Exogenous fibronectin is not required for organogenesis in vitro

The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50 micrograms/ml) was a strong mitogen for kidney cells, whereas the addition of soluble fibronectin (50 to 250 micrograms/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. However, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 micrograms/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250 micrograms/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro.     

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.     

Transferrin receptor induction is required for human B-lymphocyte activation but not for immunoglobulin secretion

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. In the experiments reported here we have examined the regulation of transferrin receptor expression on activated human B cells and whether or not these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We have determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B-cell growth factor). This expression is required for proliferation to occur, since antibody to transferrin receptor (42/6) blocks B-cell proliferation. Induction of immunoglobulin secretion, however, although dependent on PHA-treated T-cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by antitransferrin receptor antibody. In addition, we have demonstrated that IgM messenger RNA induction following mitogen stimulation is unaffected by antitransferrin receptor antibody. These findings support a model for B-cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B-cell growth factor and transferrin receptor expression, for proliferation, and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.     

Caspase activity is required for nephrogenesis in the developing mouse metanephros

Programmed cell death is a mechanism through which organisms get rid of unwanted cells and is thought to be an important process in organogenesis. Although large-scale cell death is observed in the developing kidney, the precise roles of cell death in kidney organogenesis remain to be elucidated. To address this question, we prevented cell death in metanephric explants by applying caspase inhibitors. Administration of caspase inhibitors (Z-D-CH2DCB and Ac-DEVD-CHO) effectively prevented the cell death that is normally observed in nondifferentiating mesenchymal cells. Both ureteric bud branching and nephrogenesis were prevented by caspase inhibition. Our results suggest that caspases are crucial in kidney organogenesis and cell death in the nondifferentiating mesenchyme.     

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.     

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.     

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.     

Fibronectin is not a serum spreading factor for HeLa cells

The spreading of HeLa cells on plastic substratum is mediated by fibronectin-depleted foetal calf serum but not by fibronectin isolated by gelatin-Sepharose affinity chromatography. The same is true for freshly explanted chick embryonic chondrocytes. In contrast, BHK cell spreading exceeds 67% after 120 min at 37 degrees C in fibronectin-supplemented (10 micrograms ml-1) serum-free medium. Long-term cultivation of HeLa cells in Eagle's MEM supplemented with fibronectin-free serum is associated with the accumulation of cells in mitosis or before cytokinesis; many cells die and the remaining living cells, characterized by marked changes in morphology, multiply very slowly. It can be concluded therefore that fibronectin does not produce spreading in HeLa cells but forces them into mitosis.     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

A method for the quantitative analysis of nerve growth in vitro

Summary A method is described for the quantitative analysis of the nerve-growth-promoting activity of biological molecules in tissue culture. The criteria used for the evaluation of this activity is based on the neurite length as well as the total number of neurites produced by the explant of whole dorsal root ganglia from 12-d-old chick embryos. A nerve growth index (NGI) is given to each ganglion during each of a 5-d culture period. The NGI is defined as the product of average neurite length in millimeters and the total number of neurites. We report that with increasing concentrations of fetal bovine serum, there was a proportional increase in NGI due to increased neurite density while the neurite length was not greatly affected. The NGI of several proteins with known nerve growth promoting activity, namely nerve growth factor, insulin, transferrin, and fibronectin were investigated for their activity and compared with that of fetal bovine serum. This work was supported in part by grant GM-10374 from the National Institutes of Health, Bethesda, MD.     

Initiation of fibronectin fibrillogenesis is an enzyme-dependent process

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MACCHI-BOU 2 is required for early embryo patterning and cotyledon organogenesis in Arabidopsis

The phytohormone auxin is a key regulator of organogenesis in plants and is distributed asymmetrically via polar transport. However, the precise mechanisms underlying auxin-mediated organogenesis remain elusive. Here, we have analyzed the macchi-bou 2 (mab2) mutant identified in a pinoid (pid) enhancer mutant screen. Seedlings homozygous for either mab2 or pid showed only mild phenotypic effects on cotyledon positions and/or numbers. In contrast, mab2 pid double mutant seedlings completely lacked cotyledons, indicating a synergistic interaction. We found that mab2 homozygous embryos had defective patterns of cell division and showed aberrant cotyledon organogenesis. Further analysis revealed that the mab2 mutation affected auxin response but not auxin transport in the embryos, suggesting the involvement of MAB2 in auxin response during embryogenesis. MAB2 encodes an Arabidopsis ortholog of MED13, a putative regulatory module component of the Mediator complex. Mediator is a multicomponent complex that is evolutionarily conserved in eukaryotes and its regulatory module associates with Mediator to control the interaction of Mediator and RNA polymerase II. MAB2 interacts with a regulatory module component in yeast cells. Taken together, our data suggest that MAB2 plays a crucial role in embryo patterning and cotyledon organogenesis, possibly through modulating expression of specific genes such as auxin-responsive genes.     

A combinatorial approach for directing the amount of fibronectin fibrils assembled by cells that uses surfaces derivatized with mixtures of fibronectin and cell binding domains

Fibrillar fibronectin (FN) has the crucial role of attracting and attaching cells as well as molecules that mediate tissue repair during wound healing. A previous study demonstrated higher extracellular staining of FN fibrils in cells cultured on surfaces tethered with an equimolar mixture of a FN binding domain and FN's cell binding domain, III1-2 and III9-10 respectively, than on surfaces with III9-10 alone. The effect of varying surface amounts of III1-2 and III9-10 on the quantity of FN fibrils formed by NIH-3T3 fibroblasts was examined. GST tagged III1-2 and III9-10 were conjugated to polyurethane surfaces and ELISAs were used to identify the experimental design space or the range of concentrations of GST-III1-2 and GST-III9-10 that demarcated the limits of protein loading on the surface. When GST-III1-2 was fixed and GST-III9-10 varied within the design space, the amount of FN fibrils measured by immunoblotting detergent insoluble cell lysates was dependent on the ratio of III9-10 to III1-2. When the total protein concentration was fixed and the mixture composition of GST-III1-2 and GST-III9-10 varied such that it optimally covered the design space, a parabolic relationship between FN fibril amount and the ratio of III9-10 to III1-2 was obtained. This relationship had a maximum value when the surface was bonded to equal amounts of III1-2 and III9-10 (P<0.05). Thus the ratio of III9-10 to III1-2 can be utilized to direct the quantity of FN fibrils formed on surfaces.     

The BMP antagonist follistatin-like 1 is required for skeletal and lung organogenesis

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development.     

Preproenkephalin is a Th2 cytokine but is not required for Th2 differentiation in vitro.

Preproenkephalin (PPNK) mRNA expression has been detected in many cells of the immune system, including monocytes and lymphocytes. In the present paper, the expression of PPNK mRNA in purified CD4+ Th1 and Th2 lymphocyte subpopulations is investigated and correlated with the presence of the opioid neuropeptides leu- and met-enkephalin. We found that PPNK mRNA and met-enkephalin were present at higher levels in the Th2 cultures compared with the Th1 cultures. Lymphocytes from PPNK-deficient mice were then used to look at the role of PPNK in Th2 lymphocyte differentiation. Lymphocytes from these mice could be driven into a Th2 phenotype, suggesting that cultures containing IL-4 do not require PPNK for Th2 differentiation.     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.