bluuebunny/crossref_metadata_2025_split huggingface dataset:
Basement membrane and interstitial matrix components form separate matrices in heterokaryons of PYS-2 cells and fibroblasts
Authors: Laurila Pekka , Leivo Ilmo 
Published: April 2021

ABSTRACT
In order to gain further understanding of the spatial organization of interstitial and basement membrane matrices, we studied the expression of the interstitial matrix protein, fibronectin, and the basement membrane protein, laminin, in heterokaryons formed by the fusion of normal fibroblasts and teratocarcinoma-derived epithelial PYS-2 cells. These heterokaryons showed various distributions of the matrix proteins depending on the proportions of the different parental cell nuclei within the cytoplasm of the cell. Heterokaryons containing equal numbers of fibroblast and PYS-2 cell nuclei showed an abundant laminin matrix subcellularly and only minor amounts of fibronectin matrix at the periphery of the cells. Similar results were obtained in heterokaryons containing an excess of epithelial cell nuclei. In heterokaryons containing an excess of fibroblast nuclei, on the other hand, laminin matrix was reduced and a fibrillar fibronectin matrix was seen also on top of the cell body. The results suggest a gene dosage-type of effect on the expression of these proteins. Furthermore, extracellular laminin and fibronectin matrices did not codistribute around the heterokaryons but the two proteins were assembled into separate structures. The lack of codistribution of fibronectin and laminin matrices in heterokaryons suggests that the molecular interactions, which determine the assembly of basement membrane and interstitial matrices in these cells are highly type-specific. Similar mechanisms may also operate in the assembly of extracellular matrices in vivo.
Tenascin expression in the mouse: in situ localization and induction in vitro by bFGF
Authors: Tucker Richard P. , Hammarback James A. , Jenrath David A. , Mackie Eleanor J. , Xu Yue 
Published: April 2021

ABSTRACT
The glycoprotein tenascin is found in the extracellular matrix in regions of cell motility, cell proliferation, and tissue modelling. We have used novel tenascin cDNA probes to localize tenascin transcripts in the developing mouse and to study the regulation of tenascin expression by growth factors in vitro. At postnatal day 1 tenascin mRNAs are abundant in regions of bone and cartilage formation, as well as in the ependymal layer of the central nervous system. Previous studies have demonstrated that transforming growth factor-β type 1 (TGF-β1) can induce tenascin expression in vitro. As TGF-β1 is absent or scarce in the developing brain, it is likely that other growth factors, alone or in addition to TGF-β1, may regulate tenascin expression during development. Therefore, we have compared the effects of TGF-β1 and a growth factor that is found in both developing connective tissue and the central nervous system, basic fibroblast growth factor (bFGF), on tenascin expression in a mouse embryo fibroblast cell line (Swiss 3T3 cells). Immuno-slot blot analysis of Swiss 3T3 cell-conditioned culture medium demonstrates that bFGF is a more potent inducer of tenascin expression than TGF-β1. Furthermore, bFGF and TGF-β1 have an additive effect on levels of tenascin, but not fibronectin, in the conditioned medium. Western blots revealed that different forms of tenascin are induced by bFGF and TGF-β1: the tenascin induced by the former has a molecular mass of approximately 250 kDa, the latter induces an approximately 200 kDa form of tenascin. The induction of large tenascin by bFGF was confirmed by northern blot analysis, which revealed increased levels of an 8 kb tenascin transcript after 24 h by as little as 4 ng/ml of bFGF in serum-free medium. Thus bFGF, alone or in combination with TGF-β1, is a potential regulator of tenascin expression in vitro. bFGF may alter not only the relative abundance of tenascin and fibronectin in the extracellular matrix, but also the splice variant of tenascin expressed by a given cell type.
Characterization of the nuclear translocation of acidic fibroblast growth factor
Authors: Cao Yihai , Ekström Maria , Pettersson Ralf F. 
Published: April 2021

ABSTRACT
The subcellular localization of human acidic FGF (aFGF; FGF-1) expressed to high levels by using a bacteriophage T7 RNA polymerase-driven vaccinia virus expression system was studied in BHK21 and HeLa cells. Acidic FGF was detected by immunoblotting or immunofluorescence using an affinity-purified rabbit polyclonal antibody. The nuclei of most transfected cells, but not nuclei of control cells, were strongly immunoreactive. The nuclear accumulation of aFGF was confirmed by subcellular fractionation and immunoblotting, indicating that about 50% of the expressed protein was located in the nuclei at 12 h after transfection. It has previously been reported that a putative N-terminal nuclear localization sequence (NLS) in aFGF is required for full mitogenic activity (Imamura et al., Science 249, 1567-1570, 1990). We found that deletion of the first 27 residues including the putative NLS did not prevent the nuclear translocation of aFGF in either cell type. This observation suggests that the putative NLS sequence is not essential for targeting aFGF to the cell nucleus. To analyze further the mechanism of nuclear import, purified aFGF was microinjected into the cytoplasm of growing BHK21 cells under various conditions. In chilled (4°C) or ATP-depleted cells, the injected aFGF entered the nucleus with similar efficiency to that in control cells at 37°C. This suggests that aFGF, which has a molecular mass of only 16,500, enters the cell nucleus by free diffusion, and possibly becomes trapped by binding to some nuclear structures. When added exogenously to growing BHK21 cells, aFGF was not localized to the nucleus. Instead, a punctate staining pattern in the cytosol was observed, reminiscent of that in the endosomal-lysosomal compartments. In addition, a diffuse extracellular surface-staining was evident. This result demonstrates that receptor-mediated endocytosis of aFGF does not result in its translocation to the nucleus, as has been reported for basic FGF.
A novel member of the dynamin family of GTP-binding proteins is expressed specifically in the testis
Authors: Nakata Takao , Takemura Reiko , Hirokawa Nobutaka 
Published: April 2021

ABSTRACT
Dynamin is a member of a new GTPase family, which includes the mouse Mx protein, the yeast VPS1 and the Drosophila shibire gene product. A high homology with the shibire product suggests a role for dynamin in the endocytotic process, but it is expressed only in mature neurons. We identified two additional dynamin-like pro-teins in rats, by using the polymerase chain reaction with degenerate primers corresponding to the GTP-binding areas conserved between dynamin and VPS1. The full coding sequence of one of them, dynamin-2, revealed that it has 848 amino acids and has great sim-ilarity with brain dynamin and the shibire product.
Northern blot analysis and in situ hybridization revealed its expression to be specific to the seminiferous tubules in the testis. Dynamin-2 (testis type dynamin) was expressed in germ-cell-depleted testis as well, indi-cating its expression in Sertoli cells. Our data imply that a number of dynamin family proteins, which are prod-ucts of distinct genes, may play different roles specific to each cell type in the same rat.
γ-Tubulin is present in acentriolar MTOCs during early mouse development
Authors: Gueth-Hallonet Catherine , Antony Claude , Aghion Joël , Santa-Maria Angelica , Lajoie-Mazenc Isabelle , Wright Michel , Maro Bernard 
Published: April 2021

ABSTRACT
γ-Tubulin, a recently discovered member of the tubulin superfamily, is a peri-centriolar component considered to be essential for microtubule nucleation. Mouse oocytes and early embryos lack centrioles until the blas-tocyst stage. Thus, early mouse embryos allowed us to study the location of γ-Tubulin in animal cells in the absence of centrioles. For this, we used an antiserum directed against a specific peptide of the γ-Tubulin sequence, which is conserved among species. This serum has been characterised both in PtK2 and mouse cells. We found that it specifically stained the spindle poles and the cytoplasmic microtubule organizing centers in metaphase II oocytes and the spindle poles in mitosis during the cleavage stages. In contrast, no interphase staining could be detected during cleavage. Since the overall level of γ-Tubulin did not decrease during inter-phase, as shown by immunoblotting experiments, this absence of staining during interphase is probably due to a cytoplasmic dispersion of γ-Tubulin. A single dot-like interphase reactivity appeared at the 32-cell stage. In parallel, electron microscopy studies allowed us to detect centrioles for the first time at the 64-cell stage. The possible roles of γ-Tubulin in microtubule nucleation and in centrosome maturation are discussed.
5-Bromo-2-deoxyuridine regulates invasiveness and expression of integrins and matrix-degrading proteinases in a differentiated hamster melanoma cell
Authors: Thomas Linda , Chan Patti Walter , Chang Sunghea , Damsky Caroline 
Published: April 2021

ABSTRACT
Cell interactions with the extracellular matrix play a critical role in regulating complex processes such as ter-minal differentiation and tumor progression. In these studies we describe a melanoma cell system that should be useful in addressing the regulation of cell-matrix interactions and the roles they play in regulating differ-entiation and cell invasiveness. CS (suspension)-1 melanoma cells are relatively well differentiated: they are melanotic, responsive to melanocyte-stimulating hormone, and express TA99, a melanosome membrane differentiation marker. Their repertoire of integrin receptors for extracellular matrix ligands is limited; in particular, they lack receptors for vitronectin, account-ing for the observation that they are nonadherent when cultured in the presence of serum. CS-1 cells are non-invasive as well, and express low levels of both metal-loproteinases and activated plasminogen activators. Treatment of these cells with melanocyte-stimulating hormone causes them to increase melanin production and assume an arborized phenotype, suggesting that it promotes their further differentiation. In contrast, treatment of CS-1 with the thymidine analog 5-bromo-deoxyuridine, converts them to a highly invasive cell population (termed BCS-1) that loses its differentiated properties and responsiveness to melanocyte-stimulat-ing hormone, acquires a broad integrin repertoire (including vitronectin receptors), and expresses elevated levels of metalloproteinases and activated urokinase. From these observations and findings of others on BrdU treatment of other developmental lineages, we hypoth-esize that BrdU both suppresses differentiation and pro-motes invasiveness of CS-1 cells. The demonstrated manipulability of CS-1 cells should make them extremely useful for studying the regulation of both ter-minal differentiation and tumor progression in the melanocyte lineage.
Growth cone guidance and neuron morphology on micropatterned laminin surfaces
Authors: Clark Peter , Britland Stephen , Connolly Patricia 
Published: April 2021

ABSTRACT
Neurite growth cones detect and respond to guidance cues in their local environment that determine stereo-typed pathways during development and regeneration. Micropatterns of laminin (which was found to adsorb preferentially to photolithographically defined hydro-phobic areas of micropatterns) were here used to model adhesive pathways that might influence neurite exten-sion. The responses of growth cones were determined by the degree of guidance of neurite extension and also by examining growth cone morphology. These parame-ters were found to be strongly dependent on the geom-etry of the patterned laminin, and on neuron type. Decreasing the spacing of multiple parallel tracks of laminin alternating with non-adhesive tracks, resulted in decreased guidance of chick embryo brain neurons. Single isolated 2 μm tracks strongly guided neurite extension whereas 2 μm tracks forming a 4 μm period multiple parallel pattern did not. Growth cones appear to be capable of bridging the narrow non-adhesive tracks, rendering them insensitive to the smaller period multiple parallel adhesive patterns. These observations suggest that growth cones would be unresponsive to the multiple adhesive cues such as would be presented by oriented extracellular matrix or certain axon fascicle structures, but could be guided by isolated adhesive tracks. Growth cone morphology became progressively simpler on progressively narrower single tracks. On narrow period multiple parallel tracks (which did not guide neurite extension) growth cones spanned a number of adhesive/non-adhesive tracks, and their mor-phology suggests that lamellipodial advance may be independent of the substratum by using filopodia as a scaffold. In addition to acting as guidance cues, laminin micropatterns also appeared to influence the production of primary neurites and their subsequent branching. On planar substrata, dorsal root ganglion neurons were multipolar, with highly branched neurite outgrowth whereas, on 25 μm tracks, neurite branching was reduced or absent, and neuron morphology was typi-cally bipolar. These observations indicate the precision with which growth cone advance may be controlled by substrata and suggest a role for patterned adhesiveness in neuronal morphological differentiation, but also high-light some of the limitations of growth cone sensitivity to substratum cues.
Angiogenic oligosaccharides of hyaluronan enhance the production of collagens by endothelial cells
Authors: Rooney P. , Wang M. , Kumar P. , Kumar S. 
Published: April 2021

ABSTRACT
The present study demonstrates a relationship between angiogenic oligosaccharides of hyaluronan (HA) and the production of collagens during the process of angiogen-esis in vivo and in vitro. The addition of angiogenic oligosaccharides of HA to the chorioallantoic membrane of the chick embryo induced a deposition of collagen fib-rils. The treatment of sub-confluent cultures of bovine aortic endothelial cells with the same oligosaccharides (1 μg/ml) increased the uptake of [3H]proline by approx-imately 60%. SDS-polyacrylamide gel electrophoresis of treated cultures demonstrated the enhanced synthesis of type I and type VIII collagens. The production of type VIII collagen was confirmed by western blotting and immunocytochemistry using antibodies to sheep and bovine type VIII collagen. Type VIII collagen is a short chain collagen that has a high degree of homology to cartilage-specific type X collagen. The biological func-tions of type VIII and type X collagens are unknown. We have suggested that the two collagens play a role in the process of angiogenesis.
Characterisation of an epithelium-like layer of cells in the multicellular Dictyostelium discoideum slug
Authors: Fuchs Manuela , Jones Malcolm K. , Williams Keith L. 
Published: April 2021

ABSTRACT
Ultrarapid freezing (RF) followed by freeze-substitution (FS) provide superior preservation of the Dictyostelium discoideum multicellular slug tissue over conventional methods of chemical fixation at room temperature. The peripheral cells of slugs prepared by RF and FS form a tight layer of flattened cells. This cell layer resembles epithelia of other multicellular organisms in that it has close junctional contact between cells associated with the extracellular matrix (ECM, slime sheath). This is the first report that clearly demonstrates the existence of such peripheral cellular specialisation in this otherwise well-studied model system. Junctional contacts between adjacent cells mean that there is no intercellular space evident between apical membranes of apposing cells, and basally the intermembraneous space between peripheral cells is less than 10 nm. By contrast, the inter-cellular space between internal cells is approximately 10–25 nm. The shape of the peripheral cells varies with their location around the slug. In the posterior prespore zone, the peripheral cells are squamous and exhibit polarity along their antero-posterior axis. In the ante-rior prestalk zone, peripheral cells are less flattened, project irregular filipodia between internal cells, and are polarised along their apical-basal axis. Colloidal gold immunocytochemistry with the markers MUD1, MUD50 and MUD62 demonstrated that the peripheral layer is formed of prestalk cells in the anterior region and ventrum, and mostly prespore cells along the dorsum. Thus, the peripheral layer, while having specific cell classes in different regions, is not differen-tiation-specific. Rather, it appears that the structure of these epithelium-like cells is influenced by interaction with molecules of the ECM (sheath).
A tau-like protein interacts with stress fibers and microtubules in human and rodent cultured cell lines
Authors: Cross Daniel , Vial Clarisa , Maccioni Ricardo B. 
Published: April 2021

ABSTRACT
The cytoskeletal integrity of human and rodent cell lines was analyzed using site-directed monoclonal antibodies prepared from hybridomas. Secreting hybridomas were produced by immunizing mice with synthetic peptides from the C-terminal domain of the II-tubulin isotype, II(422-434), YQQYQDATADEQG, and the first imperfect repeat from brain tau, Tau-I(187-204), VRSKIGSTENLKHQPGGG. Two hybridomas were selected for this work: MTB6.22, an anti-idiotypic mon-oclonal antibody, which was obtained from a mouse immunized with the II-peptide and recognizes specific tubulin-binding domains on MAP-2 and tau; and Tau-I/1, which recognizes the repetitive binding sequences on tau and MAP-2. Immunoblots of cytoskeletal protein preparations from the five different tumor cell lines studied, showed the interaction of the site-directed anti-bodies MTB6.22 and Tau-I/1 with a group of proteins that co-migrate with brain tau. Immunoreactive tau components were also identified using an anti-tau mon-oclonal antibody (clone Tau-2), and several polyclonal anti-tau antibodies that interact with tau epitopes, other than those of the tubulin-binding domains. These tau-like proteins bound to a calmodulin-Sepharose affinity column and were eluted using 2 mM EGTA. Interestingly, washing the extracted cytoskeleton pellet with 5 10−3 M Ca2+for short periods of time selectively released the tau-like protein components, whilst most of the other cytoskeletal proteins remained in the pellet. On the other hand, immunofluorescence microscopy of detergent-extracted cells showed immunostaining of MAP components that appear to be co-localized in a dis-crete dot-like distribution along the stress fibers, which were revealed using rhodamine-phallacidin. Further support for the specificity of tau interaction with sites on tubulin and actin polymers was obtained with double-immunofluorescence, using the MAP-reactive monoclonal antibody MTB6.22 and a polyclonal anti-body to a tubulin peptide containing part of the tau-binding domain on tubulin.
Considering the anti-idiotypic nature of the MTB6.22 monoclonal antibody, our studies indicate that, in all the cell lines analyzed, a tau-like protein component is involved in mediating the interaction of both actin and tubulin polymers.
Expression of E- or P-cadherin is not sufficient to modify the morphology and the tumorigenic behavior of murine spindle carcinoma cells: Possible involvement of plakoglobin
Authors: Navarro Pilar , Lozano Encarnación , Cano Amparo 
Published: April 2021

ABSTRACT
Transfection of E- and P-cadherin cDNA has been carried out in murine spindle carcinoma cells previously shown to be deficient in both cadherins (Navarro et al., J. Cell Biol. 115, 517-533, 1991). High levels of expression of E- or P-cadherin do not significantly affect the fibroblastic morphology of the parental spindle cells. In addition, the tumorigenic behavior of these highly malignant cells is not influenced by the ectopic expression of either cadherin. Nevertheless, a fraction of the exogenous cadherins is able to associate to detergent-insoluble components of the transfectant cells, and the expression of the exogenous E-cadherin confers Ca2+-dependent aggregation on the spindle transfectants in an in vitro assay. Immunoprecipitation analysis of the cadherin-catenin complex of the transfectants revealed that the ectopic E-cadherin associates with the ơ- and β-catenin proteins. However, the γ-catenin/plakoglobin component could not be detected in the E-cadherin immunocomplexes of the spindle transfectant cells, in contrast to the epithelial cells where the three catenins appeared to be associated with E-cadherin. The lack of association of γ-catenin is correlated with very low levels of plakoglobin in whole cell extracts of the parental spindle cells. These results indicate that the association of E-cadherin with the α- and β-catenin components is not sufficient to promote a fibroblastoid-epithelial conversion of highly malignant spindle cells. The presence of plakoglobin could be required for the proper organization of E-cadherin in the transfectant cells in order to acquire an epithelioid phenotype.
Novel structural organisation of a Mus musculus dba/2 chromosome shows a fixed position for the centromere
Authors: Mitchell Arthur R. , Nicol Linda , Malloy Patricia , Kipling David 
Published: April 2021

ABSTRACT
Chromosome 1 of the inbred mouse strain DBA/2 shows an unusual polymorphism associated with its centromeric satellite DNA sequences. The minor satellite array has undergone amplification and is present as two blocks separated by major satellite sequences. Both minor satellite blocks appear to carry the sequence motif necessary for CENP-B protein binding. Despite this apparent similarity the functional centromere, as defined by the location of CREST antigens, appears to form only within the more terminal block. The two blocks also vary in that sister chromatid association only occurs with this more terminal block.
The role of isoprenylation in membrane attachment of nuclear lamins: A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties
Authors: Hennekes Hartwig , Nigg Erich A. 
Published: April 2021

ABSTRACT
Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.