bluuebunny/crossref_metadata_2025_split huggingface dataset:
The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility
Authors: Gagnon Claude , White Daniel , Cosson Jacky , Huitorel Philippe , Eddé Bernard , Desbruyères Elisabeth , Paturle-Lafanechère Laurence , Multigner Luc , Job Didier , Cibert Christian 
Published: April 2021

ABSTRACT
To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly α-tubulin in sea urchin sperm axonemes and equally well brain α- and β-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of α-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated α-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of α-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate.
Taken together, the data reported here indicate that the polyglutamylated lateral chain of α-tubulin plays a dynamic role in a dynein-based motility process.
Regulation of cell migration by the integrin β subunit ectodomain
Authors: Filardo Edward J. , Deming Sandra L. , Cheresh David A. 
Published: April 2021

ABSTRACT
CS-1 melanoma cells transfected with cDNAs encoding either the β3 or β5 integrin subunit protein express αvβ3 or αvβ5, respectively, enabling them to adhere to vitronectin yet only αvβ3 promotes cell spreading and migration on this substrate. Following exposure to insulin or insulin-like growth factor, αvβ5-expressing CS-1 cells gain the ability to migrate on vitronectin. To identify structural regions in β3 or β5 that account for these distinct biological properties, CS-1 cells were transfected with one of two chimeric β subunit proteins, in which the ectoand cytoplasmic domains of β3 and β5 were exchanged (termed αvβ3/5 or αvβ5/3). Surprisingly, αvβ3/5 expressing cells spread and migrate on vitronectin while cells expressing αvβ5/3 do not unless they are exposed to cytokine. These findings suggest that the distinct migratory properties mediated by integrins αvβ3 and αvβ5 and their response to cytokine activation is determined by a sequence(s) within the ectodomain of the integrin β subunit.
Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells
Authors: Mayer Arie , Ivanov Ivan E. , Gravotta Diego , Adesnik Milton , Sabatini David D. 
Published: April 2021

ABSTRACT
An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15-30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-γ-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane.
Deficiency of the integrin β4 subunit in junctional epidermolysis bullosa with pyloric atresia: consequences for hemidesmosome formation and adhesion properties
Authors: Niessen Carien M. , van der Raaij-Helmer Liesbeth M. H. , Hulsman Esther H. M. , van der Neut Ronald , Jonkman Marcel F. , Sonnenberg Arnoud 
Published: April 2021

ABSTRACT
Junctional epidermolysis bullosa (JEB) comprises a group of inherited autosomal recessive blistering disorders characterized by dermo-epidermal separation through the lamina lucida of the basement membrane. We identified a patient with JEB associated with pyloric atresia (PA), in whom the integrin β4 subunit was completely absent. At the ultrastructural level, the hemidesmosomes were reduced in number, appeared rudimentary and lacked a subbasal dense plate and frequently an inner attachment plaque. However, keratin filaments were still anchored to the cytoplasmic plaque of the hemidesmosome. Immunofluorescence analysis showed that the β4 subunit was absent in the skin of the PA-JEB patient, whereas the α6 subunit appeared to be normally distributed along the basement membrane zone, as were the other hemidesmosomal components BP230, BP180 and HD1. Furthermore, the α3 and β1 subunits were not only detected at the lateral membranes of basal cells in PA-JEB skin, as in normal skin, but also along the basement membrane zone. The few hemidesmosome-like structures found in cultured keratinocytes from the PA-JEB patient contained the hemidesmosomal components BP230, BP180 and HD1, but not the integrin α6 subunit. Like α3, this subunit was colocalized with vinculin in focal contacts at the ends of actin stress fibers. Immunoprecipitation analysis revealed that α6 was associated with β1 on PA-JEB keratinocytes, whereas normal human keratinocytes (NHKs) exclusively express α6β4 on their cell surface. The initial adhesion of PA-JEB and normal keratinocytes to laminin-1 and laminin-5, both ligands for α6β1 and α6β4, was similar. In migration assays, the PA-JEB keratinocytes were more motile on laminin-5 than normal keratinocytes. Our observations indicate that the integrin α6β4 plays a crucial role in the proper assembly of hemidesmosomes and in the stabilization of the dermal-epidermal junction. The fragility of the skin and the blistering in this patient appear to have been due to the deficiency of the integrin β4 subunit, which results in the formation of too few and structurally abnormal hemidesmosomes.
Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system
Authors: Soussi-Yanicostas Nadia , Hardelin Jean-Pierre , Arroyo-Jimenez Maria del Mar , Ardouin Olivier , Legouis Renaud , Levilliers Jacqueline , Traincard François , Betton Jean-Michel , Cabanié Lucien , Petit Christine 
Published: April 2021

ABSTRACT
The KAL gene is responsible for the X-chromosome linked form of Kallmann’s syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.
Transport of insulin and albumin by the microvascular endothelium of the rete mirabile
Authors: Bendayan Moïse , Rasio Eugenio A. 
Published: April 2021

ABSTRACT
Vascular permeability for albumin and insulin in the continuous capillary network of the rete mirabile of the eel swimbladder was evaluated by ultrastructural immunocytochemistry and countercurrent perfusion experiments. Upon perfusion of the rete capillaries with a buffer solution containing albumin and insulin, these serum proteins were revealed at the electron microscope level, by the Protein A-gold immunocytochemical technique on a post-embedding step. For the simultaneous detection of both proteins, the double labeling technique with different sized gold particles was used. Furthermore, labeling was performed with the mixture of anti-albumin and anti-insulin antibodies. The labelings obtained were morphometrically evaluated and demonstrate that: (1) serum proteins such as albumin and insulin are transported by the endothelial cells through their plasmalemmal vesicular system; (2) insulin is transported preferentially to albumin; and (3) this transport involves different populations of plasmalem-mal vesicles. Measurements of diffusion permeability coefficients have confirmed the preferential transport of insulin, its coefficient being higher than that of albumin. Conversely, when compared to that of inulin or sucrose, which are assumed to be markers of the paracellular diffusion, it was found to be much lower, indicating that transcytosis through the vesicular system is less efficient than diffusion along the intercellular junctions. These results indicate that transcytosis of insulin and albumin occurs via different sets of plasmalemmal vesicles, probably through receptor-mediated mechanisms, and that the overall rate of transport across the rete capillaries, with respect to paracellular diffusion, is higher for insulin than for albumin.
The Schizosaccharomyces pombe pyp1 protein tyrosine phosphatase negatively regulates nutrient monitoring pathways
Authors: Santo Paola Dal , Blanchard Barbara , Hoffman Charles S. 
Published: April 2021

ABSTRACT
The Schizosaccharomyces pombe pyp1+ gene, encoding a protein tyrosine phosphatase (pyp1), was isolated as a high copy number suppressor of a mutation that results in reduced cAMP-dependent protein kinase (PKA) activity. Overexpression of pyp1+ inhibits both transcription of the fbp1 gene, which is negatively regulated by a glucoseinduced activation of PKA, and sexual development, which is negatively regulated by PKA through a nitrogen- and glucose-monitoring mechanism. Overexpression of a catalytically inactive form of pyp1 has little effect on either process. Previous studies suggest that overexpression of pyp1+ results in a mitotic delay by positively regulating wee1 activity. We show that pyp1 repression of fbp1 transcription is independent of wee1. The direct role of the pyp1 protein is to dephosphorylate and inactivate the sty1/spc1 mitogen-activated protein kinase (MAPK) that is activated by the wis1 MAPK kinase. As overexpression of pyp1+ has no further effect upon the mitotic delay observed in a wis1 deletion strain, the role of pyp1 appears to be restricted to negative regulation of the sty1/spc1 MAPK. This study indicates that pyp1 negatively regulates fbp1 transcription, sexual development and mitosis by inactivation of the sty1/spc1 MAPK, but that bifurcations downstream of the MAPK separate these processes as seen by the differential role for the wee1 gene.
The wis1 signal transduction pathway is required for expression of cAMP-repressed genes in fission yeast
Authors: Stettler Sophie , Warbrick Emma , Prochnik Simon , Mackie Shaun , Fantes Peter 
Published: April 2021

ABSTRACT
The wis1 protein kinase of Schizosaccharomyces pombe is a member of the MAP kinase kinase family. Loss of wis1 function has previously been reported to lead to a delay in the G2-mitosis transition, loss of viability in stationary phase, and hypersensitivity to osmotic shock. It acts at least in part by activating the MAP kinase homologue sty1; loss-of-function sty1 mutants share many phenotypes with wis1 deletion mutants.
We show here that, in addition, loss of wis1 function leads to defective conjugation, and to suppression of the hyperconjugation phenotype of the pat1-114 mutation. Consistent with this, the induction of the mei2 gene, which is normally induced by nitrogen starvation, is defective in wis1 mutants. In wild-type cells, nitrogen starvation leads to mei2 induction through a fall in intracellular cyclic AMP (cAMP) level and activity of the cAMP-dependent protein kinase. We show here that wis1 function is required for mei2 induction following nitrogen starvation. Expression of the fbp1 gene is negatively regulated by cAMP in response to glucose limitation: induction of fbp1 also requires wis1 and sty1 function. Loss of wis1 is epistatic over increased fbp1 expression brought about by loss of adenylate cyclase (git2/cyr1) or cAMP-dependent protein kinase (pka1) function. These observations can be explained by a model in which the pka1 pathway negatively regulates the wis1 pathway, or the two pathways might act independently on downstream targets. The latter explanation is supported, at least as regards regulation of cell division, by the observation that loss of function of the regulatory subunit of the cAMP-dependent protein kinase (cgs1) brings about a modest increase in cell length at division in both wis1+ and wis1Δ genetic backgrounds.
Calcium mobilization and influx during sperm exocytosis
Authors: Spungin Ben , Breitbart Haim 
Published: April 2021

ABSTRACT
We have previously shown that two intracellular events which occur during capacitation of bovine sperm are the formation of actin filaments on the plasma and outer acrosomal membranes and the attachment of a PIP2-specific phospholipase C (PLC) to this membrane bound F-actin. This PLC plays an essential role in sperm exocytosis (acrosome reaction). In the present report, we further elucidated the role of this PLC using a PIP2-specific PLC of bacterial origin. This PLC is different from the endogenous sperm PLC in that it is calcium independent and not inhibited by neomycin. Here we report using bovine sperm that this bacterial PLC can restore actin release from extracted membranes as well as membrane fusion in a cellfree assay when the endogenous PLC is inhibited by neomycin. The sperm PLC requires 2 μM calcium for half maximal activation, while half maximal actin release from extracted plasma membranes occurs at 80 μM. Extracted sperm membranes were examined for calcium pumps and channels. Sperm plasma membranes were found to possess a thapsigargin insensitive calcium pump and calcium channels which are opened by phosphorylation by protein kinase C. The acrosomal membrane possesses a calcium pump which is inhibited by thapsigargin and calcium channels which are opened by cAMP. These observations are discussed in terms of a model of acrosomal exocytosis which involves a calcium rise that occurs in two stages resulting from calcium mobilization from internal stores followed by influx of extracellular calcium.
Migration of highly aggressive melanoma cells on hyaluronic acid is associated with functional changes, increased turnover and shedding of CD44 receptors
Authors: Goebeler Matthias , Kaufmann Dieter , Bröcker Eva-Bettina , Klein C. Eberhard 
Published: April 2021

ABSTRACT
Recent evidence indicates that CD44, a multifunctional adhesion receptor involved in cell-cell as well as in cell-matrix interactions, plays an important role in local progression and metastasis of malignant tumors. We have studied a set of human melanoma cell lines differing in their metastatic potential in nude mice as well as in normal melanocytes for changes in CD44 expression and function. All melanocytes and melanoma cell lines tested highly expressed the CD44 standard form (CD44s, 85 kDa) but variants at low levels only. With respect to one of the CD44-associated functions primarily involved in tumor progression we found that two highly metastatic tumor cell lines, MV3 and BLM, showed fivefold higher migration rates towards hyaluronate than melanomas with low metastatic potential and normal melanocytes. Moreover, the highly metastatic cell lines expressed fourto sixfold higher levels of the CD44 epitope involved in hyaluronic acid-binding (monoclonal antibody Hermes-1) than less aggressive melanomas and melanocytes. Hermes-1 efficiently blocked haptotaxis to hyaluronate, supporting the functional relevance of this epitope. In contrast, expression levels of other CD44s epitopes recognized by seven different anti-CD44 monoclonal antibodies were unchanged, suggesting that the migratory behaviour of the cells depends on the formation of the hyaluronate-binding Hermes-1 epitope rather than on the overall CD44s surface expression, which was virtually identical in all melanoma and melanocyte cell lines tested. Differences in the accessibility of the hyaluronate-binding epitope defined by Hermes-1 correlated with the phosphorylation state of CD44s, probably reflecting different activation states of the receptor. Furthermore, immunoprecipitation and pulse/chase studies revealed a threeto fivefold increase in CD44 synthesis in the highly aggressive melanoma cells as compared to the other cell lines and the melanocytes, indicating a reduction of CD44 half-life and up-regulation of turnover. Moreover, highly aggressive melanoma cell lines were found to shed significant amounts of CD44 from the cell surface and to secrete its ligand hyaluronic acid, which may refer to an ‘autocrine’ mechanism mediating melanoma cell motility.
Changes in expression of monoclonal antibody epitopes on laminin-5r induced by cell contact
Authors: Plopper George , Falk-Marzillier Jutta , Glaser Scott , Fitchmun Mark , Giannelli Gianluigi , Romano Tracy , Jones Jonathan C. R. , Quaranta Vito 
Published: April 2021

ABSTRACT
Laminin-5r is a basement membrane component that promotes rapid adhesion and hemidesmosome formation in epithelial cells. We raised monoclonal antibodies and identified their corresponding epitopes on the constituent chains of laminin-5r by western blotting. Using a combination of immunoprecipitation and ELISA assays, we determined that these epitopes are differentially exposed on two forms of the laminin-5r heterotrimer: soluble (passively adsorbed onto plastic) and cell-associated. Antibody 5C5 epitope is exposed on the cell-associated form, but not the soluble/passively adsorbed form of laminin-5r. Epitopes reactive with antibodies CM6, FM3, and TR1 are also preferentially exposed on cell-associated laminin-5r, such that reactivity of these antibodies with the cell-associated form is fourfold higher than with the soluble/passively adsorbed form in ELISA assays. Incubation of passively adsorbed laminin-5r with the human epithelial cell line SCC12 induced exposure of 5C5 and CM6, FM3, or TR1 epitopes. These data suggest that cells actively modify laminin-5r, perhaps during matrix assembly, and that the 5C5 epitope may serve as a marker for assembled laminin-5r matrix.
v-Src induces constitutive macropinocytosis in rat fibroblasts
Authors: Veithen Alex , Cupers Philippe , Baudhuin Pierre , Courtoy Pierre J. 
Published: April 2021

ABSTRACT
The role of v-Src as regulator of fluid-phase pinocytosis was investigated in Rat-1 cells expressing a stable (Rat-1/BB16) or a thermosensitive (Rat-1/tsLA29) v-Src protein. In the second cell line, this protein is inactive when cells are cultured at 40°C but recovers its tyrosine kinase activity upon transfer to 34°C, resulting into a transformed phenotype. The rate of fluid-phase pinocytosis of the tracer horseradish peroxidase was 2-fold higher in v-Src-transformed fibroblasts (Rat-1/BB16, Rat-1/tsLA29 cultured at 34°C) as compared to nontransformed cells (Rat-1, Rat-1/tsLA29 kept at 40°C). In contrast, receptor-mediated endocytosis of transferrin was poorly affected, suggesting that structures distinct from clathrin-coated pits are involved in pinocytosis stimulation. By light and electron microscopy, transformed cells frequently contained large peroxidase-labeled pinocytic vesicles located near to membrane ruffles, demonstrating that stimulation of pinocytosis corresponds to induction of constitutive macropinocytosis.
Stimulation of pinocytosis occurred more than 8 hours after transfer to the permissive temperature, whereas transfer to the non-permissive temperature partially reversed the stimulation within 2 hours. Protein synthesis inhibition for 6 hours abrogated pinocytosis stimulation in transformed cells, indicating that constitutive macropinocytosis induced by v-Src depends on continuous synthesis of a short-lived regulatory machinery.