Extracellular matrix fibrils and cell contacts in the chick embryo

Summary The migration of neural crest and sclerotome cells and the extension of ventral root axons in chick embryos at stages 16–20 were studied by light microscopy as well as scanning and transmission electron microscopy at the leg bud level of fixed specimens. Extensive cellular movements take place in association with an extracellular matrix consisting of microfibrils. The neural crest and sclerotome cells migrate into the large matrix-filled extracellular space surrounding the neural tube and notochord, apparently using microfibril bundles as substratum. The cells exhibit pseudopodia which are closely associated with the matrix fibrils. The fibrils around the notochord show a spatial arrangement indicating that the sclerotome cells are contact-guided to their subsequent positions. Mutual cell contacts, including those established by cell processes, frequently show cytoplasmic electron dense plaques at adjacent membranes. These small plaque contacts might be correlated to contact inhibition of locomotion between the cells and participate in the guidance of cells. The growth cones of extending axons exhibit filopodia contacting both surrounding mesenchyme cells and extracellular fibrils. The orientation of the axons might thus be affected by contacts with cell surfaces as well as with extracellular material.Technical assistance was given by Mrs. Kerstin Ahlfors, Mrs. Charlotte Fällström, Mrs. Annika Kylberg and Mrs. Stine SöderströmSupported by grants from The Swedish Natural Science Research Council     

Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

Physiological and biochemical contributions to the taxonomy of the genera Ankistrodesmus and Scenedesmus

The 17 Ankistrodesmus strains studied do not produce extracellular amylolytic enzymes. 20 out of 28 Scenedesmus strains, however, are able to hydrolyse starch.The groups of strains previously characterized physiologically and biochemically were assigned to established species of the genera Ankistrodesmus (including Monoraphidium) and Scenedesmus.Abbreviation GC Guanine + cytosine     

Rapid isolation of plant peroxidase. Purification of peroxidase a from Petunia

A rapid isolation procedure was developed for purification of peroxidase a from Petunia hybrida . Rapid isolation was possible since about 15% of the extracellular protein from stem tissue obtained by vacuum infiltration followed by centrifugation of the tissue represents peroxidase. Purification of peroxidase a from intercellular fluid was achieved by two acetone precipitation steps followed by DEAE-cellulose chromatography.
Three different forms of peroxidase were eluted from DEAE-cellulose at different NaCl concentrations. Isoelectric focusing showed, however, a pI of 3.8 for all three forms of peroxidase a . Only part of the peroxidase a enzymes bound to Concanavalin A indicating heterogeneity in the carbohydrate part. Homology of peroxidase a to the peroxidase G₁ group from tobacco is discussed.     

Ground cover and slope exposure effects on micro- and mesobiota in forest soils

The interrelation of Alpine topography with the micro − and mesobiota is still poorly understood. We investigated the effects of ground cover type and slope exposure on the soil microbial biomass (double-stranded DNA, dsDNA) and abundances (real time PCR, qPCR); hydrolytic enzyme activities; and enchytraeid community structure in top soils (2.5-cm increments depth) in subalpine forests in the Italian Alps. Dominant ground covers were grass, moss, litter and woody debris at the north- and the south-facing slopes. The autochthonous soil microbiota (bacteria, fungi and archaea) was quantified by qPCR in the extracellular (eDNA) and intracellular fraction (iDNA) of the total soil DNA pool. A higher eDNA/iDNA ratio indicative of lower microbial activity was recorded in the deepest layer of the grass plots at the north-facing slope. This can be related to a lower degradation of eDNA and/or to an accumulation of eDNA with increasing depth as a result of leaching. The exposure effect was enzyme-specific and higher activities occurred under woody debris primarily at the south-facing slope. These plots also showed a higher nutrient content and a greater microbial biomass assessed as dsDNA yields. Total microannelid abundance was elevated on north-facing slopes on account of strong acidity indicator species. This was related to soil pH being one unit lower compared to the south-facing slope. The thickness of the organic layer (OL + OF + OH) was elevated at the north-facing slope due to a considerably thicker OH-horizon. The vast majority of microannelids at this slope occurred in the organic layer, while at south exposure they were almost evenly distributed between the organic layer and the mineral soil (A-horizon). Exposure was found to be more determinative for the composition of microannelid assemblages than the ground cover type.     

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

Clustering or overexpression of the transmembrane form of the extracellular matrix proteoglycan agrin in neurons results in the formation of numerous highly motile filopodia-like processes extending from axons and dendrites. Here we show that similar processes can be induced by overexpression of transmembrane-agrin in several non-neuronal cell lines. Mapping of the process-inducing activity in neurons and non-neuronal cells demonstrates that the cytoplasmic part of transmembrane agrin is dispensable and that the extracellular region is necessary for process formation. Site-directed mutagenesis reveals an essential role for the loop between β-sheets 3 and 4 within the Kazal subdomain of the seventh follistatin-like domain of TM-agrin. An aspartic acid residue within this loop is critical for process formation. The seventh follistatin-like domain could be functionally replaced by the first and sixth but not by the eighth follistatin-like domain, demonstrating a functional redundancy among some follistatin-like domains of agrin. Moreover, a critical distance of the seventh follistatin-like domain to the plasma membrane appears to be required for process formation. These results demonstrate that different regions within the agrin protein are responsible for synapse formation at the neuromuscular junction and for process formation in central nervous system neurons and suggest a role for agrin''s follistatin-like domains in the developing central nervous system.     

Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix

Extracellular matrix plays a critical role in cellular development by providing signaling cues that direct morphogenesis. In order to study both the cues that natural matrix provides and endothelial cell responses to that information, human fetal lung fibroblasts were used to produce a fibrous three-dimensional matrix. Following the removal of the fibroblasts by detergent extraction, protein and proteoglycan constituents of the remaining matrix were identified by immunofluorescence and immunoblotting. Matrix components included fibronectin, tenascin-C, collagen I, collagen IV, collagen VI, versican, and decorin. Colocalization analysis suggested that fibronectin was a uniquely distributed matrix protein. Morphology, three-dimensional matrix adhesions, and integrin-mediated signaling during vasculogenesis were then studied in human endothelial cells seeded onto the fibroblast-derived matrix. Elongated morphology and decreased cell area were noted, as compared with cells on fibronectin-coated coverslips. Cell-matrix adhesions contained vinculin, pY397-FAK, and pY410-p130Cas, and all of these colocalized more with fibronectin than tenascin-C, collagen I, or collagen VI. Additionally, the endothelial cells remodeled the fibroblast-derived matrix and formed networks of tubes with demonstrable lumens. Matrix adhesions in these tubes also predominantly colocalized with fibronectin. The pattern of membrane type 1 matrix metalloprotease expression in the endothelial cells suggested its involvement in the matrix remodeling that occurred during tubulogenesis. These results indicated that information in fibroblast-derived matrix promoted vasculogenic behavior.