an attractive model to study eukaryotic cytokinesis. In S. pombe, positioning of the actomyosin ring depends on the anillin-related protein Mid1p. Mid1p arrives first at the medial cortex and recruits actomyosin ring components to node-like structures, although how this is achieved is unknown. Here we show that the IQGAP-related protein Rng2p, an essential component of the actomyosin ring, is a key element downstream of Mid1p. Rng2p physically interacts with Mid1p and is required for the organization of other actomyosin ring components into cortical nodes. Failure of localization of Rng2p to the nodes prevents medial retention of Mid1p and leads to actomyosin ring assembly in a node-independent manner at nonmedial locations. We conclude that Mid1p recruits Rng2p to cortical nodes at the division site and that Rng2p, in turn, recruits other components of the actomyosin ring to cortical nodes, thereby ensuring correct placement of the division site.
Asunto(s)
Actomiosina/metabolismo , Proteínas de Ciclo Celular/fisiología , División Celular/fisiología , Proteínas Activadoras de GTPasa/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/fisiología , Sustitución de Aminoácidos , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Inmunoprecipitación , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación/genética , Proteínas de Schizosaccharomyces pombe/genética , Análisis de Secuencia de ADN , Imagen de Lapso de TiempoRESUMEN
A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random mutations are introduced precisely into the gene of interest, together with the reconstitution of the latter selective marker by homologous recombination. This approach was successfully tested for several genes in the fission yeast Schizosaccharomyces pombe. It circumvents the problems encountered with other methods and should be adaptable to any organism that incorporates exogenous DNA by homologous recombination.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genética Microbiana/métodos , Mutagénesis , Schizosaccharomyces/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Schizosaccharomyces/fisiologíaRESUMEN
The expression of three BH3-only proteins, Bad, Bid and Bim, were knocked down in HEK 293 cells using vectors that express corresponding siRNAs. When cultured in the presence of 10% (v/v) serum and a diminished glucose/nutrients environment, cells lacking any one of these BH3-only proteins showed delayed cell death compared to wild type cells. Remarkably, the culture life of Bad (-) cells was extended for an additional 5 days compared to WT HEK 293 cells. In the absence of serum, the suppression of either Bad, Bim or Bid expression delayed cell death under several stress conditions. Results presented in this paper offer an insight into the functions of BH3-only proteins in mediating the death signals under different stress conditions.