bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges
Authors: Kruse Michael , Steffen Renate , Batel Renato , Müller Isabel M. , Müller Werner E. G. 
Published: April 2021

ABSTRACT
Very recently, Porifera (sponges) have been proven to be suitable model systems to study auto- and allograft recognition at the molecular level. Several potential immune molecules have been isolated from the marine sponges Suberites domuncula and Geodia cydonium, among them those which comprise Ig-like domains in their extracellular part. Here we report on the isolation of two cDNAs from S. domuncula that code for molecules involved in mammals in cytokine-mediated graft response; a putative allograft inflammatory factor 1 (AIF-1) and a non-selenium glutathione peroxidase (GPX). Both polypeptides share high similarity with the corresponding mammalian proteins. The expression of the two genes during auto- and allograft recognition in S. domuncula and G. cydonium was determined. It is shown that the expression of the AIF-1-related gene is upregulated only in allografts, while the GPX-related gene is expressed in the fusion zones formed between auto-as well as allografts. Taken together, these findings suggest that besides cell-mediated defense reactions a cytokine-dependent immune response is also elicited during graft recognition in sponges.
Sec61β, a subunit of the protein translocation channel, is required during: Drosophila development
Authors: Valcárcel Rafael , Weber Ursula , Jackson David B. , Benes VladimÍr , Ansorge Wilhelm , Bohmann Dirk , Mlodzik Marek 
Published: April 2021

ABSTRACT
We have identified and isolated mutations in the first Drosophila gene encoding a subunit of the Sec61 protein translocation channel, DSec61β. While neither the Saccharomyces cerevisiae Sec61β nor its functional Escherichia coli homologue are essential for viability or for protein translocation, we show that DSec61β is essential for embryonic development. Homozygous mutant embryos die at the end of embryogenesis and are impaired in the secretion of cuticle proteins from the epidermis. DSec61β germ line clones, result in defects in dorso-ventral patterning of the egg and are consistent with affected secretion of the protein Gurken from the oocyte to the follicle cells. Clonal analyses in the imaginal discs reveal defects in adult structures, including rhabdomere morphogenesis and a reduction of the size of tarsal segments in the leg. This is the first in vivo study of a component of the protein translocation machinery in higher eukaryotes, and illustrates how a protein that has an inessential, kinetic function in single-cell organisms can become critical for the complex development of a multicellular organism.
Detection of cytokeratin dynamics by time-lapse fluorescence microscopy in living cells
Authors: Windoffer Reinhard , Leube Rudolf E. 
Published: April 2021

ABSTRACT
To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells. Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin 13 and is therefore a reliable in vivo tag for this polypeptide and the structures formed by it. Time-lapse fluorescence microscopy reveals that the cytokeratin 13-containing network is in constant motion, resulting in continuous restructuring occurring in single and migratory cells, as well as in desmosome-anchored cells. Two major types of movement are distinguished: (i) oscillations of mostly long filaments, and (ii) an inward-directed flow of fluorescence originating as diffuse material at the cell periphery and moving in the form of dots and thin filaments toward the deeper cytoplasm where it coalesces with other filaments and filament bundles. Both movements are energy dependent and can be inhibited by nocodazole, but not by cytochalasin D. Finally, disassembly and reformation of cytokeratin filament networks are documented in dividing cells revealing distinct and rapidly occurring stages of cytokeratin organisation and distribution.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs0841.html http://www.uni-mainz.de/FB/Medizin/Anatomie/Leube/
Inhibition of cadherin function differentially affects markers of terminal differentiation in cultured human keratinocytes
Authors: Hines Michelle D. , Jin Hong C. , Wheelock Margaret J. , Jensen Pamela J. 
Published: April 2021

ABSTRACT
Cadherin function is required for normal keratinocyte intercellular adhesion and stratification. In the present study, we have investigated whether cadherin-cadherin interactions may also modulate keratinocyte differentiation, as evidenced by alterations in the levels of several differentiation markers. Confluent keratinocyte cultures, propagated in low Ca2+medium in which cadherins are not active, were pre-incubated with antibodies that block the function of E-cadherin and/or P-cadherin; Ca2+was then elevated to 1 mM to activate the cadherins and induce differentiation. In control cultures (incubated with no antibody or with antibodies to other cell surface molecules), Ca2+elevation induced an increase in type 1 transglutaminase, profilaggrin, and loricrin, as measured by western blotting and in agreement with previous results. However, the concurrent addition of antibodies against both E- and P-cadherin prevented this increase in transglutaminase 1 protein. Incubation with either antibody alone had no consistent effect. Profilaggrin and loricrin, which are later markers of keratinocyte differentiation, responded differently from transglutaminase 1 to addition of antibodies. In the presence of anti-E-cadherin antibody, both loricrin and profilaggrin levels were dramatically enhanced compared to the high Ca2+control cells, while addition of antibody to P-cadherin slightly attenuated the Ca2+-induced increase. In the presence of both antibodies, loricrin and profilaggrin protein levels were intermediate between those observed in the presence of either antibody alone. The expression of involucrin, however, was unaffected by addition of antibodies. In addition, effects of the anti-cadherin antibodies were not secondary to alterations in proliferation or programmed cell death, as determined by several independent assays of these processes. Thus, the consequences of cadherin inhibition depend upon both the particular cadherin and the differentiation marker under study. Taken together, these data suggest that E-cadherin and P-cadherin contribute to the orderly progression of terminal differentiation in the epidermis in multiple ways.
Identification of PA2.26 antigen as a novel cell-surface mucin-type glycoprotein that induces plasma membrane extensions and increased motility in keratinocytes
Authors: Scholl Francisco G. , Gamallo Carlos , Vilaró Senén , Quintanilla Miguel 
Published: April 2021

ABSTRACT
PA2.26 antigen was identified as a cell-surface protein induced in epidermal carcinogenesis and skin remodeling processes. PA2.26 is expressed in carcinoma cell lines and cultured fibroblasts but absent in nontumorigenic keratinocytes. In tissues, PA2.26 is present in epithelial cells of the choroid plexus, ependyma, glomerulus and alveolus, in mesothelial cells, and in endothelia of lymphatic vessels. Biochemical characterization of PA2.26 protein and sequence analysis of the isolated cDNA demonstrate that PA2.26 antigen is a mucin-like transmembrane glycoprotein. Confocal and immunoelectron microscopy analysis in cultured cells reveal that PA2.26 is concentrated in actin-rich microvilli and plasma membrane projections, such as filopodia, lamellipodia and ruffles, where it colocalizes with members of the ERM (ezrin, radixin, moesin) family protein. Ezrin and moesin, but not radixin, can be coimmunoprecipitated together with PA2.26 from cell lysates. Ectopic expression of PA2.26 in immortalized, nontumorigenic, keratinocytes induces an epithelial-fibroblastoid morphological conversion with increased plasma membrane extensions, concomitantly to a major reorganization of the actin cytoskeleton, redistribution of ezrin to cell-surface projections, and enhanced motility. These findings suggest an involvement of PA2.26 in cell migration.
Vimentin contributes to human mammary epithelial cell migration
Authors: Gilles Christine , Polette Myriam , Zahm Jean-Marie , Tournier Jean-Marie , Volders Laure , Foidart Jean-Michel , Birembaut Philippe 
Published: April 2021

ABSTRACT
Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound’s edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound’s edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound’s edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.
Characterization of a coiled coil protein present in the basal body of Trypanosoma brucei
Authors: Dilbeck Vincent , Berberof Magali , Cauwenberge Anne Van , Alexandre Henri , Pays Etienne 
Published: April 2021

ABSTRACT
TBBC(for Trypanosoma bruceibasal body component) is a unique gene transcribed in a 4.8 kb mRNA encoding a 1,410 amino acid protein that consists almost entirely of a coiled coil structure. This protein appeared to localize in the basal body, with an accessory presence at the posterior end of the cell, the nucleus and over the flagellum. Since the two other known components of the trypanosome basal body are γ-tubulin and an uncharacterized component termed BBA4 we performed double immunofluorescence experiments with anti-TBBC and either anti-BBA4 or anti-γ-tubulin antibodies. These three components did not colocalize but were very closely associated, BBA4 being the most proximal to the kinetoplast DNA. Anti-TBBC antibodies detected a 170 kDa protein in western blots of total HeLa cell extracts. Moreover, these antibodies stained the centriole of HeLa and COS cells as well as the centriole of mouse spermatozoa, indicating that a TBBC-like centriolar component has been conserved during the evolution of eukaryotes.
Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the β1 and β3 families
Authors: Testaz Sandrine , Delannet Muriel , Duband Jean-Loup 
Published: April 2021

ABSTRACT
Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely α3β1, α4β1, α5β1, α8β1, αvβ1, αvβ3 and a β8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only αvβ1 and αvβ3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the β1 and β3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by αvβ1 and α8β1, migration involved primarily α4β1, αvβ3 and α8β1 and, more indirectly, α3β1. α5β1 and the β8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of α4β1 and αvβ3, individually or in combination using antibodies or recombinant VCAM-1 and PECAM-1 molecules as a substratum, was required for migration but was not sufficient to produce migration of the cell population as efficiently as with fibronectin. In conclusion, our study indicates that neural crest cells express a multiplicity of fibronectin-binding integrins and suggests that dispersion of the cell population requires cooperation between distinct integrins regulating different events of cell adhesion, locomotion and, possibly, proliferation and survival.
Integrin subunits β1C-1 and β1C-2 expressed in GD25T cells are retained and degraded intracellularly rather than localised to the cell surface
Authors: Svineng Gunbjørg , Johansson Staffan 
Published: April 2021

ABSTRACT
We have previously identified the integrin β1C-2 and characterised the distribution of β1C-1 and β1C-2 transcripts in various cell lines and normal cells. In this study we have investigated the expression of the two β1C-variants in integrin β1 deficient mouse GD25T cells. After stable transfection of the GD25T cells with cDNAs coding for β1A, β1C-1 and β1C-2, the cell surface expression of the β1C-1 and β1C-2 variants was found to be very low while the β1A variant was expressed at high levels. Northern blot analysis showed that the level of β1-transcript in the β1C-1 and β1C-2 clones was equal or higher than in the β1A clones. Metabolic labelling and deglycosylation by endoglycosidase H treatment clearly demonstrated that the majority of the β1C-1 and β1C-2 chains did not become maturely glycosylated, nor did they dimerise with α subunits. After 20 hours of chase, the labelled β1C-1 and β1C-2 chains had been gradually degraded, whereas immature β1A was converted into the maturely glycosylated form during the same period of time. Immunostaining showed intracellular β1 localisation in the β1C-1 and β1C-2 expressing clones, while in the β1A expressing clones the β1 chains were mainly localised to focal adhesion sites and along fibronectin fibres. Taken together, we have shown that expression of both integrin β1C-1 and β1C-2 in GD25T cells result in very low cell surface expression compared with the normal β1A isoform. Instead, both β1C-1 and β1C-2 chains remain in the endoplasmic reticulum until they are intracellularly degraded.
A contractile activity that closes phagosomes in macrophages
Authors: Swanson Joel A. , Johnson Melissa T. , Beningo Karen , Post Penny , Mooseker Mark , Araki Nobukazu 
Published: April 2021

ABSTRACT
Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes. The sequential processes of pseudopod extension and contraction can explain how macropinosomes and spacious phagosomes form without guidance from a particle surface.
Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors
Authors: Sorkina Tatiana , Bild Andrea , Tebar Francesc , Sorkin Alexander 
Published: April 2021

ABSTRACT
Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with µ1 as well as µ2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by coimmunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.
Antagonistic effects of NES and NLS motifs determine S. cerevisiae Rna1p subcellular distribution
Authors: Feng Wenqin , Benko Ann L. , Lee Jia-Hai , Stanford David R. , Hopper Anita K. 
Published: April 2021

ABSTRACT
Nucleus/cytosol exchange requires a GTPase, Ran. In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF). RanGAP is primarily cytosolic and GEF is nuclear. Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus. Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange. We provide three lines of evidence that Rna1p enters and leaves the nuclear interior. (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location. (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus. (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins. The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function.
Intracellular processing of endocytosed triglyceride-rich lipoproteins comprises both recycling and degradation
Authors: Heeren Jörg , Weber Wilfried , Beisiegel Ulrike 
Published: April 2021

ABSTRACT
The current study was performed to investigate the intracellular fate of triglyceride-rich lipoproteins. Triglyceride-rich lipoproteins are responsible for the delivery of lipids to various tissues, however, their intracellular pathway has not yet been elucidated. Here radiolabeled triglyceride-rich lipoproteins, associated with lipoprotein lipase, were used for the quantitative evaluation of the intracellular metabolism. Pulse chase experiments showed that after 90 minutes approximately 60% of the labeled protein, mainly apoproteins E and C, was released intact into the medium, where it re-associates with lipoproteins. Apoprotein B, in contrast, was degraded, following the same pathway as the apoprotein B from low density lipoproteins. In kinetic experiments uptake and intracellular fate of triglyceride-rich lipoproteins was compared to that of transferrin and low density lipoproteins. These experiments revealed that apoproteins were retained inside the cell much longer than transferrin, and unlike low density lipoproteins were not degraded. Using immunofluorescence it was shown that apoprotein E and lipoprotein lipase follow a distinct route from the sorting compartment to the surface, which is clearly distinguishable from the perinuclear transferrin recycling compartment. In contrast, the fluorescence labeled lipids were delivered to lysosomal compartments. The data presented here show that surface proteins of triglyceride-rich lipoproteins, such as apoproteins E and C and lipoprotein lipase follow a recycling pathway, whereas lipids and high molecular mass core proteins are degraded.