Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes

Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an “antiadhesive” matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.

Keratocytes Generate Traction Forces in Two PhasesV⃞

Forces generated by goldfish keratocytes and Swiss 3T3 fibroblasts have been measured with nanonewton precision and submicrometer spatial resolution. Differential interference contrast microscopy was used to visualize deformations produced by traction forces in elastic substrata, and interference reflection microscopy revealed sites of cell-substratum adhesions. Force ranged from a few nanonewtons at submicrometer spots under the lamellipodium to several hundred nanonewtons under the cell body. As cells moved forward, centripetal forces were applied by lamellipodia at sites that remained stationary on the substratum. Force increased and abruptly became lateral at the boundary of the lamellipodium and the cell body. When the cell retracted at its posterior margin, cell-substratum contact area decreased more rapidly than force, so that stress (force divided by area) increased as the cell pulled away. An increase in lateral force was associated with widening of the cell body. These mechanical data suggest an integrated, two-phase mechanism of cell motility: (1) low forces in the lamellipodium are applied in the direction of cortical flow and cause the cell body to be pulled forward; and (2) a component of force at the flanks pulls the rear margins forward toward the advancing cell body, whereas a large lateral component contributes to detachment of adhesions without greatly perturbing forward movement.

Characterization of Cell–Matrix Adhesion Requirements for the Formation of Fascin Microspikes

Cell adhesion to thrombospondin-1 (TSP-1) correlates with assembly of cell–substratum contact structures that contain fascin microspikes. In this analysis, cell-matrix requirements for assembly of fascin microspikes were examined in detail. In six cell lines, cell spreading on a TSP-1 substratum correlated with expression of fascin protein and formation of fascin microspikes. Microspikes were not formed by H9c2 cells adherent on fibronectin, vitronectin, collagen IV, or platelet factor 4. However, both fascin microspikes and focal contacts were assembled by cells adherent on laminin-1. Using mixed substrata containing different proportions of TSP-1, and fibronectin, fascin microspike formation by H9c2 and C2C12 cells was found to be reduced on substrata containing 25% fibronectin and abolished on substrata containing 75% fibronectin. Adhesion to intermediate mixtures of TSP-1 and fibronectin resulted in coassembly of fascin microspikes and focal contacts, colocalization of fascin with actin stress fiber bundles and altered distributions of β1 integrins, cortical α-actinin, and tropomyosin. In cells adherent on 50% TSP-1:50% fibronectin, GRGDSP peptide treatment decreased focal contact assembly and altered cytoskeletal organization but did not inhibit microspike assembly. Treatment with chondroitin sulfate A or p-nitrophenol β-d-xylopyranoside decreased microspike formation and modified cytoskeletal organization but did not inhibit focal contact formation. In polarized migratory and postmitotic C2C12 cells, fascin microspikes and ruffles were localized at leading edges and TSP matrix deposition was also concentrated in this region. Depletion of matrix TSP by heparin treatment correlated with decreased microspike formation and cell motility. Thus, the balance of adhesive receptors ligated at the cell surface during initial cell–matrix attachment serves to regulate the type of substratum adhesion contact assembled and subsequent cytoskeletal organization. A role for fascin microspikes in cell motile behavior is indicated.

Integrin-mediated Adhesion Regulates Cell Polarity and Membrane Protrusion through the Rho Family of GTPasesV⃞

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through α5β1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.

How amoeboids self-organize into a fruiting body: Multicellular coordination in Dictyostelium discoideum

When individual amoebae of the cellular slime mold Dictyostelium discoideum are starving, they aggregate to form a multicellular migrating slug, which moves toward a region suitable for culmination. The culmination of the morphogenesis involves complex cell movements that transform a mound of cells into a globule of spores on a slender stalk. The movement has been likened to a “reverse fountain,” whereby prestalk cells in the upper part form a stalk that moves downwards and anchors to the substratum, while prespore cells in the lower part move upwards to form the spore head. So far, however, no satisfactory explanation has been produced for this process. Using a computer simulation that we developed, we now demonstrate that the processes that are essential during the earlier stages of the morphogenesis are in fact sufficient to produce the dynamics of the culmination stage. These processes are cAMP signaling, differential adhesion, cell differentiation, and production of extracellular matrix. Our model clarifies the processes that generate the observed cell movements. More specifically, we show that periodic upward movements, caused by chemotactic motion, are essential for successful culmination, because the pressure waves they induce squeeze the stalk downwards through the cell mass. The mechanisms revealed by our model have a number of self-organizing and self-correcting properties and can account for many previously unconnected and unexplained experimental observations.

Tissue spreading on implantable substrates is a competitive outcome of cell–cell vs. cell–substratum adhesivity

While the interactions of cells with polymeric substrata are widely studied, the influence of cell–cell cohesivity on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or “tug-of-war” between cell–cell and cell–substratum adhesions. We have generated both a “library” of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesivity. Cell–substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell–cell cohesivity was varied through the expression of the homophilic cohesion molecules N- and R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell–cell cohesivity on a series of copolymeric substrata having different levels of cell–substratum adhesivity. In these experiments, cell–cell cohesivity was measured by tissue surface tensiometry, and cell–substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescence-labeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell–cell cohesivity dramatically slowed the spreading rate of cell aggregates.

Direct observation of extension and retraction of type IV pili

Type IV pili are thin filaments that extend from the poles of a diverse group of bacteria, enabling them to move at speeds of a few tenths of a micrometer per second. They are required for twitching motility, e.g., in Pseudomonas aeruginosa and Neisseria gonorrhoeae, and for social gliding motility in Myxococcus xanthus. Here we report direct observation of extension and retraction of type IV pili in P. aeruginosa. Cells without flagellar filaments were labeled with an amino-specific Cy3 fluorescent dye and were visualized on a quartz slide by total internal reflection microscopy. When pili were attached to a cell and their distal ends were free, they extended or retracted at rates of about 0.5 μm s−1 (29°C). They also flexed by Brownian motion, exhibiting a persistence length of about 5 μm. Frequently, the distal tip of a filament adsorbed to the substratum and the filament was pulled taut. From the absence of lateral deflections of such filaments, we estimate tensions of at least 10 pN. Occasionally, cell bodies came free and were pulled forward by pilus retraction. Thus, type IV pili are linear actuators that extend, attach at their distal tips, exert substantial force, and retract.

Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan.

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.

Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor.

Cell adhesion has a fundamental role in the proliferation and motility of normal cells and the metastasis of tumor cells. To identify signaling pathways activated by the adherence of tumor cells, we analyzed the tyrosine phosphorylation of proteins in mouse melanoma cells before and after attachment to substrata. We discovered that cellular adherence activated the protein-tyrosine kinase of the cell surface receptor Met, whose ligand is hepatocyte growth factor and scatter factor. The activation was exceedingly prompt, affected the great majority of Met in the cells, persisted so long as the cells remained adherent, and was rapidly reversed as soon as the cells were detached from substrata. Activation of Met required that cells be adherent but not that they spread on the substratum, and it occurred in the absence of any apparent ligand for the receptor. Ligand-independent activation of Met occurred in several varieties of tumor cells but not in normal endothelial cells that express the receptor. The activation of Met described here may represent a means by which cells respond to mechanical as opposed to biochemical stimuli.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Remoção de metil paration e atrazina em reatores de bancada com fungos

Neste estudo foi avaliada a remoção de metil paration - inseticida e atrazina - herbicida presentes em água, em reatores de bancada, com fungos. A pesquisa foi dividida em quatro etapas: operação em batelada com metil paration e micélio fúngico, com e sem glicose; teste de toxicidade em placas com Aspergillus niger AN400; operação em batelada com os pesticidas atrazina e metil paration e esporos de Aspergillus niger AN400, com e sem glicose; e operação em reatores de leito fixo e fluxo ascendente. Na primeira etapa, a remoção de metil paration foi de 97% nos reatores sem glicose e 94% nos reatores com glicose com 32 dias de reação. Na operação em batelada, com esporos, um modelo cinético de primeira ordem representou bem a velocidade de decaimento de metil paration nesta fase, principalmente, nos reatores que continham glicose. Para os experimentos sem adição de glicose, a constante cinética foi de 0,063 ± 0,005/h, enquanto que para os experimentos com glicose a constante foi de 0,162 ± 0,014/h. Dessa forma, a adição de glicose resultou efetivamente em aumento na velocidade de conversão do inseticida. Na fase experimental, com atrazina e esporos de Aspergillus niger AN400, a presença do substrato primário (glicose) não teve influência na remoção de atrazina, sendo que os percentuais de remoção foram muito próximos aos percentuais encontrados nos reatores sem glicose. O estudo cinético, nessa fase com atrazina e esporos, revelou que para os experimentos sem a adição de glicose, o valor da velocidade de conversão de atrazina (RATZo) foi de 0,023/d, enquanto que para os experimentos com glicose (RATZo) foi 0,022/d. Portanto, a adição de glicose parece não ter influenciado significativamente a velocidade de remoção do herbicida por Aspergillus niger AN400. O teste de toxicidade demonstrou que metil paration e atrazina não inibiram o crescimento do fungo nas várias concentrações testadas, inclusive nas mais elevadas, que foram 60 mg/L e 25 mg/L para metil paration e atrazina, respectivamente. No reator de leito fixo a remoção de metil paration foi de 40% com 12 h de tempo de detenção hidráulica, e 0,5 g glicose/L. Porém, quando a concentração de glicose foi duplicada a remoção de metil paration diminuiu para 35%. Neste reator o pH se manteve na faixa ácida 3,4 a 5,2, considerada ideal para os fungos. Os resultados encontrados mostram a viabilidade dos fungos para remoção desses pesticidas, considerados persistentes no ambiente.

Culture and World Vision: The Cognitive Mythical Mode

Culture is the system of knowledge, from whose meanings the human being screened and selected their understanding of Reality in the broad sense, and interprets and regulates the facts and data of social behavior. In this sense, culture is a program for social action and acting in humans during the process of socialization and social interaction. The meanings of each culture are the cumulative product of collective and individual thinking, in ecological economic, social and political specific situations, so are the expression of each particular cultural historical conjuncture. Moreover, the universal cognitive structure for the apprehension of cultural reality is the World Vision (WV). Due to its importance and significance as substratum of religious and political belief systems, we will gird our study to mythical cognitive mode or mythical WV.

Geological risk assessment of Amtoudi Agadir in southern Morocco: a key case for sustainable cultural heritage

Medieval fortified granaries known as “agadirs” are very common in southern Morocco, being catalogued as world cultural heritage by United Nations. These Berber buildings (made of stones and tree trunks) usually located on rocky promontories, constitute historical testimonials related to the origin of Morocco, and, as tourist attractions, have a positive impact on the local economy. The sustainability of these ancient monuments requires geological-risk evaluations of the massif stability under the agadir with the proposal of stabilization measures, and an architectonic analysis with appropriate maintenance of the structural elements. An interdisciplinary study including climate, seismicity, hydrology, geology, geomorphology, geotechnical surveys of the massif, and diagnosis of the degradation of structural elements have been performed on the Amtoudi Agadir, selected as a case study. The main findings from this study are that the prevalent rocks used for construction (coming from the underlying substratum) are good-quality arkosic sandstones; the SW cliffs under the agadir are unstable under water saturation; some masonry walls are too thin and lack interlocking stones and mortar; and failures in the beams (due to flexure, fracture, and exhaustion in the resistance due to insect attacks or plant roots) are common. The basic risk assessment of ancient buildings of cultural heritage and their geologic substratum are needed especially in undeveloped areas with limited capacity to implement durable conservation policies. Therefore, recommendations have been provided to ensure the stability and maintenance of this important archaeological site.

Aggregation dynamics and foraging behaviour of striped red mullet Mullus surmuletus in the western Mediterranean Sea

Habitat-related heterogeneity of striped red mullet Mullus surmuletus heterospecific foraging assemblages was examined off the coast of Spain. Video-based focal-follows conducted on 122 M. surmuletus assemblages (446 total individuals) revealed an array of attendant species (n = 7) with composition linked to benthic habitat complexity; bare sandy substrata were characterized by homospecific groups of M. surmuletus, while habitats with rock and vegetation attracted a variety of scrounging labrids and sparids. Although the nature of the relationship between M. surmuletus and attendants requires further exploration, the present study indicates that substratum composition can be a driving factor explaining the dynamics of this heterospecific assemblage.

Modernist Ecology: Modernism and the Reimagination of Nature

Countering the trend in contemporary ecocriticism to advance realism as an environmentally responsible mode of representation, this essay argues that the anti-realist aesthetics of literary modernism were implicitly “ecological.” In order to make this argument I distinguish between contemporary and modernist ecological culture (both of which I differentiate in turn from ecological science); while the former is concerned primarily with the practical reform characteristic of what we now call “environmentalism,” the latter demanded an all-encompassing reimagination of the relationship between humanity and nature. “Modernist ecology,” as I call it, attempted to envision this change, which would be ontological or metaphysical rather than simply social, through thematically and formally experimental works of art. Its radical vision, suggestive in some ways of today’s “deep” ecology, repudiated modern accounts of nature as a congeries of inert objects to be manipulated by a sovereign subject, and instead foregrounded the chiasmic intertexture of the subject/object relationship. In aesthetic modernism we encounter not “objective” nature, but “nature-being” – a blank substratum beneath the solid contours of what philosopher Kate Soper calls “lay nature” – the revelation of which shatters historical constructions of nature and alone allows for radical alternatives. This essay looks specifically at modernist ecology as it appears in the works of W. B. Yeats, D. H. Lawrence, and Samuel Beckett, detailing their attempts to envision revolutionary new ecologies, but also their struggles with the limited capacity of esoteric modernist art to effect significant ecological change on a collective level.