bluuebunny/crossref_metadata_2025 huggingface dataset:
the role of α4β1 integrin in cell motility and fibronectin matrix assembly
Authors: Wu Chuanyue , Fields Andrew J. , Kapteijn Barbara A. E. , McDonald John A.
Published: April 2021

The α4β1 integrin has been suggested to play important roles in embryogenesis and pathogenesis of many diseases which involve both cell adhesion and cell migration. Previous studies using anti-α4β1 antibodies and fibronectin (Fn) fragments have suggested that α4β1 integrins may be involved in cell motility on Fn and vascular cell adhesion molecule-1 (VCAM-1). However, the cells used in these studies also express other Fn integrin receptors including α5β1 integrin, which is known to function in cell motility on Fn. To test whether α4β1 integrins mediate cell motility on Fn and VCAM-1 in the absence of α5β1 integrin, we expressed human α4 integrin in a Chinese hamster ovary (CHO) cell line that is deficient in α5β1 integrin (CHO B2). The parental α5 deficient CHO B2 cells were unable to adhere, spread or migrate on Fn, nor could they assemble a fibrillar Fn matrix. Expression of α4β1 integrin in the CHO B2 cells enabled the cells to adhere, spread and migrate on Fn and on VCAM-1 but not to assemble a fibrillar Fn matrix. The cellular processes mediated by the interaction of α4β1 with Fn or VCAM-1 were inhibited by the CS1 peptide derived from the major α4β1 binding site on Fn. These findings demonstrate that α4β1 integrins not only function as cell adhesion receptors but also as cell motility receptors for Fn and VCAM-1 independent of α5β1. Moreover, they reveal important functional differences between Fn binding integrins. The α4-positive, α5-negative CHO cells described in this report will be useful tools in studying the mechanism of molecular signalling during integrin mediated cellular processes.
Modulation of epidermal differentiation by epiligrin and integrin α3β1
Authors: Symington Banu Erhan , Carter William Gene
Published: April 2021

We previously reported that integrin α3β1 mediates epidermal intercellular adhesion as well as cell-substrate adhesion. P1B5, an anti-α3β1 specific monoclonal antibody, is a potent in vitro trigger of epidermal cell-cell adhesion and an inhibitor of cell-substrate adhesion. We now show that P1B5 specifically induces the intercellular localization of integrins α2β1 and α3β1, consistent with its role in inducing intercellular adhesion via these two integrins. P1F2, another anti-α3β1 antibody, does not induce either intercellular adhesion or intercellular accumulation of α3β1 and α2β1.

Growth of epidermal cells in high calcium, known to induce epidermal differentiation, also induces intercellular accumulation of α3β1 and α2β1 and increased cell-cell adhesion. We therefore asked whether P1B5 treatment induces epidermal differentiation. P1B5 treatment induces changes consistent with epidermal differentiation, including increased involucrin expression, stratification, and production of squames. P1F2 treatment has none of these effects. In vivo, epidermal basal cells are in close contact with the epithelial basement membrane component epiligrin. Growth of keratinocytes on purified epiligrin but not other matrix components specifically reduces involucrin expression by P1B5-treated keratinocytes. These results suggest that integrin α3β1 has a unique role in epidermal differentiation, that the epitope recognized by P1B5 is involved in triggering this differentiation, and that keratinocyte adhesion to epiligrin inhibits α3β1-mediated differentiation. Taken together, these data suggest that α3β1-mediated intercellular adhesion may be part of the terminal differentiation process in epidermal cells.
Overexpressing cell surface β1,4-galactosyltransferase in pc12 cells increases neurite outgrowth on laminin
Authors: Huang Qiuling , Shur Barry D. , Begovac Paul C.
Published: April 2021

Neurite outgrowth on cellular and extracellular matrices is mediated by a variety of cell surface receptors. Some of these receptors recognize peptide determinants, whereas others bind oligosaccharide ligands. Previous studies have suggested that cell surface β1,4-galactosyltransferase functions as one of these receptors during neurite outgrowth on basal lamina by binding to N-linked oligosaccharides in the E8 domain of laminin. However, these previous investigations have been limited to the use of galactosyltransferase inhibitory reagents to block neurite formation. Therefore, in this study, we investigated whether the level of surface galactosyltransferase directly affects the efficiency of neurite outgrowth, or rather, is incidental to neurite formation. Northern blot analysis and cell surface galactosyltransferase assays were used to select two stable PC12 transfectants that overexpress surface galactosyltransferase by approximately four-fold. Radiolabeled antibody binding to intact cells and indirect immunofluorescence confirmed the higher expression of surface galactosyltransferase on transfected cells, compared to controls. Both galactosyltransferase transfected cell lines exhibited markedly enhanced neurite initiation, neurite formation, and rates of neurite elongation by two- to three-fold. These studies demonstrate that the expression of laminin receptors can be rate-limiting during neurite outgrowth, and that the level of surface galactosyltransferase can modulate the frequency and rate of neurite formation from PC12 cells on laminin.
Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation
Authors: Kiley Susan C. , Parker Peter J.
Published: April 2021

U937 human promonocytic leukemia cells express PKC isozymes β1, β2, ε and ζ. Indirect immunocytofluorescence using affinity-purified PKC-specific antibodies indicates that each of the endogenous PKC isozymes in U937 cells display a unique compartmentalization within the intact cell. PKC-β1 is distributed between two identifiable pools: a cytoplasmic pool which redistributes to the plasma membrane upon activation with acute phorbol ester-treatment, and a membrane-bound pool associated with intracellular vesicles containing β2-integrin adhesion molecules, cd11b and cd11c. The vesicle-associated PKC-β1 translocates with the secretory granules to the plasma membrane upon agonist-stimulated activation. PKC-β2 is associated with the microtubule cytoskeleton in resting cells. PKC overlay assays indicate that PKC-β2 binds to proteins associated with microtubules, and not directly to tubulin. PKC-ε is associated with filamentous structures in resting cells and redistributes to the perinuclear region upon activation with phorbol esters. In differentiated U937 cells, PKC-β1 remains associated with vesicles translocat-ing from the trans-Golgi region to the plasma membrane and PKC-ε is primarily associated with perinuclear and plasma membranes. PKC-ζ, which does not respond to phorbol ester treatment, is primarily cytosolic in undiffer-entiated cells and accumulates in the nucleus of differenti-ated cells blocked in the G2 phase of the cell cycle. The data clearly demonstrate that individual PKCs localize to different subcellular compartments and promote the hypothesis that PKC subcellular localization is indicative of unique functions for individual PKC isozymes.
Characterisation of shrimp haemocytes and plasma components by monoclonal antibodies
Authors: Rodriguez J. , Boulo V. , Mialhe E. , Bachere E.
Published: April 2021

Various haemolymph components of the shrimp Penaeus japonicus were identified and characterised by monoclonal antibodies. Three groups of monoclonal antibodies were raised. Their reactivity to haemocyte types and/or secreted molecules was determined by immunofluorescence and the molecular masses of the antigens were analysed by western-blotting. A 170 kDa protein, in reducing conditions, was recognized by four panhaemocytic monoclonal antibodies from group 1. This protein was present both in the plasma and in the haemocytes from which it appears to be secreted. The shrimp haemocytes were separated by isopycnic cen-trifugation on a Percoll gradient and the different subpop-ulations were antigenically analysed using the two mono-clonal antibodies, 40E2-2A and 40E10-2B, from group 2. The granular cells were labelled by 40E2-2A which was specific for a protein of 142 kDa also present in plasma. By comparison, the 40E10-2B monoclonal antibody was assumed to be the marker for small hyaline and semi-granular cells since the granular ones were not labelled. Moreover, the antigen immunoprecipitated by this mono-clonal antibody was shown to have different molecular masses of 250, 150, 66 and 27 kDa under nonreducing con-ditions. It appeared to be secreted by the haemocytes. Some plasma proteins were recognized by the third group of monoclonal antibodies. The antibodies, designated 41D11-3A, 42C11-3B and 42E8-3C, all immunoprecipitated a protein with an apparent molecular mass of 180 kDa under reduced conditions. The 44E6-3D antibody was specific for a 75 kDa protein under reduced conditions and was shown to be immunoreactive against P. japonicus haemocyanin extract. An antigenic relationship was studied for the haemolymph components of P. japonicus and two other shrimp species, P. vannamei and P. indicus. Hypotheses about the possible nature of the proteins, antigenically identified, are advanced.
A cytokeratin 8-like protein with plasminogen-binding activity is present on the external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines
Authors: Hembrough Todd A. , Vasudevan Jayanand , Allietta Margaretta M. , Glass William F. , Gonias Steven L.
Published: April 2021

Plasminogen binding to cell surfaces may be important for tumor invasion and other processes that involve cellular migration. In this investigation, the principal plasminogen-binding protein was identified in the plasma membrane fraction of rat hepatocytes. The protein had an apparent mass of 59 kDa, was insoluble in a spectrum of detergents, and was identical to cytokeratin 8 (CK 8) as determined by sequence analysis of nine amino acids at the N terminus of two cyanogen bromide fragments. The 59 kDa protein bound CK 8-specific antibody in western blot analyses. These studies demonstrate that CK 8 or a CK 8-like protein binds plasminogen. Given this newly determined and potentially important CK 8 function, immunofluorescence and immunoelectron microscopy studies were performed to determine whether CK 8 may be present on the external surfaces of unpermeabilized, viable hepatocytes. All of the cells in each preparation were immunopositive with two separate CK 8-specific antibodies. A punctate pattern of immunofluorescence was detected on the cell surface with approximately even intensity from cell to cell. By immuno-electron microscopy, CK 8 was preferentially associated with microvilli. In order to determine whether other epithelial cells express cell-surface CK 8, immunofluores-cence and immunoelectron microscopy studies were performed with HepG2 hepatocellular carcinoma cells and with BT20 and MCF-7 breast carcinoma cells. The pattern of antigen expression was equivalent with each cell type and comparable to that observed with hepatocytes. These studies support the hypothesis that CK 8 is associated with the external cell surface where it may express important proteinase receptor function.
Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule A new model for parietal cell transformation
Authors: Pettitt John M. , Humphris Danielle C. , Barrett Simon P. , Toh Ban-Hock , Driel Ian R. van , Gleeson Paul A.
Published: April 2021

The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic ‘tubulovesicles’ as the apical surface of the cell progres-sively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesi-cles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimu-lated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with α-and β-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progres-sive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; conse-quently, they cannot have a role in parietal cell transfor-mation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is com-patible with the localization of cytoskeletal components in these cells.
p80-coilin: a component of coiled bodies and interchromatin granule-associated zones
Authors: Puvion-Dutilleul Francine , Besse Sylvie , Chan Edward K. L. , Tan Eng M. , Puvion Edmond
Published: April 2021

We investigated at the electron microscope level the fate of the three intranuclear structures known to accumulate snRNPs, and which correspond to the punctuate immuno-fluorescent staining pattern (the coiled bodies, the clusters of interchromatin granules and the interchromatin granule-associated zones) after exposure to either a low salt medium which induces a loosening and partial spreading of nucleoprotein fibers or a high ionic strength salt medium and subsequent DNase I digestion, in order to obtain DNA-depleted nuclear matrices. The loosened clusters of inter-chromatin granules and the coiled bodies could no longer be distinguished from surrounding nucleoprotein fibers solely by their structure, but constituents of the clusters of interchromatin granules could be detected by in situ hybridization with both U1 and U2 DNA probes, and con-stituents of the coiled bodies were detectable mainly with the U2 DNA probe. The interchromatin granule-associated zones, the electron-opacity and compactness of which were preserved despite the loosening treatment, remained labeled with the U1 DNA probe only. In DNA-depleted nuclear matrices, the snRNA content of the coiled bodies, the clusters of interchromatin granules and their associated zones, which were all easily recognizable within the residual nuclear ribonucleoprotein network, was unmodi-fied. The data indicate, therefore, that the loosening procedure as well as the high salt extraction procedure preserve the snRNA content of all three spliceosome component-accumulation sites and reveal that interchro-matin granule-associated zones are elements of the nuclear matrix. The p80-coilin content of coiled bodies was also preserved whatever the salt treatment used. An intriguing new finding is the detection of abundant p80-coilin within the interchromatin granule-associated zones, both before and after either low or high salt treatment of cells. Therefore, p80-coilin is an integral constituent of the inter-chromatin granule-associated zones.
Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes
Authors: Guénette Suzanne , Magendantz Margaret , Solomon Frank
Published: April 2021

To identify proteins that regulate microtubule assembly in Saccharomyces cerevisiae, we screened for multicopy sup-pressors of a conditional mutation in α-tubulin. Cells expressing the recessive allele tub1-729 as their sole α-tubulin gene grow normally at permissive temperature. However, at 15°C the cells lose viability and arrest primarily with large buds and quantitatively diminished microtubule structures. Transformation of mutant cells with genomic libraries repeatedly identified three different suppressors: the two wild-type α-tubulin genes, TUB1 and TUB3; and BUB3. BUB3 is a checkpoint gene that permits entry into mitosis depending upon the assembly state of microtubules. Excess BUB3 rescues both the loss of viability and microtubule defects but not the benomyl supersensitivity associated with tub1-729. The suppression is specific for the mutation ALA422VAL in TUB1, and does not affect several other mutations in TUB1 that produce the ‘no microtubule’ phenotype. Overexpression of BUB1, which interacts genetically with BUB3 and which is involved in the same checkpoint pathway, also rescues the cold sensitivity of tub1-729, but another checkpoint gene, MAD2, does not. Overexpression of BUB3 in wild-type cells has no detectable growth or microtubule defect, but dis-ruption of the BUB3 gene produces slow growth and benomyl supersensitivity. Our results suggest that BUB1 and BUB3 overexpression modulate an event required for mitotic spindle function which is rate limiting for tub1-729 cells at the restrictive temperature.
Regulation of Cdc2/cyclin B activation by Ran, a Ras-related GTPase
Authors: Clarke Paul R. , Klebe Christian , Wittinghofer Alfred , Karsenti Eric
Published: April 2021

During the cell cycle, a checkpoint prevents the initiation of mitosis until S-phase is completed. The molecular mechanism may involve the RCC1 protein, which catalyses guanine nucleotide exchange on the Ras-related nuclear protein, Ran (or TC4). Genetic studies have suggested that RCC1 may be involved in sensing the replication state of DNA and controlling the activation of Cdc2/cyclin B protein kinase through Ran. In this report, we present direct biochemical evidence for the post-translational control of Cdc2/cyclin B activation by Ran. In a cell-free system of concentrated Xenopus egg extracts supplemented with nuclei, a mutant form of Ran (T24N) analogous to dominant inactive mutants of other Ras-related GTPases inhibits Cdc2/cyclin B activation in the presence of repli-cating nuclear DNA. This role for Ran is mediated through control of the tyrosine phosphorylation state of Cdc2 and appears to be distinct from other effects on nuclear import, nuclear formation and DNA replication. When extracts were supplemented with RCC1 protein prior to addition of Ran T24N, inhibition of Cdc2/cyclin B by Ran T24N was relieved. This suggests that Ran T24N may act in a dominant manner by sequestering RCC1 in an inactive form. In contrast to Ran T24N, a mutant of Ran (Q69L) defective in GTPase activity and hence locked in the GTP-bound state has no inhibitory effect on Cdc2/cyclin B acti-vation. In the light of these results, we propose that gener-ation of the GTP-bound form of Ran is required for Cdc2/cyclin B activation and entry into mitosis when this process is coupled to the progression of S-phase.
Collagen matrices attenuate the collagen-synthetic response of cultured fibroblasts to TGF-β
Authors: Clark Richard A. F. , Nielsen Larry D. , Welch Marshall P. , McPherson John M.
Published: April 2021

Transforming growth factor-β, a potent modulator of cell function, induces fibroblasts cultured on plastic to increase collagen synthesis. In 5- and 7-day porcine skin wounds, which have minimal to moderate collagen matrix, respectively, transforming growth factor-β and type I procollagen were coordinately expressed throughout the granulation tissue. However, in 10-day collagen-rich granulation tissue type I procollagen expression diminished despite persistence of transforming growth factor-β. To investigate whether collagen matrix attenuates the collagen-synthetic response of fibroblasts to transforming growth factor-β, we cultured human dermal fibroblasts in conditions that simulate collagen-rich granulation tissue. Therefore, human dermal fibroblasts were suspended in attached collagen gels and collagen and noncollagen production was assayed in the absence and presence of transforming growth factor-β. Although transforming growth factor-β stimulated collagen synthesis by fibroblasts cultured in the collagen gels, these fibroblasts consistently produced less collagen than similarly treated fibroblasts cultured on plastic. This phenomenon was not secondary to nonspecific binding of transforming growth factor-β to the collagen matrix. Fibroblasts cultured in a fibrin gel responded to transforming growth factor-β similarly to fibroblasts cultured on plastic. Using immunofluorescence probes to type I procollagen, we observed that transforming growth factor-β increased type I procollagen expression in most fibroblasts cultured on plastic, but only in occasional fibroblasts cultured in collagen gels. From these data we conclude that collagen matrices attenuate the collagen synthetic response of fibroblast to transforming growth factor-β in vitro and possibly in vivo.
Putative myosin heavy and light chains in Tetrahymena: co-localization to the basal body-cage complex and association of the heavy chain with skeletal muscle actin filaments in vitro
Authors: Garcés Jorge A. , Hoey John G. , Gavin R. H.
Published: April 2021

The basal body cage is a fibrillar chamber which surrounds each basal body in the ciliate cytoskeleton. The function of this chamber is unknown. In Tetrahymena, the cage contains actin filaments which connect the cage to triplet microtubules. In this study, we have examined the cage for the presence of myosin. Skeletal muscle myosin-II heavy and light chains were used to affinity-purify anti-MHC and anti-MLC antibodies, respectively, from an antiserum raised against Tetrahymena oral apparatus proteins. On western immunoblots of ATP-solubilized Tetrahymena proteins, the anti-MHC antibody detected a putative myosin heavy (180 kDa) chain, and the anti-MLC antibody detected a putative myosin light (18 kDa) chain. The anti-MHC antibody specifically labeled the AI zone of sarcomeres. In cosedimentation assays with an ATP-solubilized protein fraction, the 180 kDa polypeptide associated with skeletal muscle actin filaments in an ATP-dependent manner. The sedimented actin filaments appeared to be organized into bundles. Immunodepletion of the 180 kDa rendered the ATP-solubilized protein fraction ineffective in bundling actin filaments in a cosedimentation assay. ATP-solubilized Tetrahymena proteins, which included the 180 kDa polypeptide, exhibited F-actin-stimulated, Mg2+ ATPase activity and K+, EDTA ATPase activity which are characteristic of myosin ATPases. Immunodepletion of the 180 kDa polypeptide reduced the F-actin, Mg2+ ATPase activity of the ATP-solubilized protein fraction by more than 80%. Based on these various observations, we conclude that the 180 kDa polypeptide is a putative myosin heavy chain, probably a myosin-II and that the 18 kDa polypeptide is probably a myosin-II light chain. We have used the affinity-purified, anti-myosin antibodies with immunofluorescence microscopy and immunogold electron microscopy to map the location of the putative myosin heavy and light chains in Tetrahymena. Immunofluores-cence microscopy showed that the anti-myosin antibodies localized to Tetrahymena somatic and oral region basal bodies. At the ultrastructural level, the anti-myosin anti-bodies localized to filaments in the basal body-cage complex. The labeling patterns with both anti-myosin anti-bodies were identical to the labeling pattern observed with an anti-actin antibody reported in a previous study. The co-localization of myosin and actin argue for a motility system within the basal body-cage complex.
Liver-regulating protein (LRP) is a plasma membrane protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes
Authors: Gérard Nadine , Corlu Anne , Kneip Bernard , Kercret Henri , Rissel Maryvonne , Guguen-Guillouzo Christiane , Jégou Bernard
Published: April 2021

We have identified a liver-regulating protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes in rat testis. Liver-regulating protein was studied using monoclonal antibody L8 prepared from rat primitive biliary epithelial cells. This molecule was located in vivo at the interface of Sertoli cells and spermatocytes, and expressed in a stage-dependent manner (expression peaked on leptotene-zygotene sperma-tocytes). In vitro, the liver-regulating protein was found on Sertoli cell, spermatocyte and early spermatid membranes. Immunoaffinity procedures revealed two peptides of 85 and 73 kDa for Sertoli cells, while spermatocytes and sper-matids displayed a single smaller peptide of 56 kDa. The involvement of the liver-regulating protein in cell interac-tion-mediated regulation of Sertoli cell was assessed in vitro by tracing Sertoli cell transferrin and inhibin secretion, as well as mRNA synthesis in spermatocyte-Sertoli cell cocul-tures and in rat liver biliary epithelial cell-Sertoli cell cocultures, performed in the presence or absence of monoclonal antibody L8. Inhibition of the spermatocyte-and liver biliary epithelial cell-stimulated secretion of transferrin and inhibin by Sertoli cells was observed in the presence of antibody, whereas spermatocyte adhesiveness was unchanged. Using northern blot analysis, the steady state levels of transferrin mRNA decreased when the anti-liver-regulating protein antibody was added to the Sertoli cell-spermatocyte cocultures or to the Sertoli cell-liver biliary epithelial cell cocultures. The data demonstrate the role of the liver-regulating protein in cell-cell contact-mediated regulation of Sertoli function by primary spermatocytes and the important implications of this cell contact-dependent control in testicular activity.
Vimentin and CP49/filensin form distinct networks in the lens which are independently modulated during lens fibre cell differentiation
Authors: Sandilands Aileen , Prescott Alan R. , Carter Jane M. , Hutcheson Aileen M. , Quinlan Roy A. , Richards John , FitzGerald Paul G.
Published: April 2021

The cells of the eye lens contain the type III intermediate filament protein vimentin, as well as two other intermediate filament proteins, CP49 and filensin. These two proteins appear to be unique to the differentiated lens fibre cell. Immunoblotting and confocal microscopy were used to describe changes which occur in these three intermediate filament proteins and the networks they form during fibre cell differentiation and maturation. The vimentin network was present in both epithelial cells and some fibre cells. Fibre cells were vimentin positive up to a specific point 2-3 mm in from the lens capsule where the vimentin signal was drastically reduced. The CP49/filensin network was not present in the undifferentiated epithelial cells but emerged in the differentiating fibre cells. This latter network exhibited a principally plasma membrane localization in younger fibre cells but became more cytoplasmic in older fibre cells. This change also occurred at a distinct point in fibre cell differentiation, much earlier than the observed loss of the vimentin network. The subcellular changes in the distributions of these cytoskeletal networks were correlated to the loss of the fibre cell nucleus, another feature of fibre cell differentiation. No correlation was found to changes in the vimentin network but nuclear loss did coincide with changes in the CP49/filensin network. Concomitant with nuclear pyknosis, there were also changes in the nuclear lamina as well as infringement of the nuclear compartment by CP49, as shown by confocal microscopy. This study demonstrates vimentin and the CP49/filensin network to be independent in the lens but both networks undergo dramatic changes in subcellular distribution during the differentiation/maturation of the fibre cell. Only changes in the CP49/filensin network can be correlated to nuclear loss. Thus in the lens, unlike mammalian erythropoiesis which is also characterized by nuclear loss, the vimentin network does not appear linked to nuclear retention.
Heparin-binding vitronectin up-regulates latent TGF-β production by bovine aortic endothelial cells
Authors: Ribeiro Solange M. F. , Schultz-Cherry Stacey , Murphy-Ullrich Joanne E.
Published: April 2021

Vitronectin, a serum and extracellular matrix protein, is present in vivo in two different conformations: a native form, which does not bind heparin, and a heparin-binding conformer, which results from interactions of native vitronectin with either the thrombin-antithrombin III complex or the terminal complement complex, C5b-9. We found that vitronectin stimulates the activity of the growth regulatory peptide, TGF-β, in the conditioned media of bovine aortic endothelial cells as a result of increased production of latent TGF-β. This effect is specific for the denatured, heparin-binding, form of vitronectin, since native vitronectin has no effect on the production of latent TGF-β by those cells. Stimulation is time and concentration-dependent, but is independent of protease activity. Stimulation is dependent on the presence of cells, since there was no increase in TGF-β activity observed when vit-ronectin was added to the conditioned media after removal from cells. Furthermore, incubation of recombinant latent

TGF-β with vitronectin in a cell-free system does not result in increased TGF-β activity. Assays of total TGF-β levels in heat-treated conditioned media showed that vitronectin treatment elevates the levels of total TGF-β in the conditioned media. These results were further confirmed by western blot analysis of the conditioned media with anti-bodies specific for latent TGF-β. These data suggest that vitronectin regulates expression and/or secretion of TGF-β by bovine aortic endothelial cells. This cellular response to the heparin-binding form of vitronectin seems to be mediated by αvβ3 integrins. Since in vivo situations in which heparin-binding vitronectin is present correlate well with those in which TGF-β activity is increased, we propose that vitronectin may be a significant regulator of TGF-β activity in vivo.
Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts
Authors: Chou Laisheng , Firth James D. , Uitto Veli-Jukka , Brunette Donald M.
Published: April 2021

The regulation of cell shape, fibronectin mRNA level, secretion and assembly by substratum surface topography was investigated in early passage human gingival fibroblasts cultured on titanium-coated smooth or V-shaped grooved substrata produced by micromachining. Cells on grooved surfaces were significantly elongated and orientated along the grooves of the substratum, while cell height, measured using confocal scanning laser microscopy, was ∼1.5-fold greater than that of cells on smooth surfaces. Northern hybridization analysis revealed that on a per cell basis the grooved surface increased the amounts of fibronectin mRNA/cell ∼3.5-fold at 16 hours, ∼1.9-fold at 40 hours and ∼2.2-fold at 90 hours, while the mRNA levels of the house-keeping gene glyceraldehyde-3-phosphate dehy-drogenase (GAPD) were constant. The amounts of secreted fibronectin on the grooved surface were increased ∼2-fold for all time points. The stability of fibronectin mRNA was also altered by substratum surface topography. The half-life of fibronectin mRNA on smooth surfaces was estimated to be ∼5 hours, but on the grooved surfaces the half-life of fibronectin mRNA showed a two-phase response: a rapid 60% reduction in the first half-life (tG ∼2 hours) and a 2.4-fold increase in the second half-life (tG ∼12 hours) relative to that observed on the smooth surface. The GAPD mRNA half-lives were essentially unaffected by the surface topography of the substrata. The grooved surface was also found to alter the amount of fibronectin assembled into the extracellular matrix, producing a ∼2-fold increase in the cultures at all time points. It thus appears that substratum surface topography alters cell shape and modulates fibronectin at the transcriptional and post-transcriptional levels, as well as the amount of fibronectin assembled into extracellular matrix. Micromachining, which has the ability to precisely control surface topography over a wide range of dimensions and shapes, appears to be a useful technique in investigating the relationship between cell shape and function.
Fibronectin fibril growth in the extracellular matrix of the Xenopus embryo
Authors: Winklbauer Rudolf , Stoltz Cornelia
Published: April 2021

We show that the mechanism of fibronectin fibril formation on the blastocoel roof of the Xenopus embryo is comparable to that in other systems. Fibril assembly is inhibited by RGD peptide, by an amino-terminal fragment of fibronectin, and by cytochalasin B. When added exogeneously, intact fibronectin, but not a 110 kDa cell binding fragment of fibronectin, is incorporated into fibrils. Thus, the blastocoel roof of Xenopus represents a valid model system for the study of fibronectin fibril formation in situ. Moreover, we show that fibril formation can be induced experimentally in this system. Examination of fibril elongation by double-labelling experiments reveals that individual, unbranched fibronectin fibrils grow only at one end, i.e. in a unipolar fashion. The rate of elongation is 4.7 μm/min. Most fibrils grow only for a short time, and the increase in total fibril length per cell is driven by the repeated initiation of new fibrils. Assembly of fibronectin into fibrils precedes cross-linking of fibronectin into multimers in this system.
The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells
Authors: Darchen François , Senyshyn Jan , Brondyk William H. , Taatjes Douglas J. , Holz Ronald W. , Henry Jean-Pierre , Denizot Jean-Pierre , Macara Ian G.
Published: April 2021

Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine β-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.
Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells
Authors: Van Itallie Christina M. , Balda Maria S. , Anderson James Melvin
Published: April 2021

Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells.
Hormone-induced meiotic maturation in Xenopus oocytes occurs independently of p70s6k activation and is associated with enhanced initiation factor (eIF)-4F phosphorylation and complex formation
Authors: Morley Simon J. , Pain Virginia M.
Published: April 2021

Hormone-induced meiotic maturation of the Xenopus oocyte is regulated by complex changes in protein phosphorylation. It is accompanied by a stimulation in the rate of translation, manifest at the level of polypeptide chain initiation. At later times in the maturation process, this reflects an increased ability for mRNA to interact with the 40 S ribosomal subunit. In mammalian cells there is growing evidence for the regulation of translation by phosphorylation of ribosomal protein S6 and of initiation factors responsible for the binding of mRNA to ribosomes. In this report, we show that although the 70 kDa form of S6 kinase is activated within 1.5 hours in response to progesterone or insulin, a time critical for protein synthesis, its activation is not required for hormone-induced stimulation of translation rates or maturation. In response to progesterone, activation of translation occurs in parallel with enhanced phosphate labelling of eIF-4α and eIF-4γ and eIF-4F complex formation, events which are thought to facilitate the interaction of eIF-4F with the mRNA cap structure. However, with insulin, activation of translation occurs prior to detectable de novo phosphorylation of eIF-4F, although a small enhancement of turnover of phosphate on eIF-4α may occur at this early time. With either hormone, enhanced phosphate labelling of eIF-4α is shown to reflect activation of eIF-4α kinase(s), which co-incides temporally with activation of p42 MAP and p90rsk kinases. The possible role of initiation factor modification on increased translation rates during meiotic maturation is discussed.
Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum
Authors: Hidalgo Josefina , Muñiz Manuel , Velasco Angel
Published: April 2021

Streptolysin O-permeabilized cells incubated with a high concentration (5-10 mg/ml) of cytosolic proteins and ATP-generating system exhibit redistribution into the endoplasmic reticulum (ER) of Golgi integral proteins (mannosidase II, galactosyltransferase, TGN 38), detected by immuno-fluorescence. In addition, mannosidase II is detected in the ER of cells exposed to a high concentration of cytosolic proteins and processed for immunolectron microscopy by immunoperoxidase. The redistribution process requires ATP and is not affected by previous microtubule depolymerization. Ultrastructural observations indicate that Golgi disassembly occurs by budding of coated vesicles. This stage of the process is inhibited by GTPγS, AlF(3-5), transducin βγ subunits, and mastoparan, indicating the involvement of trimeric G proteins. At a later stage, vesicles lose their coats and fuse with the ER. This part of the process does not occur in cells incubated at either 15°C or 20°C, or exposed to N-ethylmaleimide. In cells treated with either cholera or pertussis toxin Golgi redistribution into the ER shows a 50-fold lower requirement for cytosolic factors than in untreated cells. These data suggest a regulatory role for both αs and αi trimeric G proteins in the normal Golgi-ER retrograde transport taking place in intact cells.
Characterisation of a desmocollin isoform (bovine dsc3) exclusively expressed in lower layers of stratified epithelia
Authors: Yue K. K. M. , Holton J. L. , Clarke J. P. , Hyam J. L. M. , Hashimoto T. , Chidgey M. A. J. , Garrod D. R.
Published: April 2021

Desmocollins are cadherin-like glycoproteins involved in cell adhesion and plaque formation in desmosome junctions. Three distinct isoforms, the products of different genes, have been found in bovine tissues. We have reported previously that one of these, DSC3, is expressed only in basal and lower suprabasal layers of stratified epithelia. Using RT-PCR we have now obtained the complete cDNA coding sequence of mature bovine DSC3. It has alternatively spliced ‘a’ and ‘b’ forms found in other desmocollins but is unique in having a 43 instead of a 46 base pair exon. We have characterised a monoclonal antibody, 07-4G, which is specific for the Dsc3 protein, recognising an epitope in the extracellular domain. Immunofluorescent staining with 07-4G confirms that this isoform is found only in stratified epithelia, being strongly expressed in the basal cell layers of these tissues. The intensity of expression fades gradually in the suprabasal layers and disappears completely below the upper limit of desmosome expression. These results suggest that Dsc3 plays an important role in cell epithelial differentiation.
Immunomorphological characterization and effects of 12-(s)-hete on a dynamic intracellular pool of the αiibβ3-integrin in melanoma cells
Authors: Tímár J. , Bazaz R. , Kimler V. , Haddad M. , Tang D. G. , Robertson D. , Tovari J. , Taylor J. D. , Honn K. V.
Published: April 2021

In metastatic B16a murine melanoma cells, αIIbβ3 integrin was shown to be one of the key adhesion molecules responsible for matrix adhesion and spreading. Upon stimulation, αIIbβ3 can be upregulated at the cell surface due to translocation of the receptor to the plasma membrane from an intracellular pool. Here we have characterized this integrin pool as a tubulovesicular structure (TVS) corresponding to endosomes. TVS was found to be associated temporarily with microtubules and intermediate filaments especially after protein kinase C (PKC) stimulation with a lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. After PKC stimulation, the predominantly vesicular TVS became elongated and αIIbβ3 appeared at the apical plasma membrane and microvilli. Disruption of either the microtubules or intermediate filaments prevented the 12-(S)-HETE effect both on vesicular to tubular transition of TVS as well as on surface expression of this integrin. The connection with the Golgi system of the integrin-containing TVS was proved by a Golgi-inhibitor (brefeldin A) pretreatment, which prevented the PKC-stimulation-induced TVS elongation and subsequent receptor-upregulation at the cell surface. After a soluble ligand binding (mAb to the αIIbβ3 complex) the surface receptor endocytosed back to the TVS indicating the presence of a dynamic, cytoskeleton associated integrin pool in melanoma cells.
Apoptosis precedes necrosis of dystrophin-deficient muscle
Authors: Tidball James G. , Albrecht Douglas E. , Lokensgard Beth E. , Spencer Melissa J.
Published: April 2021

The current view that death of dystrophin-deficient muscle fibers is a necrotic process relies primarily upon the histological appearance of the tissue after the degenerative process is well advanced. Here, we tested this view by examining the possibility that apoptosis is a component of dystrophin-deficient muscle cell death. Three assays for apoptosis were employed in analyzing prenecrotic, peak necrotic and regenerated hindlimb muscle of mdx mice: (1) terminal deoxynucleotidyl transferase (TdT) mediated end-labeling of DNA in nuclei in tissue sections; (2) assays for DNA ladders; and (3) electron microscopic assays for the presence of organelles undergoing structural changes characteristic of apoptosis. At all ages sampled, mdx muscle contained apoptotic nuclei, according to TdT-mediated dUTP labeling of tissue sections. Nuclei in regenerated mdx muscle fibers did not display apoptosis. dUTP-labeled nuclei in control C57 muscles were rare or absent at all ages sampled. DNA from 4-week-old mdx mice was found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. Electron microscopic analysis showed that organelle structural changes indicating apoptosis appear before pathological changes diagnostic of necrosis. For example, condensed mitochondria, fragmented sarcoplasmic reticulum and nuclei with chromatin condensations resembling apoptosis appear in fibers that otherwise possess normal morphology. Together, the findings show that apoptosis precedes any detectable necrotic change in mdx muscle, and that apoptotic events continue into the stage of dystrophic pathology that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in dystrophin-deficient muscle which is followed secondarily by necrotic processes.
Localisation of mRNA for collagenase in osteocytic, bone surface and chondrocytic cells but not osteoclasts
Authors: Fuller K. , Chambers T.J.
Published: April 2021

Osteoclasts resorb the extracellular matrix of bone by secreting protons and enzymes into a circumpherentially sealed compartment between the osteoclast and the bone surface. Although the lysosomal cysteine proteinases play a major role in matrix degradation by osteoclasts, collagenase (matrix metalloproteinase-1, EC 3.4.24.7) is also required for osteoclastic bone resorption, and may be directly involved in collagen degradation in the hemivacuole. We assessed the effects of inhibitors of cysteine proteinases and collagenase on bone resorption by osteoclasts isolated from rodent bone. We found that while inhibition of cysteine proteinases strongly suppressed osteoclastic resorption, inhibitors of collagenase were without effect on the number, size, or demineralised fringe of excavations. We could find no evidence of expression of mRNA for collagenase in rat osteoclasts by in situ hybridisation, but found that it was expressed by chondrocytes, bone surface cells and osteocytes adjacent to osteoclasts. The distribution of these cells, and the correlation between increased collagenase production and increased stimulation of osteoclastic resorption in vitro by bone cells, suggests that these cells might be involved in the regulation of bone resorption in situ, and that collagenase production might play a role in this process.