bluuebunny/crossref_metadata_2025 huggingface dataset:
Vinblastine induces an interaction between FtsZ and tubulin in mammalian cells
Authors: Yu Xuan-chuan , Margolin William , Gonzalez-Garay Manuel L. , Cabral Fernando
Published: April 2021

The Escherichia coli cell division protein FtsZ was expressed in Chinese hamster ovary cells, where it formed a striking array of dots that were independent of the mammalian cytoskeleton. Although FtsZ appears to be a bacterial homolog of tubulin, its expression had no detectable effects on the microtubule network or cell growth. However, treatment of the cells with vinblastine at concentrations that caused microtubule disassembly rapidly induced a network of FtsZ filaments that grew from and connected the dots, suggesting that the dots are an active storage form of FtsZ. Cells producing FtsZ also exhibited vinblastine- and calcium-resistant tubulin polymers that colocalized with the FtsZ network. The FtsZ polymers could be selectively disassembled, indicating that the two proteins were not copolymerized. The vinblastine effects were readily reversible by washing out the drug or by treating the cells with the vinblastine competitor, maytansine. These results demonstrate that FtsZ assembly can occur in the absence of bacterial chaperones or cofactors, that FtsZ and tubulin do not copolymerize, and that tubulin-vinblastine complexes have an enhanced ability to interact with FtsZ.
Integrin α and β subunit contribution to the kinetic properties of α2β1 collagen receptors on human keratinocytes analyzed under hydrodynamic conditions
Authors: Masson-Gadais Bénédicte , Pierres Anne , Benoliel Anne-Marie , Bongrand Pierre , Lissitzky Jean-Claude
Published: April 2021

The adhesion of keratinocytes to type I collagen or laminin 5 was studied in a laminar flow chamber. These experiments provided an insight into the binding kinetics of integrins in their natural environment and the effects of monoclonal antibodies specific for α and β chains. Cells driven by a force too low to alter the natural lifetime of a single bond displayed multiple arrests. Studying the frequency and duration of these arrests yielded fairly direct information on the rate of bond formation (on-rate) and dissociation (off-rate). Off-rate values obtained on collagen or laminin 5 (0.06 seconds−1) were tenfold lower than values determined on selectins. Bond stability was strongly regulated by anti-β1 chain antibodies since the off-rate was decreased sixfold by activating antibody TS2/16 and increased fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibody K20 had no effect on this parameter. Binding frequencies were not significantly changed by all these antibodies. In contrast, both binding frequency and off-rate were altered by antibodies specific for the α2 chain, suggesting that these antibodies interfered with ligand recognition and also with the ligand-β1 chain interactions responsible for bond stabilization. The latter hypothesis was supported by the finding that the partial alteration of α2 chain function by inhibiting antibodies was corrected by anti-β1 chain antibody TS2/16. These results could not be ascribed to allosteric changes of the functional region of β1 integrin subunits regulated by TS2/16 since there was no competition between the binding of TS2/16 and anti-α2 chain antibodies.

Interpreted within the framework of current concepts of integrin-ligand binding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the formation of the ligand-β1 subunit bonding involved in stabilization of the ligand-integrin complex by regulating its dissociation rate.
A human intracellular apyrase-like protein, LALP70, localizes to lysosomal/autophagic vacuoles
Authors: Biederbick Annette , Rose Scott , Elsässer Hans-Peter
Published: April 2021

Using antibodies against autophagic vacuole membrane proteins we identified a human cDNA with an open reading frame of 1848 bp, encoding a protein of 70 kDa, which we named lysosomal apyrase-like protein of 70 kDa (LALP70). Sequence analysis revealed that LALP70 belongs to the apyrase or GDA1/CD39 family and is almost identical to a human uridine diphosphatase, with the exception of nine extra amino acids in LALP70. Members of this family were originally described as ectoenzymes, with some intracellular exceptions. Transfected LALP70 fused to the green fluorescent protein localized in the cytoplasm with a punctate pattern in the perinuclear space. These structures colocalized with the autophagic marker monodansylcadaverine and the lysosomal protein lamp1.

Hydrophobicity analysis of the encoded protein revealed a transmembrane region at the N and C termini. Most of the sequence is arranged between these transmembrane domains, and contains four apyrase conserved regions. In vitro transcription/translation in the presence of microsomes showed that no signal sequence is cleaved off and that the translation product is protected from trypsin treatment. Our data indicate that LALP70 is a type III lysosomal/autophagic vacuole membrane protein with the apyrase conserved regions facing the luminal space of the vacuoles.
A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat
Authors: Kleene Ralf , Zdzieblo Joachim , Wege Konstanze , Kern Horst-Franz
Published: April 2021

Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas a cDNA coding for the 29 kDa protein (ZG29p) was identified by immunoscreening of a hormonally stimulated pancreas cDNA library. Western blot analysis suggests that ZG29p is a pancreas-specific protein and immunofluorescence shows that ZG29p is mainly associated with zymogen granules. Analysis of subcellular fraction applying immunoblotting revealed that ZG29p was localized mainly in the soluble fraction of zymogen granules and in a Golgi- andRER-enriched fraction, but was absent from the cytosol. In isolated zymogen granule content ZG29p was associated with protein complexes containing amylase as main constituent.

The cDNA coding for ZG29p is homologous to the C-terminal region of the candidate metastasis-associated gene mta1. Northern blot analysis and RT-PCR showed that no MTA1 mRNA is present in pancreas from fasted rats and in the rat pancreas carcinoma cell line AR4-2J in its protodifferentiated state. Although no ZG29p specific mRNA was seen in the northern blot analysis, RT-PCR showed that ZG29p was expressed under both non-stimulated and stimulated conditions. The expression of MTA1 was up-regulated in the pancreas by endogenous cholecystokinin release and in AR4-2J after induction of cellular differentiation by dexamethasone. Western blotting and immunofluorescense studies indicated that MTA1p is localized in the nucleus in all tissues studied.

Using genomic DNA in PCR analysis it was shown that two short introns are present flanking the sequences of the 5′end of ZG29p cDNA. One intron contains consensus elements required for pancreas specific transcription initiation, suggesting that MTA1 and ZG29 are differentially expressed by alternative transcription initiation in the pancreas. The localisation of MTA1p in the nucleus of most cell types could signify a general role in gene regulation, while the cell type specific and exclusive expression of ZG29p in pancreatic acinar cells could indicate a role in granule formation.
Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion
Authors: Aballay Alejandro , Stahl Philip D. , Mayorga Luis S.
Published: April 2021

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.
Role of endothelial cell extracellular signal-regulated kinase1/2 in urokinase-type plasminogen activator upregulation and in vitro angiogenesis by fibroblast growth factor-2
Authors: Giuliani Roberta , Bastaki Maria , Coltrini Daniela , Presta Marco
Published: April 2021

Downstream signaling triggered by the binding of fibroblast growth factor-2 (FGF2) to its tyrosine-kinase receptors involves the activation of mitogen-activated protein kinase kinase (MEK) with consequent phosphorylation of extracellular signal-regulated kinases (ERKs). Here we demonstrate that FGF2 induces ERK1/2 activation in bovine aortic endothelial (BAE) cells and that the continuous presence of the growth factor is required for sustained ERK1/2 phosphorylation. This is prevented by the MEK inhibitors PD 098059 and U0126, which also inhibit FGF2-mediated upregulation of urokinase-type plasminogen activator (uPA) and in vitro formation of capillary-like structures in three-dimensional type I collagen gel.

Various FGF2 mutants originated by deletion or substitution of basic amino acid residues in the amino terminus or in the carboxyl terminus of FGF2 retained the capacity to induce a long-lasting activation of ERK1/2 in BAE cells. Among them, K128Q/R129Q-FGF2 was also able to stimulate uPA production and morphogenesis whereas R129Q/K134Q-FGF2 caused uPA upregulation only. In contrast, K27,30Q/R31Q-FGF2, K128Q/K138Q-FGF2 and

R118,129Q/K119,128Q-FGF2 exerted a significant uPA-inducing and morphogenic activity in an ERK1/2-dependent manner only in the presence of heparin. Furthermore, no uPA upregulation and morphogenesis was observed in BAE cells treated with the deletion mutant Δ27-32-FGF2 even in the presence of soluble heparin. Thus, mutational analysis of FGF2 dissociates the capacity of the growth factor to induce a persistent activation of ERK1/2 from its ability to stimulate uPA upregulation and/or in vitro angiogenesis.

In conclusion, the data indicate that ERK1/2 phosphorylation is a key step in the signal transduction pathway switched on by FGF2 in endothelial cells. Nevertheless, a sustained ERK1/2 activation is not sufficient to trigger uPA upregulation and morphogenesis. FGF2 mutants may represent useful tools to dissect the signal transduction pathway(s) mediating the complex response elicited by an angiogenic stimulus in endothelial cells.
Chromatid cohesion during mitosis: lessons from meiosis
Authors: Rieder Conly L. , Cole Richard
Published: April 2021

The equal distribution of chromosomes during mitosis and meiosis is dependent on the maintenance of sister chromatid cohesion. In this commentary we review the evidence that, during meiosis, the mechanism underlying the cohesion of chromatids along their arms is different from that responsible for cohesion in the centromere region. We then argue that the chromatids on a mitotic chromosome are also tethered along their arms and in the centromere by different mechanisms, and that the functional action of these two mechanisms can be temporally separated under various conditions. Finally, we demonstrate that in the absence of a centromeric tether, arm cohesion is sufficient to maintain chromatid cohesion during prometaphase of mitosis. This finding provides a straightforward explanation for why mutants in proteins responsible for centromeric cohesion in Drosophila (e.g. ord, mei-s332) disrupt meiosis but not mitosis.
Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade
Authors: Agnew Brian J. , Duman Joseph G. , Watson Charles L. , Coling Donald E. , Forte John G.
Published: April 2021

Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H,K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H,K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H,K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.
Induced differentiation in HT29, a human colon adenocarcinoma cell line
Authors: Cohen Esther , Ophir Ilana , Shaul Yehuda Ben
Published: April 2021

The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma.
Metalloproteinase-mediated release of the ectodomain of L1 adhesion molecule
Authors: Beer Sandra , Oleszewski Matthias , Gutwein Paul , Geiger Claudia , Altevogt Peter
Published: April 2021

The L1 adhesion molecule is an approx. 200-220 kDa type I membrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. L1 can bind in a homotypic fashion and was shown to support integrin-mediated binding via RGDs in the 6th Ig-like domain. In addition to its cell-surface expression, L1 can occur in the extracellular matrix (ECM). Here we demonstrate that L1 is constitutively released from the cell surface by membrane-proximal cleavage. L1 shed from B16F10 melanoma cells remains intact and can serve as substrate for integrin-mediated cell adhesion and migration. The release of L1 occurs in mouse and human cells and is blocked by the metalloproteinase inhibitor TAPI (Immunex compound 3). This compound has been shown previously to block release of L-selectin and TNF-α which is mediated by the membrane-bound metalloproteinase TNF-α converting enzyme (TACE). Using CHO cells that are low in TACE expression and do not release L-selectin we demonstrate that L1 release is distinct from L-selectin shedding. We propose that cell-surface release may be necessary for the conversion of L1 from a membrane into an ECM protein.
Functional characterisation of tetanus and botulinum neurotoxins binding domains
Authors: Lalli Giovanna , Herreros Judit , Osborne Shona L. , Montecucco Cesare , Rossetto Ornella , Schiavo Giampietro
Published: April 2021

Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial neurotoxins.
Matrix expression and proliferation of primary gingival fibroblasts in a three-dimensional cell culture model
Authors: Hillmann Georg , Gebert Andreas , Geurtsen Werner
Published: April 2021

The growth of cultured primary human gingival fibroblasts and the three-dimensional arrangement of the extracellular matrix in a polyester carrier system was investigated using various histological techniques. The results were compared with monolayer cultures. Collagen types I, III, V, and VI were investigated by conventional and fluorescence microscopy, scanning and transmission electron microscopy, and confocal laser scanning microscopy. Human gingival fibroblasts were obtained from tissue biopsies of five donors and were cultivated up to 5 weeks under three-dimensional culture conditions. The cells displayed an elongated, spindle-like or stellate morphology resembling the in vivo situation. Collagen type I revealed thick fiber bundles, and collagens type III and V were distributed as fine fibrils or small bundles throughout the culture system. Frequently, the fibers were oriented parallel to the long axis of the cells. Type VI collagen formed thin fibers and revealed a reticular pattern. In histological sections the cultured cells exhibited a morphology clearly different from that of cells cultured in monolayers. Their shape and spatial distribution resembled that of cells in tissue biopsies more closely. The culture system presented here promotes a dynamic model for performing studies for instance on the interactions of cultured cells with extracellular matrix molecules, on the pathogenesis of inflammatory processes or on the interactions with biomaterials, thus providing qualitative and quantitative information.
Dynamic rearrangement of the spectrin membrane skeleton during the generation of epithelial polarity in Drosophila
Authors: Thomas Claire M. , Williams Janice A.
Published: April 2021

The origin of epithelial cell polarity during development is a fundamental problem in cell biology. Central to this process is the establishment of asymmetric membrane domains that will ultimately form the apical and basolateral surfaces. The spectrin-based membrane skeleton has long been thought to participate in the generation of this asymmetry. Drosophila melanogaster contains two known β-spectrin isoforms: a conventional β-spectrin chain, and the novel isoform βHeavy-spectrin. These two proteins are restricted to the basolateral and apical membrane domains, respectively. To assay for the emergence of membrane asymmetry, we have characterized the distribution of these two β-spectrins during the formation of the primary epithelium in the fly embryo. Our results show that the syncytial embryo contains a maternally established apical membrane skeleton containing βHeavy-spectrin into which the basolateral β-spectrin membrane skeleton is added. We have called this process basolateral interpolation. Although basolateral membrane skeleton addition begins during cellularization, it does not become fully established until the formation of a mature zonula adherens at mid to late gastrulation. The behavior of β-spectrin is consistent with a primary role in establishing and/or maintaining the basolateral domain while the behavior of βHeavy-spectrin suggests that its primary role is associated with a specialized DE-cadherin complex associated with the furrow canals and with the maturation of the zonula adherens. Thus, the apical spectrin membrane skeleton appears to play a distinct rather than analogous role to the basolateral spectrin membrane skeleton, during the emergence of cell polarity. We find that there are several parallels between our observations and previous studies on the establishment of primary epithelial polarity in vertebrates, suggesting that basolateral interpolation of the membrane skeleton may be a common mechanism in many organisms.
α3β1 integrin regulates epithelial cytoskeletal organization
Authors: Wang Zemin , Symons Jordan M. , Goldstein Stuart L. , McDonald Alice , Miner Jeffrey H. , Kreidberg Jordan A.
Published: April 2021

Epithelial cell morphology and cytoskeletal organization are determined by interactions, with both adjacent cells and the extracellular matrix, which are mediated by integrins and cadherins. Little is known, however, of the relative contributions of integrins and cadherins to maintaining the sub-cortical cytoskeleton characteristic of epithelial cells. Since most studies that utilize integrin-blocking antibodies result in a loss of both cell-cell adhesion and sub-cortical cytoskeletal organization, it has been difficult to distinguish whether integrins and cadherins both mediate cytoskeletal assembly in epithelial cells. Therefore, cells derived from kidney collecting ducts of α3β1 integrin-deficient mice were used to examine the role of integrins in epithelial cell morphology and cytoskeletal organization. In primary cell culture, α3β1 integrin-deficient kidney collecting duct cells maintain cadherin-mediated cell-cell adhesions but fail to form the sub-cortical cytoskeleton that is characteristic of epithelial cells, and instead assemble actin stress fibers. Moreover, the cell-cell junctions in mutant cells were irregular, rather than being uniformly oriented perpendicular to the culture substrate. These results demonstrated that integrins have an primary and essential function in establishing and maintaining the sub-cortical cytoskeleton that is characteristic of epithelial cells. To further study the role of α3β1 integrin in establishing and maintaining cytoskeletal organization in tubular epithelial cells, we derived immortalized cell lines from wild-type and α3β1 integrin-deficient kidney collecting ducts that duplicated the cytoskeletal and cadherin organization observed in primary cells. E-cadherin and α- and β-catenin were complexed together in equal amounts in membranes of wild-type and α3β1 integrin-deficient cells. However, association of the cadherin:catenin complex with α-actinin was greatly decreased in mutant cells, indicating that integrin-mediated assembly of the sub-cortical cytoskeleton is essential for subsequent association of the cytoskeleton with the cadherin:catenin complex. These results present direct evidence for integrin:cadherin cross-regulation in which cadherin function is dependent on the presence of an integrin.
Activation of the small GTPase Cdc42 by the inflammatory cytokines TNFα and IL-1, and by the Epstein-Barr virus transforming protein LMP1
Authors: Puls Axel , Eliopoulos Aristides G. , Nobes Catherine D. , Bridges Tina , Young Lawrence S. , Hall Alan
Published: April 2021

Cdc42, a Rho-family GTPase, has been implicated in several signal transduction pathways, including organization of the actin cytoskeleton, activation of the c-Jun N-terminal MAP kinase (JNK) and stimulation of the nuclear transcription factor kappa B (NFκB). We report here that exposure of fibroblasts to the inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) triggers the activation of Cdc42 leading first to filopodia formation and subsequently to Rac and Rho activation. Inhibition of Cdc42 completely suppresses cytokine-induced actin polymerization, but not activation of JNK or NFκB. The latent membrane protein 1 of Epstein-Barr virus, LMP1, is thought to mimic constitutively activated TNF family receptors. When expressed in fibroblasts, LMP1 stimulates Cdc42-dependent filopodia formation as well as JNK and NFκB activation. Using LMP1 mutants, we show that activation of Cdc42 and JNK/NFκB occur through distinct pathways and that Cdc42 activation is independent of LMP1’s interaction with TRADD and TRAF proteins.
Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/β-catenin
Authors: Hiscox Stephen , Jiang Wen G.
Published: April 2021

Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. The function of these proteins is to link the plasma membrane to the actin cytoskeleton. Merlin is defective or absent in schwannomas and meningiomas and has been suggested to function as a tumour suppressor. In this study, we have examined the role of ezrin as a potential regulator of the adhesive and invasive behaviour of tumour cells. We have shown that following inhibition of ezrin expression in colo-rectal cancer cells using antisense oligonucleotides, these cells displayed a reduced cell-cell adhesiveness together with a gain in their motile and invasive behaviour. These cells also displayed increased spreading over matrix-coated surfaces. Immunofluorescence studies revealed that antisense-treated cells also displayed an increased staining of paxillin in areas representing focal adhesions. Furthermore, coprecipitation studies revealed an association of ezrin with E-cadherin and β-catenin. Induction of the phosphorylation of ezrin by orthovanadate and hepatocyte growth factor/scatter factor resulted in changes similar to those seen with antisense treatment, together with a marked decrease in the association of ezrin with both β-catenin and E-cadherin. It is concluded that ezrin regulates cell-cell and cell-matrix adhesion, by interacting with cell adhesion molecules E-cadherin and β-catenin, and may thus play an important role in the control of adhesion and invasiveness of cancer cells.
Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms
Authors: Valdenaire Olivier , Barret Alain , Schweizer Anja , Rohrbacher Elisabeth , Mongiat Françoise , Pinet Florence , Corvol Pierre , Tougard Claude
Published: April 2021

Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting endothelins. Three ECE-1 isoforms, differing by their N-terminal cytoplasmic tails, are generated from a single gene. When expressed in CHO cells, they display comparable enzymatic activity but whereas ECE-1a is strongly expressed at the cell surface, ECE-1b is exclusively intracellular and ECE-1c presents an intermediate distribution. In the present study these different localizations were further described at the ultrastructural level, by electron microscope immunocytochemistry. To characterize the motifs responsible for the intracellular localization of ECE-1b we constructed chimeric proteins and point mutants. Two di-leucine-based motifs, contained in the N-terminal part of ECE-1b, were thus identified. One of these motifs (LV), displayed by both ECE-1b and ECE-1c, accounts for the reduced surface expression of ECE-1c as compared to ECE-1a. Mutation of both motifs (LL and LV) induces a very strong appearance of ECE-1b at the cell surface indicating that their presence in the N-terminal extremity of ECE-1b is critical for its exclusively intracellular localization.
The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression
Authors: Christopher Renee A. , Judge Stephen R. , Vincent Peter A. , Higgins Paul J. , McKeown-Longo Paula J.
Published: April 2021

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin’s amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin’s amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.
Rapid recruitment of late endosomes and lysosomes in mouse macrophages ingesting Candida albicans
Authors: Káposzta Rita , Maródi László , Hollinshead Michael , Gordon Siamon , da Silva R. P.
Published: April 2021

Candida albicans is an important opportunistic pathogen, whose interaction with cells of the immune system, in particular macrophages (MØ), is poorly understood. In order to learn more about the nature of the infectious mechanism, internalisation of Candida albicans was studied in mouse MØ by confocal immunofluorescence and electron microscopy in comparison with latex beads of similar size, which were coated with mannosyl-lipoarabinomannan (ManLAM) to target the MØ mannose receptor (MR). Uptake of Candida yeasts had characteristics of phagocytosis, required intact actin filaments, and depended on the activity of protein kinase C (PKC). Candida phagosomes rapidly attracted lysosome-associated membrane protein (Lamp)-rich vacuoles, indicative of fusion with late endosomes and lysosomes. Rapid recruitment of late endosomes and lysosomes could be observed regardless of heat-inactivation or serum-opsonisation of Candida, but did not follow binding of the mannosylated-beads to MØ, which suggest that this phenotype is not MR-specific. The yeasts developed germ tubes within phagolysosomes, distended their membranes and escaped, destroying the non-activated MØ. The filamentous form of Candida could penetrate intact MØ even when phagocytosis was blocked, and also attracted Lamp-rich organelles. Inhibition of lysosomal acidification and associated lysosomal fusion reduced germ tube formation of Candida within the phagolysosomes. These data suggest that rapid recruitment of late endocytic/ lysosomal compartments by internalising C. albicans favours survival and virulence of this pathogen.
Tensional forces in fibrillar extracellular matrices control directional capillary sprouting
Authors: Korff Thomas , Augustin Hellmut G.
Published: April 2021

During angiogenesis, anastomosing capillary sprouts align to form complex three-dimensional networks of new blood vessels. Using an endothelial cell spheroid model that was developed to study endothelial cell differentiation processes, we have devised a novel collagen gel-based three-dimensional in vitro angiogenesis assay. In this assay, cell number-defined, gel-embedded endothelial cell spheroids act as a cellular delivery device, which serves as a focal starting point for the sprouting of lumenized capillary-like structures that can be induced to form complex anastomosing networks. Formation of capillary anastomoses is associated with tensional remodeling of the collagen matrix and directional sprouting of outgrowing capillaries towards each other. To analyze whether directional sprouting is dependent on cytokine gradients or on endothelial cell-derived tractional forces transduced through the extracellular matrix, we designed a matrix tension generator that enables the application of defined tensional forces on the extracellular matrix. Using this matrix tension generator, causal evidence is presented that tensional forces on a fibrillar extracellular matrix such as type I collagen, but not fibrin, are sufficient to guide directional outgrowth of endothelial cells. RGD peptides but not control RAD peptides disrupted the integrity of sprouting capillary-like structures and induced detachment of outgrowing endothelial cells cultured on top of collagen gels, but did not inhibit primary outgrowth of endothelial cells. The data establish the endothelial cell spheroid-based three-dimensional angiogenesis technique as a standardized, highly reproducible quantitative assay for in vitro angiogenesis studies and demonstrate that integrin-dependent matrix tensional forces control directional capillary sprouting and network formation.
Interaction between the Ret finger protein and the int-6 gene product and co-localisation into nuclear bodies
Authors: Morris-Desbois Christelle , Bochard Valérie , Reynaud Caroline , Jalinot Pierre
Published: April 2021

The mouse int-6 gene was identified in mammary tumors as an integration site for the mouse mammary tumor virus. Its human counterpart encodes a product that interacts with the Tax viral oncoprotein of the human T cell leukaemia virus type 1. This interaction impedes the localisation of over-expressed Int-6 in nuclear bodies containing the promyelocytic leukaemia gene product (PML). In this study, Int-6 is characterised as a 52 kDa protein that is localised within nuclear bodies in primary lymphocytes. Screening of a human B cell cDNA library for proteins that interact with Int-6 led to isolation of four clones coding for the p110 subunit of eIF3, in accordance with previous detection of Int-6 in purified forms of this translation initiation factor. Another clone was interesting with respect to the subcellular localisation of Int-6. It encodes the Ret finger protein (Rfp) which interacts with PML and localises within a subset of PML nuclear bodies. The interaction of Rfp with Int-6 is mediated through a region in Rfp designated ‘Rfp domain’, distinct from that involved in the interaction with PML. Int-6 and Rfp are co-localised in certain PML nuclear bodies in lymphocytes and transfection studies in HeLa cells strongly suggest that Rfp triggers translocation of Int-6 to nuclear bodies.
Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation
Authors: Brouard Michel , Casado Mariano , Djelidi Sabri , Barrandon Yann , Farman Nicolette
Published: April 2021

The amiloride-sensitive epithelial sodium channel (ENaC) is a main determinant of sodium absorption in renal and colonic epithelial cells. Surprisingly, it is also expressed in non-transporting epithelia such as the epidermis. To gain insight into the putative role of ENaC in keratinocytes, we have evaluated its expression in human skin and in cultured human keratinocytes. Our results indicate that (1) ENaC is expressed in the epidermis and in cultured keratinocytes, at the mRNA and at the protein levels, (2) the ratio of expression of the different ENaC subunits is drastically modified at the protein level during cell growth and differentiation, with a selective upregulation of the beta subunit, (3) no transepithelial sodium transport function is apparent in cultured keratinocytes, but patch-clamp recordings indicate the existence of functional sodium channels with properties similar to those of the cloned ENaC and (4) ENaC inhibition does not alter keratinocyte proliferation, but it significantly decreases the frequency of dome formation in confluent keratinocyte cultures. These results document for the first time the characteristics of ENaC subunit expression in human keratinocytes, and suggest that ENaC may be important during differentiation.
Chromosomes as well as chromosomal subdomains constitute distinct units in interphase nuclei
Authors: Visser Astrid E. , Aten Jacob A.
Published: April 2021

Fluorescence in situ hybridization has demonstrated that chromosomes form individual territories in interphase nuclei. However, this technique is not suitable to determine whether territories are mutually exclusive or interwoven. This notion, however, is essential for understanding functional organizations in the cell nucleus. Here, we analyze boundary areas of individual chromosomes during interphase using a sensitive method based on replication labeling and immunocytochemistry. Thymidine analogues IdUrd and CldUrd were incorporated during S-phase into DNA of Chinese Hamster fibroblasts. Cells labeled with IdUrd were fused with cells labeled with CldUrd. Fused nuclei contained both IdUrd or CldUrd labeled chromosomes. Alternatively, the two labels were incorporated sequentially during successive S-phases and segregated to separate chromosomes by culturing the cells one more cell cycle. Metaphase spreads showed IdUrd-, CldUrd- and unlabeled chromosomes. Some chromatids were divided sharply in differently labeled subdomains by sister chromatid exchanges. With both methods, confocal imaging of interphase nuclei revealed labeled chromosomal domains containing fiber-like structures and unlabeled areas. At various sites, fiber-like structures were embedded in other territories. Even so, essentially no overlap between chromosome territories or between subdomains within a chromosome was observed. These observations indicate that chromosome territories and chromosomal subdomains in G1-phase are mutually exclusive at the resolution of the light microscope.
Cytoskeletal changes induced by GRAF, the

G

TPase

r

egulator

a

ssociated with

f

ocal adhesion kinase, are mediated by Rho
Authors: Taylor Joan M. , Macklem Marlene M. , Parsons J. Thomas
Published: April 2021

Graf, the GTPase regulator associated with focal adhesion kinase was previously shown to have GAP activity for Rho A and Cdc42 in vitro (Hildebrand et al 1996 Mol. Cell Biol. 16: 3169-3178). In this study we sought to determine whether Graf acted at the level of Cdc42, Rho, or both in vivo and whether Graf was a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes were observed following injection of the Rho-inhibitor, C3 into these cells. The Graf response was dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) which block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, we demonstrate that Graf blocks sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results we examined the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types.
Overexpression of protein kinase C-α in the epidermis of transgenic mice results in striking alterations in phorbol ester-induced inflammation and COX-2, MIP-2 and TNF-α expression but not tumor promotion
Authors: Wang Hui Qin , Smart Robert C.
Published: April 2021

Protein kinase Cα (PKCα) is one of six PKC isoforms expressed in keratinocytes of mouse epidermis. To gain an understanding of the role of epidermal PKCα, we have localized its expression to specific cells of normal mouse skin and examined the effect of keratin 5 (K5) promoter directed expression of PKCα in transgenic mice. In normal mouse skin, PKCα was extensively expressed in the outer root sheath (ORS) keratinocytes of the anagen hair follicle and weakly expressed in keratinocytes of interfollicular epidermis. K5-targeted expression of PKCα to epidermal basal keratinocytes and follicular ORS keratinocytes resulted in a tenfold increase in epidermal PKCα. K5-PKCα mice exhibited no abnormalities in keratinocyte growth and differentiation in the epidermis. However, a single topical treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a striking inflammatory response characterized by edema and extensive epidermal infiltration of neutrophils that formed intraepidermal microabscesses in the epidermis.

Compared to TPA-treated wild-type mice, the epidermis of TPA-treated K5-PKCα mice displayed increased expression of cyclooxygenase-2 (COX-2), the neutrophil chemotactic factor macrophage inflammatory protein-2 (MIP-2) mRNA and the proinflammatory cytokine TNFα mRNA but not IL-6 or IL-1α mRNA. To determine if K5-PKCα mice display an altered response to TPA-promotion, 7,12-dimethylbenz[a]anthracene-initiated K5-PKCα mice and wild-type mice were promoted with TPA. No differences in papilloma incidence or multiplicity were observed between K5-PKCα mice and wild-type littermates. These results demonstrate that the overexpression of PKCα in epidermis increases the expression of specific proinflammatory mediators and induces cutaneous inflammation but has little to no effect on epidermal differentiation, proliferation or TPA tumor promotion.
Evidence for the presence of an NF-κB signal transduction system in Dictyostelium discoideum
Authors: Traincard François , Ponte Eleonora , Pun Jason , Coukell Barrie , Veron Michel
Published: April 2021

The Rel/NF-κB family of transcription factors and regulators has so far only been described in vertebrates and arthropods, where they mediate responses to many extracellular signals. No counterparts of genes coding for such proteins have been identified in the Caenorhabditis elegans genome and no NF-κB activity was found in Saccharomyces cerevisiae. We describe here the presence of an NF-κB transduction pathway in the lower eukaryote Dictyostelium discoideum. Using antibodies raised against components of the mammalian NF-κB pathway, we demonstrate in Dictyostelium cells extracts the presence of proteins homologous to Rel/NF-κB, IκB and IKK components. Using gel-shift experiments in nuclear extracts of developing Dictyostelium cells, we demonstrate the presence of proteins binding to κB consensus oligonucleotides and to a GC-rich κB-like sequence, lying in the promoter of cbpA, a developmentally regulated Dictyostelium gene encoding the Ca2+-binding protein CBP1. Using immunofluorescence, we show specific nuclear translocation of the p65 and p50 homologues of the NF-κB transcription factors as vegetatively growing cells develop to the slug stage. Taken together, our results strongly indicate the presence of a complete NF-κB signal transduction system in Dictyostelium discoideum that could be involved in the developmental process.
Fibrillin assembly: dimer formation mediated by amino-terminal sequences
Authors: Ashworth Jane L. , Kelly Vicky , Wilson Richard , Shuttleworth C. Adrian , Kielty Cay M.
Published: April 2021

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9-11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin. Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.